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Sample records for fructo oligosaccharides production

  1. Fructo-oligosaccharides: Production, Purification and Potential Applications.

    PubMed

    Bali, Vandana; Panesar, Parmjit S; Bera, Manab B; Panesar, Reeba

    2015-01-01

    The nutritional and therapeutic benefits of prebiotics have attracted the keen interest of consumers and food processing industry for their use as food ingredients. Fructo-oligosaccharides (FOS), new alternative sweeteners, constitute 1-kestose, nystose, and 1-beta-fructofuranosyl nystose produced from sucrose by the action of fructosyltransferase from plants, bacteria, yeast, and fungi. FOS has low caloric values, non-cariogenic properties, and help gut absorption of ions, decrease levels of lipids and cholesterol and bifidus-stimulating functionality. The purified linear fructose oligomers are added to various food products like cookies, yoghurt, infant milk products, desserts, and beverages due to their potential health benefits. This review is focused on the various aspects of biotechnological production, purification and potential applications of fructo-oligosaccharides. PMID:24915337

  2. New Trends and Technological Challenges in the Industrial Production and Purification of Fructo-oligosaccharides.

    PubMed

    Nobre, Clarisse; Teixeira, José A; Rodrigues, Lígia R

    2015-01-01

    An increased commercial interest in fructo-oligosaccharides (FOS) has emerged in the last decade due to their prebiotic activity. At large scale, the FOS are produced by microbial enzymes from sucrose. A mixture of FOS and other saccharides is obtained in this process. The presence of such saccharides reduces the prebiotic, caloric, and cariogenic value of the final product. Therefore, many efforts have been conducted to obtain a product with increased FOS purity. This review comprises the most important technological and physicochemical aspects including FOS production and recovery processes; safety, dose and health claims concerning its intake; and commercially available FOS. PMID:24915327

  3. Studies on production of fructo-oligosaccharides (FOS) by gamma radiation processing of microbial levan.

    PubMed

    Jalan, N; Varshney, Lalit; Misra, Nilanjal; Paul, Jhimli; Mitra, D; Rairakhwada, D D; Bhathena, Z; Kumar, Virendra

    2013-07-01

    Microbial levan, a natural polymer of fructose, was produced and purified by alcohol precipitation from culture supernatants of Bacillus megaterium type 1 grown in an optimized liquid sucrose medium. GPC analysis showed that the yield of the major fraction of levan having molecular weight ~5000 D increased with increase in sucrose concentration in the broth. Levan subjected to (60)Co-gamma radiation as well as acid hydrolysis was investigated by rheometry, UV-visible spectrophotometry and gel permeation chromatography (GPC) techniques. Unlike most of the polysaccharides, levan powder exhibited good radiation degradation stability up to 150 kGy. Gamma irradiation of 10% levan aqueous solution at 250 kGy yielded 63.0% fructo-oligosaccharide (FOS) with an average molecular weight of 1250 D. Acid hydrolysis of levan using 0.5 N HCl for 60 min treatment time gave rise to the desired FOS with lower yield (23.1%) as compared to that obtained in gamma radiolysis process. PMID:23688493

  4. A one-step bioprocess for production of high-content fructo-oligosaccharides from inulin by yeast.

    PubMed

    Wang, Da; Li, Fu-Li; Wang, Shi-An

    2016-10-20

    Commercial fructo-oligosaccharides (FOS) are predominantly produced from sucrose by transfructosylation process that presents a maximum theoretical yield below 0.60gFOSgSucrose(-1). To obtain high-content FOS, costly purification is generally employed. Additionally, high-content FOS can be produced from inulin by using endo-inulinases. However, commercial endo-inulinases have not been extensively used in scale-up production of FOS. In the present study, a one-step bioprocess that integrated endo-inulinase production, FOS fermentation, and non-FOS sugars removal into one reactor was proposed to produce high-content FOS from inulin. The bioprocess was implemented by a recombinant yeast strain JZHΔS-TSC, in which a heterologous endo-inulinase gene was expressed and the inherent invertase gene SUC2 was disrupted. FOS fermentation at 40°C from 200g/L chicory inulin presented the maximun titer, yield, and productivity of 180.2±0.8g/L, 0.9gFOSgInulin(-1), and 7.51±0.03g/L/h, respectively. This study demonstrated that the one-step bioprocess was simple and highly efficient. PMID:27474673

  5. Gaseous response to ingestion of a poorly absorbed fructo-oligosaccharide sweetener.

    PubMed

    Stone-Dorshow, T; Levitt, M D

    1987-07-01

    Fructo-oligosaccharides are naturally occurring sweet substances that are poorly absorbed and have the potential to be clinically useful nonnutritive sweeteners. Because most nonabsorbed carbohydrates are fermented yielding gas, we assessed flatulent symptoms and H2 excretion during ingestion of fructo-oligosaccharide (5 g tid) for 12 d. Ten subjects had significantly greater flatulence while taking the oligosaccharide than did five subjects taking sucrose (5 g tid). Breath H2 after 10 g fructo-oligosaccharide was similar to that of 10 g lactulose, suggesting near total malabsorption of the fructo-oligosaccharide. Although previous studies found a marked diminution in breath H2 after prolonged exposure to lactulose, breath H2 response increased by 50% after a 12-d period on the oligosaccharide and gaseous symptoms did not improve. We conclude that adaptation of colonic bacteria to carbohydrate malabsorption is variable and may depend upon quantity or nature of the carbohydrate. PMID:3604970

  6. Comparison of Yacon (Smallanthus sonchifolius) Tuber with Commercialized Fructo-oligosaccharides (FOS) in Terms of Physiology, Fermentation Products and Intestinal Microbial Communities in Rats.

    PubMed

    Utami, Ni Wayan Arya; Sone, Teruo; Tanaka, Michiko; Nakatsu, Cindy H; Saito, Akihiko; Asano, Kozo

    2013-01-01

    The yacon (Smallanthus sonchifolius) tuber was examined with regard to its prebiotic effects compared with commercialized fructo-oligosaccharides (FOS). A feed containing 10% yacon tuber, which is equivalent to 5% commercialized FOS in terms of the amount of fructo-oligosaccharides (GF2, GF3 and GF4), was administrated to rats for 28 days. The yacon diet changed the intestinal microbial communities beginning in the first week, resulting in a twofold greater concentration of cecal short-chain fatty acids (SCFAs). The SCFA composition differed, but the cecal pH in rats fed yacon tuber was equal to that in rats fed FOS. Serum triglycerides were lower in rats fed yacon compared with rats fed FOS and the control diet. Cecal size was greater with the yacon tuber diet compared with the control diet. The abundant fermentation in the intestines created a selective environment for the intestinal microbiota, which included Lactobacillus acidophilus, Bifidobacterium pseudolongum, Bifidobacterium animalis and Barnesiella spp. according to identification with culture-independent analysis, 16S rRNA gene PCR-DGGE combined with cloning and sequencing. Barnesiella spp. and B. pseudolongum were only found in the rats fed the yacon diet, while L. acidophilus and B. animalis were found in abundance in rats fed both the yacon and FOS diets. The genus Barnesiella has not previously been reported to be associated with yacon or FOS fermentation. We concluded that the physiological and microbiological effects of the yacon tuber were different from those of FOS. Differences in cecal size, blood triglycerides and microbial community profiles including their metabolites (SCFAs) between the yacon tuber and FOS were shown to be more greatly affected by the yacon tuber rather than FOS. PMID:24936376

  7. Comparison of Yacon (Smallanthus sonchifolius) Tuber with Commercialized Fructo-oligosaccharides (FOS) in Terms of Physiology, Fermentation Products and Intestinal Microbial Communities in Rats

    PubMed Central

    UTAMI, Ni Wayan Arya; SONE, Teruo; TANAKA, Michiko; NAKATSU, Cindy H; SAITO, Akihiko; ASANO, Kozo

    2013-01-01

    The yacon (Smallanthus sonchifolius) tuber was examined with regard to its prebiotic effects compared with commercialized fructo-oligosaccharides (FOS). A feed containing 10% yacon tuber, which is equivalent to 5% commercialized FOS in terms of the amount of fructo-oligosaccharides (GF2, GF3 and GF4), was administrated to rats for 28 days. The yacon diet changed the intestinal microbial communities beginning in the first week, resulting in a twofold greater concentration of cecal short-chain fatty acids (SCFAs). The SCFA composition differed, but the cecal pH in rats fed yacon tuber was equal to that in rats fed FOS. Serum triglycerides were lower in rats fed yacon compared with rats fed FOS and the control diet. Cecal size was greater with the yacon tuber diet compared with the control diet. The abundant fermentation in the intestines created a selective environment for the intestinal microbiota, which included Lactobacillus acidophilus, Bifidobacterium pseudolongum, Bifidobacterium animalis and Barnesiella spp. according to identification with culture-independent analysis, 16S rRNA gene PCR-DGGE combined with cloning and sequencing. Barnesiella spp. and B. pseudolongum were only found in the rats fed the yacon diet, while L. acidophilus and B. animalis were found in abundance in rats fed both the yacon and FOS diets. The genus Barnesiella has not previously been reported to be associated with yacon or FOS fermentation. We concluded that the physiological and microbiological effects of the yacon tuber were different from those of FOS. Differences in cecal size, blood triglycerides and microbial community profiles including their metabolites (SCFAs) between the yacon tuber and FOS were shown to be more greatly affected by the yacon tuber rather than FOS. PMID:24936376

  8. Effect of fructo-oligosaccharide and isomalto-oligosaccharide addition on baking quality of frozen dough.

    PubMed

    Park, Eun Young; Jang, Sung-Bum; Lim, Seung-Taik

    2016-12-15

    The baking quality of frozen doughs containing different levels of fructo-oligosaccharides (FO) or isomalto-oligosaccharides (IMO) (3-9%, w/w flour), and stored for 0-8weeks at -18°C, was examined. The addition of FO or IMO increased the proof volume of the dough and the loaf volume of bread prepared from frozen dough. A 6% addition of FO or IMO was optimum, giving the highest proof volume and bread loaf volume, but a higher concentration than 6% induced low baking quality including lower proof volume and bread loaf volume. The bread crumb was moister and softer after the addition of FO or IMO before, and even after, frozen storage. Darker crumb colour was observed in the bread after the addition of FO or IMO. The oligosaccharides added to the frozen dough were effective in improving the quality of bread made from frozen dough, except for resulting in a darker bread crumb. PMID:27451167

  9. Production of levansucrase from Bacillus subtilis NRC 33a and enzymic synthesis of levan and Fructo-Oligosaccharides.

    PubMed

    Abdel-Fattah, Ahmed F; Mahmoud, Doaa A R; Esawy, Mona A T

    2005-12-01

    Bacillus subtilis NRC 33a was able to produce both inducible and constitutive extracellular levansucrase, respectively, using sucrose and glucose as carbon source. The optimal production of the levansucrase was at 30 degrees C. The effect of different nitrogen sources showed that baker's yeast with 2% concentration gave the highest levansucrase activity. Addition of 0.15 g/L MgSO(4) was the most favorable for levansucrase production. The enzymic synthesis of levan was studied using 60% acetone fraction. The results indicated that high enzyme concentrations produced increasing amounts of levan, and hence conversion of fructose to levan reached 84% using 1,000 microg/ml enzyme protein. Sucrose concentration was the most effective factor controlling the molecular weight of the synthesized levan. The conversion of fructose to levan was maximal at 30 degrees C. The time of reaction clearly affected the conversion of fructose to levan, which reached its maximum productivity at 18 hours (92%). Identification of levan indicated that fructose was the building unit of levan. PMID:16328628

  10. Evaluation of commercial resins for fructo-oligosaccharide separation.

    PubMed

    Nobre, C; Suvarov, P; De Weireld, G

    2014-01-25

    Fructo-oligosaccharides (FOS) produced by fermentative processes are obtained in mixtures containing significant amounts of salts and other non-prebiotic sugars. A demineralisation process using a mixture of a cationic and an anionic resin was proposed. The separation of FOS from a mixture of fructose, glucose and sucrose was evaluated. Experiments were conducted with several commercial cationic exchange resins in calcium, sodium and potassium forms packed in preparative columns (7cm×2.2cm length×diameter). Resins in potassium form obtained the higher retention factor values for sugars when compared to the other ionic forms. However, when compared to calcium and sodium ones, resins in potassium cationic forms were shown to be the less efficient separating sugar mixtures. The resin with best separation performance was the Diaion UBK535Ca. A recovery yield of 92% (w/w) of FOS with 90% (w/w) of purity was obtained from batch experiments conducted in a single column loaded with the Diaion UBK535Ca resin at 25°C. The temperature shown did not influence the separation performance significantly. By increasing the column length, the purity of FOS increased to 92% (w/w), however the recovery yield decreased to 88% (w/w). PMID:23806732

  11. Structural analyses and immunomodulatory properties of fructo-oligosaccharides from onion (Allium cepa).

    PubMed

    Kumar, V Prasanna; Prashanth, K V Harish; Venkatesh, Y P

    2015-03-01

    Onion (Allium cepa) is an immune-boosting food rich in fructans. The major aim of this study is to characterize and investigate the immunomodulatory properties of onion fructo-oligosaccharides (FOS). FOS was isolated from onion bulbs by hot 80% ethanol extraction (yield: ∼4.5 g/100 g fw) followed by gel permeation chromatography. NMR of onion FOS revealed unusual β-D-Glc terminal residue at the non-reducing end. TLC and ESI-MS analyses showed that onion FOS ranged from trisaccharides to hexasaccharides. Onion FOS (50 μg/mL) significantly increased (∼3-fold) the proliferation of mouse splenocytes/thymocytes vs. control. Further, onion FOS enhanced (∼2.5-fold) the production of nitric oxide by peritoneal exudates cells (PECs) from Wistar rats; intracellular free radicals production and phagocytic activity of isolated murine PECs were also augmented. Our structural and in vitro results indicate that onion FOS comprising of tri- to hexasaccharide units belongs to inulin-type fructans, and possess immunostimulatory activities towards murine lymphocytes and macrophages. PMID:25498616

  12. Production of inulinase, fructosyltransferase and sucrase from fungi on low-value inulin-rich substrates and their use in generation of fructose and fructo-oligosaccharides.

    PubMed

    Rawat, Hemant Kumar; Ganaie, Mohd Anis; Kango, Naveen

    2015-03-01

    Owing to applications in the food and nutraceutical industries, inulinases, fructosyltransferases and sucrases have gained considerable attention in recent times. Twenty-five fungal strains were screened for production of these enzymes on three different media formulated using inulin-rich plant extracts prepared from asparagus root, dahlia tuber and dandelion root extract. Culture filtrates of the fungi were examined for hydrolytic activities. Fungi belonging to genus Aspergillus, A. niger GNCC 2655 (11.3 U/ml), A. awamori MTCC 2879 (8.2 U/ml), A. niger ATCC 26011 (7.9 U/ml) secreted high titers of inulinase followed by Penicillium sp. NFCCI 2768 (2.6 U/ml) and Penicillium citrinum MTCC 1256 (1.1 U/ml). High sucrase activity was noticed in A. niger GNCC 2613 (113 U/ml) and A. awamori MTCC 2879 (107.8 U/ml). Analysis of end products of inulinase action by HPLC revealed that most of the enzymes were exo-inulinases liberating fructose exclusively from inulin. Five fungi, P. citrinum MTCC 1256, Penicillium rugulosum MTCC 3487, Penicillium sp. NFCCI 2768, A. fumigatus GNCC 1351 and A. niger ATCC 26011 however, produced a mixture of endo- and exo-inulinases liberating oligosaccharides (GF3 and GF2) along with fructose. High inulinase/sucrase yielding strains were evaluated for extracellular and intracellular hydrolytic and transfructosylating activities and intracellular enzyme profiles were found to be considerably different in terms of titers and end products. PMID:25559021

  13. Fructo-oligosaccharides purification from a fermentative broth using an activated charcoal column.

    PubMed

    Nobre, C; Teixeira, J A; Rodrigues, L R

    2012-02-15

    In this study, a simple and efficient process to purify fructo-oligosaccharides (FOS) from a fermentative broth was proposed using a single activated charcoal column. The FOS adsorption onto the activated charcoal was modeled by a pseudo-second order model. Several volumes and concentrations of water/ethanol were studied to optimize the selective desorption of sugars from the broth mixture at 25°C. Mixtures containing 50.6% (w/w) of FOS (FOS content in the fermentative broth) were purified to 92.9% (w/w) with a FOS recovery of 74.5% (w/w). Moreover, with the proposed process, fractions with purity up to 97% (w/w) of FOS were obtained. This purification process was also found to be efficient in the desalting of the fermentative broth. PMID:22100432

  14. Strategy for biotechnological process design applied to the enzymatic hydrolysis of agave fructo-oligosaccharides to obtain fructose-rich syrups.

    PubMed

    García-Aguirre, Mauricio; Sáenz-Alvaro, Victor A; Rodríguez-Soto, Mayra A; Vicente-Magueyal, Francisco J; Botello-Alvarez, Enrique; Jimenez-Islas, Hugo; Cárdenas-Manríquez, Marcela; Rico-Martínez, Ramiro; Navarrete-Bolaños, Jose L

    2009-11-11

    A strategy to optimize biotechnological process design is illustrated for the production of fructose-rich syrups via enzymatic hydrolysis of agave fructo-oligosaccharides. The optimization process includes ecological studies from natural fermentations leading to the selection of a strain with capacity for inulinase synthesis, and variable optimization for the synthesis, and enzymatic hydrolysis using the response surface methodology. The results lead to the selection of Kluyveromyces marxianus , endogenous strains isolated from aguamiel (natural fermented sugary sap from agave plants), as the main strain with high capacity for enzyme synthesis with inulinase activity. Production optimization at bioreactor level revealed that operation at 30.6 degrees C, 152 rpm, 1.3 VVM of aeration, and pH 6.3 leads to maximum inulinase synthesis, whereas 31 degrees C, 50 rpm, and pH 6.2 leads to maximum hydrolysis of agave fructo-oligosaccharides. HPLC analysis of the fructose-rich syrups obtained at these optimal conditions showed an average composition of 95% of fructose and 5% of glucose and the absence of sucrose. The analysis also revealed that the syrups are free of residues and toxic compounds, an undesirable occurrence often present when traditional methods based on thermal or acid hydrolysis are applied for their obtainment. Therefore, the product may be suitable for use as additive in many applications in the food and beverage industries. PMID:19827764

  15. Dietary supplementation with short-chain fructo-oligosaccharides improves insulin sensitivity in obese horses.

    PubMed

    Respondek, F; Myers, K; Smith, T L; Wagner, A; Geor, R J

    2011-01-01

    Obesity and insulin resistance are risk factors for laminitis in horses and ponies, and diet can play an important role in modulating these risk factors. Dietary supplementation with prebiotic fibers, such as short-chain fructo-oligosaccharides (scFOS), has resulted in improvement of insulin sensitivity in obese dogs and rodents. Thus, we hypothesized that scFOS may reduce insulin resistance in obese horses and designed a study to evaluate the effect of dietary supplementation with scFOS on insulin sensitivity. Eight mature Arabian geldings (BW = 523.0 ± 56.5 kg) with an average BCS of 8 were included in a crossover study. In each period, 4 horses were provided 45 g/d per horse of maltodextrin (control) and 4 horses received the same amount of scFOS for 6 wk, with a 3-wk washout between periods. Resting plasma concentrations of glucose, insulin, triglycerides, and leptin were measured. Minimal model analysis of a frequently sampled intravenous glucose tolerance test was used to evaluate insulin sensitivity, glucose effectiveness, acute insulin response to glucose, and disposition index. Without affecting BW and BCS, dietary supplementation with scFOS increased (P < 0.05) insulin sensitivity and reduced (P < 0.05) acute insulin response to glucose in comparison with maltodextrin but did not alter (P > 0.05) glucose effectiveness and disposition index. Resting serum insulin concentration also was reduced (P < 0.05) by scFOS supplementation but not by maltodextrin (P > 0.05). There was no effect (P > 0.05) of scFOS supplementation on plasma glucose or serum triglyceride and leptin concentrations. This study demonstrated that scFOS can moderately improve insulin sensitivity of obese horses, a finding that has potential relevance to the dietary management of obese, insulin-resistant horses at increased risk for laminitis. PMID:20870952

  16. Chemical characterization and prebiotic activity of fructo-oligosaccharides from Stevia rebaudiana (Bertoni) roots and in vitro adventitious root cultures.

    PubMed

    Sanches Lopes, Sheila Mara; Francisco, Mariane Grigio; Higashi, Bruna; de Almeida, Rafaela Takako Ribeiro; Krausová, Gabriela; Pilau, Eduardo Jorge; Gonçalves, José Eduardo; Gonçalves, Regina Aparecida Correia; Oliveira, Arildo José Braz de

    2016-11-01

    Stevia rebaudiana (Bertoni) is widely studied because of its foliar steviol glycosides. Fructan-type polysaccharides were recently isolated from its roots. Fructans are reserve carbohydrates that have important positive health effects and technological applications in the food industry. The objective of the present study was to isolate and characterize fructo-oligosaccharides (FOSs) from S. rebaudiana roots and in vitro adventitious root cultures and evaluate the potential prebiotic effect of these molecules. The in vitro adventitious root cultures were obtained using a roller bottle system. Chemical analyses (gas chromatography-mass spectrometry, (1)H nuclear magnetic resonance, and off-line electrospray ionization-mass spectrometry) revealed similar chemical properties of FOSs that were obtained from the different sources. The potential prebiotic effects of FOSs that were isolated from S. rebaudiana roots enhanced the growth of both bifidobacteria and lactobacilli, with strains specificity in their fermentation ability. PMID:27516323

  17. Long term ingestion of a preload containing fructo-oligosaccharide or guar gum decreases fat mass but not food intake in mice.

    PubMed

    Hadri, Zouheyr; Chaumontet, Catherine; Fromentin, Gilles; Even, Patrick C; Darcel, Nicolas; Bouras, Abdelkader Dilmi; Tomé, Daniel; Rasoamanana, Rojo

    2015-08-01

    Fermentable dietary fibre such as fructo-oligosaccharide and viscous dietary fibers such as guar gum and alginate affect energy homeostasis. The goal of this study was to compare the impact of long term intake of these three dietary fibers on food intake, meal pattern, body weight and fat accumulation in mice. Over a period of 3weeks, the mice were fed daily with a preload containing 32mg of fructo-oligosaccharide or alginate or 13mg of guar gum. Food intake and body weight were monitored weekly, while meal patterns, adiposity and the expression of hypothalamic neuropeptide genes were evaluated at the end of the study period. The 3 dietary fibers produced a similar decrease in total daily food intake (14 to 22%) at the end of the first week, and this effect disappeared over time. The 3 dietary fibers induced a slight variation in satiation parameters. Body weight and expression of hypothalamic neuropeptide genes were not affected by any of the treatment. Preload of fructo-oligosaccharide and guar gum induced a similar and substantial decrease in the development of adiposity (17% and 14%, respectively), while alginate had no effect. Our results demonstrate mainly that the inhibitory effect of dietary fiber on food intake is lost over time, and that guar gum limits fat storage. PMID:25914171

  18. Presence of Inulin-Type Fructo-Oligosaccharides and Shift from Raffinose Family Oligosaccharide to Fructan Metabolism in Leaves of Boxtree (Buxus sempervirens).

    PubMed

    Van den Ende, Wim; Coopman, Marlies; Vergauwen, Rudy; Van Laere, André

    2016-01-01

    Fructans are known to occur in 15% of flowering plants and their accumulation is often associated with stress responses. Typically, particular fructan types occur within particular plant families. The family of the Buxaceae, harboring Pachysandra terminalis, an accumulator of graminan- and levan-type fructans, also harbors boxtree (Buxus sempervirens), a cold and drought tolerant species. Surprisingly, boxtree leaves do not accumulate the expected graminan- and levan-type fructans, but small inulin fructo-oligosaccharides (FOS: 1-kestotriose and nystose) and raffinose family oligosaccharides (RFOs: raffinose and stachyose) instead. The seasonal variation in concentrations of glucose, fructose, sucrose, FOS and RFOs were followed. Raffinose and stachyose peaked during the winter months, while FOS peaked at a very narrow time-interval in spring, immediately preceded by a prominent sucrose accumulation. Sucrose may function as a reserve carbohydrate in winter and early spring leaves. The switch from RFO to fructan metabolism in spring strongly suggests that fructans and RFOs fulfill distinct roles in boxtree leaves. RFOs may play a key role in the cold acclimation of winter leaves while temporal fructan biosynthesis in spring might increase sink strength to sustain the formation of new shoots. PMID:26973663

  19. Presence of Inulin-Type Fructo-Oligosaccharides and Shift from Raffinose Family Oligosaccharide to Fructan Metabolism in Leaves of Boxtree (Buxus sempervirens)

    PubMed Central

    Van den Ende, Wim; Coopman, Marlies; Vergauwen, Rudy; Van Laere, André

    2016-01-01

    Fructans are known to occur in 15% of flowering plants and their accumulation is often associated with stress responses. Typically, particular fructan types occur within particular plant families. The family of the Buxaceae, harboring Pachysandra terminalis, an accumulator of graminan- and levan-type fructans, also harbors boxtree (Buxus sempervirens), a cold and drought tolerant species. Surprisingly, boxtree leaves do not accumulate the expected graminan- and levan-type fructans, but small inulin fructo-oligosaccharides (FOS: 1-kestotriose and nystose) and raffinose family oligosaccharides (RFOs: raffinose and stachyose) instead. The seasonal variation in concentrations of glucose, fructose, sucrose, FOS and RFOs were followed. Raffinose and stachyose peaked during the winter months, while FOS peaked at a very narrow time-interval in spring, immediately preceded by a prominent sucrose accumulation. Sucrose may function as a reserve carbohydrate in winter and early spring leaves. The switch from RFO to fructan metabolism in spring strongly suggests that fructans and RFOs fulfill distinct roles in boxtree leaves. RFOs may play a key role in the cold acclimation of winter leaves while temporal fructan biosynthesis in spring might increase sink strength to sustain the formation of new shoots. PMID:26973663

  20. Gut morphology and hepatic oxidative status of European sea bass (Dicentrarchus labrax) juveniles fed plant feedstuffs or fishmeal-based diets supplemented with short-chain fructo-oligosaccharides and xylo-oligosaccharides.

    PubMed

    Guerreiro, Inês; Couto, Ana; Pérez-Jiménez, Amalia; Oliva-Teles, Aires; Enes, Paula

    2015-12-28

    The effects of short-chain fructo-oligosaccharides (scFOS) and xylo-oligosaccharides (XOS) on gut morphology and hepatic oxidative status were studied in European sea bass juveniles weighing 60 g. Fish were fed diets including fishmeal (FM diets) or plant feedstuffs (PF diets; 30 FM:70 PF) as main protein sources (control diets). Four other diets were formulated similar to the control diets but including 1 % scFOS or 1 % XOS. At the end of the trial, fish fed PF-based diets presented histomorphological alterations in the distal intestine, whereas only transient alterations were observed in the pyloric caeca. Comparatively to fish fed FM-based diets, fish fed PF diets had higher liver lipid peroxidation (LPO), superoxide dismutase (SOD) and catalase (CAT) activities, and lower glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase activities. In fish fed the PF diets, prebiotic supplementation decreased SOD activity and XOS supplementation further decreased CAT activity. In fish fed the FM diets, XOS supplementation promoted a reduction of all antioxidant enzyme activities. Overall, dietary XOS and scFOS supplementation had only minor effects on gut morphology or LPO levels. However, dietary XOS reduced antioxidant enzymatic activity in both PF and FM diets, which indicate a positive effect on reduction of hepatic reactive oxygen species production. PMID:26435350

  1. Arabinogalactan and fructo-oligosaccharides have a different fermentation profile in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME ®).

    PubMed

    Terpend, Kathleen; Possemiers, Sam; Daguet, David; Marzorati, Massimo

    2013-08-01

    Current prebiotics, such as fructo-oligosaccharides (FOS), are limited in their persistence in the distal colon and are predominantly fermented in the proximal colon. In order to identify a potential alternative, the differences in the fermentation profile of arabinogalactan (AG) and FOS have been assessed in the Simulator of the Human Intestinal Microbial Ecosystem. The effect of each product on the composition and activity of the microbial community was analysed during a 3-week treatment period at a dose of 5 g day(-1). While FOS indeed was mainly fermented in the simulated proximal colon, AG was still available for fermentation in the simulated distal colon as shown by pH profiles, size exclusion chromatography and analyses of specific enzymatic activities. As a consequence, the main effect of the products (increase in propionate and butyrate and decrease in ammonium production) occurred in different intestinal areas. DGGE and qPCR analyses confirmed that the main modulation of the microbiota by the two products occurred in different areas of the gut. AG was associated with a statistically significant increase in the concentration of total bacteria, Bacteroidetes, Faecalibacterium prausnitzii, a delayed bifidogenic effect and a decrease of the pathogenic Clostridium perfringens. FOS led to a strong lactobacillogenic effect. PMID:23864575

  2. In vitro evaluation of the impact of human background microbiota on the response to Bifidobacterium strains and fructo-oligosaccharides.

    PubMed

    Arboleya, Silvia; Salazar, Nuria; Solís, Gonzalo; Fernández, Nuria; Gueimonde, Miguel; de los Reyes-Gavilán, Clara G

    2013-12-14

    The microbial colonisation of the infant gut begins immediately after birth and is essential for the development of the intestine, the immune system and later well-being. Important differences have been reported in the characteristics of such microbiota in different infant population groups. In the present study, we employed an in vitro faecal batch culture model using faeces from different human population groups (adults and full-term breast-fed, full-term formula-fed and preterm infants) to determine the influence that the addition of four bifidobacterial strains and fructo-oligosaccharides (FOS) exerts on the profile of SCFA measured by GC as well as on the levels of some relevant intestinal microbial groups by quantitative PCR during incubation. Differences were found in the levels of SCFA and intestinal microbial groups in the faecal cultures depending on the human group origin of the faecal samples (P< 0·05), this being a predominant factor, compared with bifidobacteria or FOS added, in determining microbiota dynamics. These results exhibit the importance of the initial characteristics of the basal intestinal microbiota in the effect exerted by bifidobacteria or FOS that are added and highlight the need to design probiotics targeting specific human population groups. PMID:23721811

  3. Metagenomic insights into the effects of fructo-oligosaccharides (FOS) on the composition of fecal microbiota in mice.

    PubMed

    Mao, Bingyong; Li, Dongyao; Zhao, Jianxin; Liu, Xiaoming; Gu, Zhennan; Chen, Yong Q; Zhang, Hao; Chen, Wei

    2015-01-28

    Fructo-oligosaccharides (FOS) are usually regarded as a type of prebiotic, favorably stimulating the growth of bifidobacteria and lactobacilli. However, they are not the specific substrates for these target species, and other bacteria, such as Streptococcus, Escherichia, and Clostridium, have been shown to be able to utilize FOS. Previous studies have mainly investigated only a few bacteria groups, and few reports analyzed the global effects of FOS on intestinal microbial communities. In this study the effects of FOS on gut bacteria in mice were investigated through a 16S rRNA metagenomic analysis. In the FOS-low group, the abundance of Actinobacteria significantly increased and that of Bacteroidetes decreased after FOS diet (5%) for 3 weeks. In the FOS-high group, Enterococcus was promoted and levels of Bifidobacterium and Olsenella both notably increased after FOS diet (25%) and the microbiota tended to revert to initial structure 2 weeks after FOS treatment ceased. The most striking observation was that Olsenella became a dominant genus comparable with Bifidobacterium after FOS treatment, and one strain of Olsenella, isolated from mice feces, was confirmed, for the first time, to be capable of using FOS. The results indicated that metagenomic analysis was helpful to reveal the FOS effects on the global composition of gut communities and new target for future studies. PMID:25598242

  4. Transfer of blood urea nitrogen to cecal microbial nitrogen is increased by fructo-oligosaccharide feeding in guinea pigs.

    PubMed

    Kawasaki, Kiyonori; Min, Xiao; Li, Xiao; Hasegawa, Ena; Sakaguchi, Ei

    2015-01-01

    The present study was conducted to determine the mechanism by which nitrogen (N) availability is improved by fructo-oligosaccharide (FOS) in guinea pigs. Adult male guinea pigs were fed a commercial pellet diet (50 g/day) with either 5% glucose or 5% FOS for 7 days in individual metabolism cages. After 7 days of feeding the diet, (15) N-urea was administered intravenously 1 h before slaughter under anesthesia. The amount and concentration of total, protein, bacterial, ammonia and urea N and the (15) N atom % excess were measured in blood, liver, gut contents and urine. The (15) N atom % excess of total and protein N, and the amount of total, protein and bacteria N and (15) N in the cecum were significantly increased by the consumption of FOS. Furthermore, the concentration and amount of short-chain fatty acids were significantly increased by the consumption of FOS. In contrast, the amount of urinary (15) N was significantly decreased by the consumption of FOS. These results suggest that consumption of FOS increases transfer of blood urea N into the large intestine for bacterial N synthesis, which is subsequently re-absorbed by cecotrophy, and contributes to the increase of N utilization in guinea pigs. PMID:24961929

  5. Effect of sucrose concentration on the composition of enzymatically synthesized short-chain fructo-oligosaccharides as determined by FTIR and multivariate analysis.

    PubMed

    Romano, Nelson; Santos, Mauricio; Mobili, Pablo; Vega, Roberto; Gómez-Zavaglia, Andrea

    2016-07-01

    Fructo-oligosaccharides (FOS) are mixtures of oligosaccharides composed of fructose and glucose units. As their composition is determined by the synthesis conditions, the goals of this work were: (a) to engineer FOS of different composition by adjusting the sucrose concentration used as initial substrate; (b) to define partial least square (PLS) based-models to quantify all the sugars present in the reaction medium directly from the FTIR spectra. The yield of each reaction was calculated as the percentage of initial sucrose converted to each oligosaccharide, as monitored by HPLC. In parallel, the reactions were followed by FTIR. Six different PLS models aiming to determine the concentration of each carbohydrate present in the reaction medium were calibrated and independently validated. The means of predicted values fitted well to those obtained by HPLC. Determining FOS composition directly from the FTIR spectra represents a useful tool to monitor enzymatic synthesis, with strong impact at both an academic and an industrial level. PMID:26920320

  6. Short-Chain Fructo-Oligosaccharides Modulate Intestinal Microbiota and Metabolic Parameters of Humanized Gnotobiotic Diet Induced Obesity Mice

    PubMed Central

    Respondek, Frederique; Gerard, Philippe; Bossis, Mathilde; Boschat, Laura; Bruneau, Aurélia; Rabot, Sylvie; Wagner, Anne; Martin, Jean-Charles

    2013-01-01

    Prebiotic fibres like short-chain fructo-oligosaccharides (scFOS) are known to selectively modulate the composition of the intestinal microbiota and especially to stimulate Bifidobacteria. In parallel, the involvement of intestinal microbiota in host metabolic regulation has been recently highlighted. The objective of the study was to evaluate the effect of scFOS on the composition of the faecal microbiota and on metabolic parameters in an animal model of diet-induced obesity harbouring a human-type microbiota. Forty eight axenic C57BL/6J mice were inoculated with a sample of faecal human microbiota and randomly assigned to one of 3 diets for 7 weeks: a control diet, a high fat diet (HF, 60% of energy derived from fat)) or an isocaloric HF diet containing 10% of scFOS (HF-scFOS). Mice fed with the two HF gained at least 21% more weight than mice from the control group. Addition of scFOS partially abolished the deposition of fat mass but significantly increased the weight of the caecum. The analysis of the taxonomic composition of the faecal microbiota by FISH technique revealed that the addition of scFOS induced a significant increase of faecal Bifidobacteria and the Clostridium coccoides group whereas it decreased the Clostridium leptum group. In addition to modifying the composition of the faecal microbiota, scFOS most prominently affected the faecal metabolome (e.g. bile acids derivatives, hydroxyl monoenoic fatty acids) as well as urine, plasma hydrophilic and plasma lipid metabolomes. The increase in C. coccoides and the decrease in C. leptum, were highly correlated to these metabolic changes, including insulinaemia, as well as to the weight of the caecum (empty and full) but not the increase in Bifidobacteria. In conclusion scFOS induce profound metabolic changes by modulating the composition and the activity of the intestinal microbiota, that may partly explain their effect on the reduction of insulinaemia. PMID:23951074

  7. Impaired barrier function by dietary fructo-oligosaccharides (FOS) in rats is accompanied by increased colonic mitochondrial gene expression

    PubMed Central

    Rodenburg, Wendy; Keijer, Jaap; Kramer, Evelien; Vink, Carolien; van der Meer, Roelof; Bovee-Oudenhoven, Ingeborg MJ

    2008-01-01

    Background Dietary non-digestible carbohydrates stimulate the gut microflora and are therefore presumed to improve host resistance to intestinal infections. However, several strictly controlled rat infection studies showed that non-digestible fructo-oligosaccharides (FOS) increase, rather than decrease, translocation of Salmonella towards extra-intestinal sites. In addition, it was shown that FOS increases intestinal permeability already before infection. The mechanism responsible for this adverse effect of FOS is unclear. Possible explanations are altered mucosal integrity due to changes in tight junctions or changes in expression of defense molecules such as antimicrobials and mucins. To examine the mechanisms underlying weakening of the intestinal barrier by FOS, a controlled dietary intervention study was performed. Two groups of 12 rats were adapted to a diet with or without FOS. mRNA was collected from colonic mucosa and changes in gene expression were assessed for each individual rat using Agilent rat whole genome microarrays. Results Among the 997 FOS induced genes we observed less mucosal integrity related genes than expected with the clear permeability changes. FOS did not induce changes in tight junction genes and only 8 genes related to mucosal defense were induced by FOS. These small effects are unlikely the cause for the clear increase in intestinal permeability that is observed. FOS significantly increased expression of 177 mitochondria-related genes. More specifically, induced expression of genes involved in all five OXPHOS complexes and the TCA cycle was observed. These results indicate that dietary FOS influences intestinal mucosal energy metabolism. Furthermore, increased expression of 113 genes related to protein turnover, including proteasome genes, ribosomal genes and protein maturation related genes, was seen. FOS upregulated expression of the peptide hormone proglucagon gene, in agreement with previous studies, as well as three other peptide

  8. Effect of Bifidobacterium thermophilum RBL67 and fructo-oligosaccharides on the gut microbiota in Göttingen minipigs.

    PubMed

    Tanner, Sabine A; Lacroix, Christophe; Del'Homme, Christophe; Jans, Christoph; Zihler Berner, Annina; Bernalier-Donadille, Annick; Chassard, Christophe

    2015-09-14

    Modulating the gut microbiota via dietary interventions is a common strategy to enhance the natural defence mechanisms of the host. Several in vitro studies have highlighted the probiotic potential of Bifidobacterium thermophilum RBL67 (RBL67) selected for its anti-Salmonella effects. The present study aimed to investigate the impact of RBL67 alone and combined with fructo-oligosaccharides (FOS) on the gut microbiota of Göttingen minipigs. Minipigs were fed a basal diet supplemented with 8 g/d probiotic powder (1×109 CFU/g in skim milk matrix) (probiotic diet (PRO)), 8 g/d probiotic powder plus 8 g/d FOS (synbiotic diet (SYN)) or 8 g/d skim milk powder (control), following a cross-sectional study design. Faecal and caecal microbiota compositions were analysed with pyrosequencing of 16S rRNA genes and quantitative PCR. Metabolic activity in the caecum and colon was measured by HPLC. 16S rRNA gene amplicon sequencing revealed that minipig faeces show close similarity to pig microbiota. During the treatments and at the time of killing of animals, RBL67 was consistently detected in faeces, caecum and colon at numbers of 105-106 16S rRNA copies/g content after feeding PRO and SYN diets. At the time of killing of animals, significantly higher Bifidobacterium numbers in the caecum and colon of SYN-fed minipigs were measured compared with PRO. Our data indicate that the Göttingen minipig may be a suitable model for gut microbiota research in pigs. Data from this first in vivo study of RBL67 colonisation suggest that the combination with FOS may represent a valuable symbiotic strategy to increase probiotic bacteria levels and survival in gastrointestinal tracts for feed and food applications. PMID:26313935

  9. Study of the effect exerted by fructo-oligosaccharides from yacon (Smallanthus sonchifolius) root flour in an intestinal infection model with Salmonella Typhimurium.

    PubMed

    Velez, Eva; Castillo, Natalia; Mesón, Oscar; Grau, Alfredo; Bibas Bonet, María E; Perdigón, Gabriela

    2013-06-01

    Beneficial effects of prebiotics like inulin and fructo-oligosaccharides (FOS) have been proven in health and nutrition. Yacon (Smallanthus sonchifolius), an Andean crop, contains FOS (50–70% of its dry weight) and, therefore, is considered a prebiotic. Commercial FOS can upregulate total secretory IgA (S-IgA) in infant mice, prevent infection with Salmonella in swine or enhance immune response for Salmonella vaccine in a mouse model. Previously, we found that administration of yacon root flour regulates gut microbiota balance and has immunomodulatory effects without inflammatory responses. The aim of the present paper is to analyse if yacon prevents enteric infection caused by a strain of Salmonella enteritidis serovar Typhimurium (S. Typhimurium) in a mouse model. BALB/c mice were supplemented with yacon flour (45 d), challenged with S. Typhimurium and killed to study pathogen translocation, total and specific IgA production by ELISA, presence of IgA and other cytokines and Toll-like receptor 4 (TLR4) and clustor of differentiation 206 (CD206) receptors positive cells by immunofluorescence and histological changes. Yacon flour administration had a protective effect from 15 to 30 d of treatment. We found a peak of total S-IgA production without translocation of the pathogen for these periods. At 30 d, there was an increase in IL-6 and macrophage inflammatory proteins-1aþ cells and expression of the receptors CD206 and TLR4. Yacon flour did not have incidence in pathogen-specific S-IgA production. Longer periods (45 d) of administration had no protective effect. Therefore, yacon can prevent enteric infection caused by S. Typhimurium when given up to 30 d; this effect would be mediated by enhancing non-specific immunity, such as total S-IgA, that improves the immunological intestinal barrier. PMID:23137694

  10. [Determination of fructo-oligosaccharides in milk powder by high performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry].

    PubMed

    Liu, Yun; Ding, Tao; Xu, Suli; Wu, Bin; Shen, Chongyu; Zhang, Rui; Wang, Yan; Fei, Xiaoqing

    2015-10-01

    A method of high performance liquid chromatography-quadrupole/electrostatic field Orbitrap high resolution mass spectrometry (HPLC-Q/Orbitrap MS) was developed to determine fructo-oligosaccharides in milk powder. The milk powder samples were dissolved in deionized water. Subsequently, an aqueous solution of zinc acetate was used to precipitate protein. After centrifugation, the final aqueous solution was filtered by a polytetrafluoroethylene (PTFE) membrane with pore size of 0.22 μm. The analytes were separated on a Carbohydrate column (100 mm x 2.1 mm, 2.6 μm) through gradient elution with the combination of acetonitrile and 0.1% formic acid aqueous solution. The target-MS/MS templates were performed at isolation window of m/z 4.0 and collision energy of 30 eV in positive mode to extract the accurate product ion mass of analytes. Under the optimal condition, 1-kestose (GF2), nystose (GF3) and 1-F-β-fructofuranosyl nystose (GF4) were well separated and the accuracy of extracted mass routinely detected was below 5 x 10(-6) (5 ppm). The whole analysis time is only ten minutes. The detection limits for GF2 and GF3 were 100 μg/kg, and the detection limit for GF4 was 55 μg/kg. Good linearities were obtained in their respective linear ranges with correlation coefficients higher than 0.998. The average recoveries at three spiked levels (5, 10 and 20 mg/kg) were in the range of 75.8%-107.3% and the relative standard deviations (RSDs) were in the range of 1.6% - 8.3%. The proposed method is simple, sensitive, fast and only in need of precipitation of proteins. The interference of matrix can be eliminated through the selection of product ion. The results were convenient and reliable and thus can be used in the large batch determination of any milk powder. PMID:26930960

  11. A Combination Supplement of Fructo- and Xylo-Oligosaccharides Significantly Abrogates Oxidative Impairments and Neurotoxicity in Maternal/Fetal Milieu Following Gestational Exposure to Acrylamide in Rat.

    PubMed

    Krishna, Gokul; Divyashri, Gangaraju; Prapulla, S G; Muralidhara

    2015-09-01

    Prebiotic oligosaccharides are demonstrated to confer a wide spectrum of physiological benefits during pregnancy. In view of this, focused attempts are being directed towards understanding their role as modulators of brain chemistry and behavior. Epidemiological studies have identified that exposure to neurotoxins during prenatal/early life can profoundly impact neurodevelopment/function. In this context, we have tested the hypothesis that a combination of prebiotic supplements during gestation has the propensity to attenuate acrylamide (ACR) induced oxidative impairments, mitochondrial dysfunction and neurotoxicity in maternal and fetal brain of rats. To achieve this, pregnant dams given oral supplements of a combination of fructo- and xylooligosaccharides (FOS + XOS, 3 g/kg/day) during gestation days (GD 0-19) were exposed to ACR (200 ppm in drinking water, GD 6-19). The behavioral analysis revealed that ACR dams fed prebiotics displayed higher exploratory behavior in the open field test. The prenatal evaluation showed that ACR-induced decrements of placental/fetal weights were markedly restored with prebiotic feeding. Prebiotics significantly offset markers of oxidative stress, restored enzymic antioxidants, cholinergic and mitochondrial function in the maternal and fetal brain. Concomitantly, prebiotics restored ACR-induced depletion in the levels of dopamine and γ-aminobutyric acid in the maternal cortex that positively correlated with cecal bacterial numbers. Collectively, these data suggest that prenatal prebiotic oligosaccharide supplements protect developing brain against oxidative stress-mediated neurotoxicity. While the underlying mechanism/s by which prebiotics abrogate the impact of neurotoxicants in the developing brain merits further studies, we speculate that it may be mediated predominantly through attenuation of oxidative stress and proliferation of enteric microbiota. PMID:26248513

  12. Effects of dietary fructo-oligosaccharide supplementation on the growth performance, haemato-immunological parameters, gut microbiota and stress resistance of common carp (Cyprinus carpio) fry.

    PubMed

    Hoseinifar, Seyed Hossein; Soleimani, Narges; Ringø, Einar

    2014-10-28

    The present study was conducted to investigate the effects of dietary fructo-oligosaccharide (FOS) (0, 1, 2 and 3%) supplementation on the growth performance, haemato-immunological parameters, cultivable autochthonous (non-adherent) intestinal microbiota and stress resistance of common carp (Cyprinus carpio) fry (3·23 (SEM 0·14) g). These parameters were measured after feeding the carp fry with the experimental diets for 7 weeks. Dietary FOS supplementation had no significant effects on the growth performance and food intake of carp fry compared with the control treatment. It also had no significant effects on the following haematological parameters: erythrocyte count; leucocyte counts (WBC); haematocrit; Hb; mean corpuscular volume; mean corpuscular Hb content; mean corpuscular Hb concentration. However, WBC and respiratory burst activity were significantly affected by dietary FOS supplementation. Evaluation of the cultivable autochthonous intestinal microbiota revealed a significant increase in the levels of total viable heterotrophic aerobic bacteria and lactic acid bacteria in fish fed diets supplemented with 2 and 3% FOS. Furthermore, dietary FOS supplementation significantly increased the survival rate and stress resistance of carp fry compared with the control treatment. These results encourage conducting further research on the administration of FOS and other prebiotics in carp fry studies. PMID:25313574

  13. A short-term ingestion of fructo-oligosaccharides increases immunoglobulin A and mucin concentrations in the rat cecum, but the effects are attenuated with the prolonged ingestion.

    PubMed

    Komura, Mika; Fukuta, Tomonori; Genda, Tomomi; Hino, Shingo; Aoe, Seiichiro; Kawagishi, Hirokazu; Morita, Tatsuya

    2014-01-01

    We examined the effects of fructo-oligosaccharides (FOS) on IgA and mucin secretion in the rat cecum after different ingestion periods. Rats were fed a control diet or a diet containing FOS for 1, 2, 4, and 8 wk. FOS ingestion greatly increased IgA and mucin concentrations at 1 and 2 wk, but the effects were disappeared or attenuated at 4 and 8 wk. After 1 wk, FOS induced higher lactobacilli and lactate concentrations and lower cecal pH in the cecum, but the alterations were moderated with the prolonged ingestion accompanying with increasing short-chain fatty acid concentrations. At 1 and 2 wk, FOS increased IgA plasma cells and polymeric immunoglobulin receptor expression in the cecal mucosa and strongly depressed fecal mucinase activities related to the lower cecal pH. These findings may explain the FOS-induced early elevation of IgA and mucin. Clearly, FOS effects on IgA and mucin secretion considerably differ depending on the ingestion period. PMID:25209509

  14. Estimation and interpretation of fermentation in the gut: coupling results from a 24 h batch in vitro system with fecal measurements from a human intervention feeding study using fructo-oligosaccharides, inulin, gum acacia, and pea fiber.

    PubMed

    Koecher, Katie J; Noack, Jackie A; Timm, Derek A; Klosterbuer, Abby S; Thomas, William; Slavin, Joanne L

    2014-02-12

    Gut bacteria ferment fiber at different rates to primarily short chain fatty acids (SCFA) and gas while proteins are metabolized to SCFA, branched chain fatty acids (BCFA), gas, and undesirable metabolites. Large volumes of gas produced in vivo may contribute to bloating and flatulence in an individual. The objectives of this trial were to (1) compare the in vitro fermentation profiles of fructo-oligosaccharides (FOS), inulin, gum acacia, and pea fiber alone or blended using a 24 h batch model and (2) relate these findings to a human study that fed enteral formula fortified with fiber blend (FB) or no fiber (FF). The in vitro fermentation of the fiber blend resulted in a delayed pH decrease and gas and SCFA production compared to the FOS and inulin. Human samples had higher SCFA on FB compared to FF (p = 0.029). BCFA were not different between formulas. By using a blend of fibers, we observed a slower fermentation in vitro but still increased fecal SCFA when fed to human subjects. PMID:24446899

  15. Fructo-oligosaccharides and iron bioavailability in anaemic rats: the effects on iron species distribution, ferroportin-1 expression, crypt bifurcation and crypt cell proliferation in the caecum.

    PubMed

    Lobo, Alexandre R; Gaievski, Eduardo H S; De Carli, Eduardo; Alvares, Eliana P; Colli, Célia

    2014-10-28

    The present study investigated the effects of fructo-oligosaccharides (FOS) on the bioavailability of Fe from ferric pyrophosphate (FP), a water-insoluble compound, in Fe-deficient anaemic rats that were subjected to a Hb repletion assay. Male Wistar rats (n 64) were fed adequate or low (8 mg/kg) Fe diets for 15 d followed by 1 or 2 weeks of Fe repletion with diets providing 35 mg Fe/kg as ferrous sulphate (FS), FP or FP that was mixed with 7·5% FOS in the form of yacon flour or Raftilose P95 (RAF), a purified source of FOS. The effects of FOS were observed within the 1st week of the repletion period. Fe bioavailability was improved by FOS supplementation, as measured by Hb regeneration efficiency and hepatic Fe stores, which were more pronounced in the RAF group. Moreover, RAF supplementation resulted in a higher biological value relative to that of the FP group. FOS supplementation resulted in caecal enlargement, in addition to acidification and Fe species redistribution in the caecal contents relative to the control rats. These effects occurred concomitantly with decreased ferroportin (FPN)-1 expression in the caecal mucosa, which was similar in magnitude to that observed in the FS group. Caecum mucosal morphometry was influenced by FOS supplementation, whereas crypt fission and cell proliferation were highest in the caecum of the RAF group. These results reinforce the effects of FOS as Fe bioavailability enhancers in anaemic rats that are sustained by early changes in their caecal environment (decreased mucosal FPN-1 expression and increased Fe absorbability, crypt fission and cellularity). PMID:25192308

  16. Effects of supplementation with a calcium-rich marine-derived multi-mineral supplement and short-chain fructo-oligosaccharides on serum lipids in postmenopausal women.

    PubMed

    Cronin, Barbara E; Allsopp, Philip J; Slevin, Mary M; Magee, Pamela J; Livingstone, M Barbara E; Strain, J J; McSorley, Emeir M

    2016-02-28

    Recent literature suggests that Ca supplements have adverse effects on cardiovascular health. The effects of a Ca-rich supplement administered alone or in combination with short-chain fructo-oligosaccharides (scFOS) on serum lipids in postmenopausal women were examined using secondary data from a 24-month double-blind randomised controlled study. A total of 300 postmenopausal women were randomly assigned to daily supplements of 800 mg of Ca (2·4 g Aquamin) (Ca), 800 mg of Ca with 3 g of scFOS (CaFOS) or control (maltodextrin) (MD). A full lipid profile, body composition, blood pressure and a range of cytokines were measured at baseline and after 24 months. Intention-to-treat ANCOVA assessed treatment effects between the groups. A significant time-by-treatment effect was observed for LDL and total cholesterol for the Ca and CaFOS groups, with both groups having lower LDL and total cholesterol concentrations compared with MD after 24 months. The control group had mean (5·2 mmol/l) total cholesterol concentrations above the normal range (≤ 5 mmol/l) at 24 months, whereas values remained within the normal range in the treatment groups. There was no significant treatment effect on HDL-cholesterol, TAG, body composition, blood pressure or cytokine concentrations at 24 months, with the exception of IL-4, where there was a significant increase in the CaFOS group compared with the placebo. This study demonstrates a lipid-lowering effect of both the Ca-rich supplement alone and the supplement with scFOS. At the 4-year follow-up, there was no significant difference between the groups for reported diagnosed cardiovascular conditions. PMID:26669430

  17. Digestive tolerance and postprandial glycaemic and insulinaemic responses after consumption of dairy desserts containing maltitol and fructo-oligosaccharides in adults

    PubMed Central

    Respondek, F; Hilpipre, C; Chauveau, P; Cazaubiel, M; Gendre, D; Maudet, C; Wagner, A

    2014-01-01

    Background/objectives: To evaluate the short-term digestive tolerance and glycaemic response of several associations of maltitol and short-chain fructo-oligosaccharides (scFOS) used to replace sugars (for example, dextrose) in foods. Subjects/methods: Thirty-six healthy subjects aged 18–60 years were recruited for the study and 32 completed it. The subjects consumed six different mixtures of dextrose, maltitol and scFOS added in a chocolate dairy dessert at a dosage of 35 g. The test days were separated by 2-week washout periods. The subjects reported the intensity of four individual gastrointestinal (GI) symptoms, number of bowel movements and stool frequency for the 48 h following consumption of the dessert. A subgroup of 18 subjects also provided blood samples 2 h after intake to evaluate the postprandial glycaemic and insulinaemic responses. Results: The composite score calculated from the intensity of flatulence, borborygmi, bloating and discomfort was significantly higher (P<0.0001) for all the desserts containing maltitol and/or scFOS than for the control dessert containing dextrose, but remains at the level of mild effects. The number of bowel movements was also slightly increased (P=0.0006) and the stools were softer (P=0.0045) for the first 24 h but not after (P=0.1373 and 0.5420, respectively). Blood glycaemic and insulinaemic responses were lower for all the sugar-free recipes containing maltitol and scFOS in comparison to the control one (P<0.0001). Conclusions: This study has shown that maltitol and scFOS can be used jointly when formulating sugar-free foods with the benefit to lower postprandial glycaemic response with only a small and transient increase in non-serious GI symptoms. PMID:24642779

  18. Fatty Acid Composition and Sensory Characteristics of Eggs Obtained from Hens Fed Flaxseed Oil, Dried Whitebait and/or Fructo-oligosaccharide.

    PubMed

    Yi, Haechang; Hwang, Keum Taek; Regenstein, Joe M; Shin, Sung Woo

    2014-07-01

    This study was conducted to assess the effects of flaxseed oil and dried whitebait as a source of ω-3 fatty acids (ω-3 FA), which could be used to produce eggs enriched with ω-3 FA, and of fructo-oligosaccharide (FOS) as a source of prebiotics on performance of hens (commercial Hy-Line Brown laying hens), and FA composition, internal quality, and sensory characteristics of the eggs. Dietary FOS increased egg weight. The amounts of α-linolenic (ALA), eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) in the eggs from the hens fed the flaxseed oil alone or flaxseed oil+dried whitebait diets were higher than those of the control. Hedonic scores for off-flavor, fishy flavor, buttery taste and overall acceptability of the eggs from the hens fed the diet containing flaxseed oil+ dried whitebait were lower (p<0.05) than those of the control. Overall acceptability of the eggs from the hens fed the diet containing soybean oil+dried whitebait was lower (p<0.05) than that of the control. However, all the sensory attributes of the eggs from the hens fed the diet containing flaxseed oil, dried whitebait and FOS were not significantly different from those of the control. These results confirmed that flaxseed oil increases the ALA content in the eggs and a combination of flaxseed oil and dried whitebait increases EPA and DHA in the eggs. Of significance was that addition of FOS to the flaxseed oil+dried whitebait diet improves the sensory characteristics of the eggs enriched with ω-3 FA. PMID:25050046

  19. Fatty Acid Composition and Sensory Characteristics of Eggs Obtained from Hens Fed Flaxseed Oil, Dried Whitebait and/or Fructo-oligosaccharide

    PubMed Central

    Yi, Haechang; Hwang, Keum Taek; Regenstein, Joe M.; Shin, Sung Woo

    2014-01-01

    This study was conducted to assess the effects of flaxseed oil and dried whitebait as a source of ω-3 fatty acids (ω-3 FA), which could be used to produce eggs enriched with ω-3 FA, and of fructo-oligosaccharide (FOS) as a source of prebiotics on performance of hens (commercial Hy-Line Brown laying hens), and FA composition, internal quality, and sensory characteristics of the eggs. Dietary FOS increased egg weight. The amounts of α-linolenic (ALA), eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) in the eggs from the hens fed the flaxseed oil alone or flaxseed oil+dried whitebait diets were higher than those of the control. Hedonic scores for off-flavor, fishy flavor, buttery taste and overall acceptability of the eggs from the hens fed the diet containing flaxseed oil+ dried whitebait were lower (p<0.05) than those of the control. Overall acceptability of the eggs from the hens fed the diet containing soybean oil+dried whitebait was lower (p<0.05) than that of the control. However, all the sensory attributes of the eggs from the hens fed the diet containing flaxseed oil, dried whitebait and FOS were not significantly different from those of the control. These results confirmed that flaxseed oil increases the ALA content in the eggs and a combination of flaxseed oil and dried whitebait increases EPA and DHA in the eggs. Of significance was that addition of FOS to the flaxseed oil+dried whitebait diet improves the sensory characteristics of the eggs enriched with ω-3 FA. PMID:25050046

  20. Normal Growth of Healthy Infants Born from HIV+ Mothers Fed a Reduced Protein Infant Formula Containing the Prebiotics Galacto-Oligosaccharides and Fructo-Oligosaccharides: A Randomized Controlled Trial

    PubMed Central

    da Costa Ribeiro, Hugo; Ribeiro, Tereza Cristina Medrado; de Mattos, Angela Peixoto; Pontes, Mariana; Sarni, Roseli Oselka Saccardo; Cruz, Maria Letícia Santos; Nogueira-de-Almeida, Carlos Alberto; Mussi-Pinhata, Marisa M; de Carvalho Norton, Rocksane; Steenhout, Philippe

    2015-01-01

    OBJECTIVE The aim of the current study was to evaluate the safety of a new reduced protein (2.1 g/100 kcal) infant formula containing 4 g/L of 90% galacto-oligosaccharides (GOS) and 10% fructo-oligosaccharides (FOS). METHODS Healthy term infants from Brazil were enrolled. Those born to human immunodeficiency virus (HIV)-positive mothers were randomized to a test (n = 65) or control (n = 63) formula group. Infants born to HIV-negative mothers were either exclusively breast-fed (n = 79) or received a mixed diet (breast milk and test formula, n = 65). Between 2 weeks and 4 months of age, infants were exclusively fed according to their assigned group. Anthropometric measurements were taken at baseline, 1, 2, 3, 4, 6, 8, 10, and 12 months. Digestive tolerance was evaluated during the first 4 months. The primary outcome was mean daily weight gain between 2 weeks and 4 months in the test formula and breast-fed groups. RESULTS Data from all infants (N = 272) were used in the intention-to-treat (ITT) analysis and data from 230 infants were used in the per-protocol (PP) analysis. The difference in mean daily weight gain between 2 weeks and 4 months in the test formula and breast-fed groups was 1.257 g/day (one-sided 95% confidence interval [CI]: −0.705 to inf, P < 0.001) in the PP analysis, showing that the lower bound of the 95% CI was above the −3.0 g/day non-inferiority margin. Results were similar in the ITT analysis. Symptoms of digestive tolerance and frequency of adverse events were similar in the two groups. CONCLUSIONS The formula containing 2.1 g/100 kcal protein and GOS and FOS was safe and tolerated well. PMID:25788839

  1. Density distribution of free fatty acid receptor 2 (FFA2)-expressing and GLP-1-producing enteroendocrine L cells in human and rat lower intestine, and increased cell numbers after ingestion of fructo-oligosaccharide.

    PubMed

    Kaji, Izumi; Karaki, Shin-Ichiro; Tanaka, Ryo; Kuwahara, Atsukazu

    2011-02-01

    Glucagon-like peptide 1 (GLP-1) is a multifunctional hormone in glucose metabolism and intestinal function released by enteroendocrine L-cells. The plasma concentration of GLP-1 is increased by indigestible carbohydrates and luminal infusion of short-chain fatty acids (SCFAs). However, the triggers and modulators of the GLP-1 release remain unclear. We hypothesized that SCFAs produced by bacterial fermentation are involved in enteroendocrine cell proliferation and hormone release through free fatty acid receptor 2 (FFA2, also known as FFAR2 or GPR43) in the large intestine. Fructo-oligosaccharide (Fructo-OS), fermentable indigestible carbohydrate, was used as a source of SCFAs. Rats were fed an indigestible-carbohydrate-free diet (control) or a 5% Fructo-OS-containing diet for 28 days. FFA2-, GLP-1-, and 5-hydroxytryptamine (5-HT)-positive enteroendocrine cells were quantified immunohistochemically in the colon, cecum, and terminal ileum. The same analysis was performed in surgical specimens from human lower intestine. The coexpression of FFA2 with GLP-1 was investigated both in rats and humans. Fructo-OS supplementation in rats increased the densities of FFA2-positive enteroendocrine cells in rat proximal colon, by over two-fold, relative to control, in parallel with GLP-1-containing L-cells. The segmental distributions of these cells in human were similar to rats fed the control diet. The FFA2-positive enteroendocrine cells were GLP-1-containing L-cells, but not 5-HT-containing EC cells, in both human and rat colon and terminal ileum. Fermentable indigestible carbohydrate increases the number of FFA2-positive L-cells in the proximal colon. FFA2 activation by SCFAs might be an important trigger for produce and release GLP-1 by enteroendocrine L-cells in the lower intestine. PMID:21113792

  2. In vitro determination of prebiotic properties of oligosaccharides derived from an orange juice manufacturing by-product stream.

    PubMed

    Manderson, K; Pinart, M; Tuohy, K M; Grace, W E; Hotchkiss, A T; Widmer, W; Yadhav, M P; Gibson, G R; Rastall, R A

    2005-12-01

    Fermentation properties of oligosaccharides derived from orange peel pectin were assessed in mixed fecal bacterial culture. The orange peel oligosaccharide fraction contained glucose in addition to rhamnogalacturonan and xylogalacturonan pectic oligosaccharides. Twenty-four-hour, temperature- and pH-controlled, stirred anaerobic fecal batch cultures were used to determine the effects that oligosaccharides derived from orange products had on the composition of the fecal microbiota. The effects were measured through fluorescent in situ hybridization to determine changes in bacterial populations, fermentation end products were analyzed by high-performance liquid chromatography to assess short-chain fatty acid concentrations, and subsequently, a prebiotic index (PI) was determined. Pectic oligosaccharides (POS) were able to increase the bifidobacterial and Eubacterium rectale numbers, albeit resulting in a lower prebiotic index than that from fructo-oligosaccharide metabolism. Orange albedo maintained the growth of most bacterial populations and gave a PI similar to that of soluble starch. Fermentation of POS resulted in an increase in the Eubacterium rectale numbers and concomitantly increased butyrate production. In conclusion, this study has shown that POS can have a beneficial effect on the fecal microflora; however, a classical prebiotic effect was not found. An increase in the Eubacterium rectale population was found, and butyrate levels increased, which is of potential benefit to the host. PMID:16332825

  3. Supplementation with calcium and short-chain fructo-oligosaccharides affects markers of bone turnover but not bone mineral density in postmenopausal women.

    PubMed

    Slevin, Mary M; Allsopp, Philip J; Magee, Pamela J; Bonham, Maxine P; Naughton, Violetta R; Strain, J J; Duffy, Maresa E; Wallace, Julie M; Mc Sorley, Emeir M

    2014-03-01

    This 24-mo randomized, double-blind, controlled trial aimed to examine whether supplementation with a natural marine-derived multi-mineral supplement rich in calcium (Ca) taken alone and in conjunction with short-chain fructo-oligosaccharide (scFOSs) has a beneficial effect on bone mineral density (BMD) and bone turnover markers (BTMs) in postmenopausal women. A total of 300 non-osteoporotic postmenopausal women were randomly assigned to daily supplements of 800 mg of Ca, 800 mg of Ca with 3.6 g of scFOS (CaFOS), or 9 g of maltodextrin. BMD was measured before and after intervention along with BTMs, which were also measured at 12 mo. Intention-to-treat ANCOVA identified that the change in BMD in the Ca and CaFOS groups did not differ from that in the maltodextrin group. Secondary analysis of changes to BTMs over time identified a greater decline in osteocalcin and C-telopeptide of type I collagen (CTX) in the Ca group compared with the maltodextrin group at 12 mo. A greater decline in CTX was observed at 12 mo and a greater decline in osteocalcin was observed at 24 mo in the CaFOS group compared with the maltodextrin group. In exploratory subanalyses of each treatment group against the maltodextrin group, women classified with osteopenia and taking CaFOS had a smaller decline in total-body (P = 0.03) and spinal (P = 0.03) BMD compared with the maltodextrin group, although this effect was restricted to those with higher total-body and mean spinal BMD at baseline, respectively. Although the change in BMD observed did not differ between the groups, the greater decline in BTMs in the Ca and CaFOS groups compared with the maltodextrin group suggests a more favorable bone health profile after supplementation with Ca and CaFOS. Supplementation with CaFOS slowed the rate of total-body and spinal bone loss in postmenopausal women with osteopenia-an effect that warrants additional investigation. This trial was registered at www.controlled-trials.com as ISRCTN63118444. PMID

  4. Enzymatic production of specifically distributed hyaluronan oligosaccharides.

    PubMed

    Yuan, Panhong; Lv, Mengxian; Jin, Peng; Wang, Miao; Du, Guocheng; Chen, Jian; Kang, Zhen

    2015-09-20

    High-molecular-mass hyaluronan (HA) was controllably depolymerized in pure aqueous solution with recombinant leech hyaluronidase (HAase). The HAase concentration per unit HA and hydrolysis time played important roles in molecular mass distribution. By modulating the concentrations of HAase and controlling the hydrolysis time, any molar-mass-defined HA oligomers could be efficiently and specifically produced on a large scale (40 g/L), such as HA oligosaccharides with weight-average molar mass of 4000, 10,000, and 30,000Da and end hydrolysates containing only HA6 and HA4. High performance liquid chromatography-size exclusion chromatography, polyacrylamide gel electrophoresis, capillary zone electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry confirmed low polydispersity of the produced molar-mass-defined HA oligosaccharides. Therefore, large-scale production of defined HA oligosaccharides with narrow molecular mass distribution will significantly promote progress in related research and its potential applications. PMID:26050905

  5. Effect of Probiotic Lactobacillus salivarius UBL S22 and Prebiotic Fructo-oligosaccharide on Serum Lipids, Inflammatory Markers, Insulin Sensitivity, and Gut Bacteria in Healthy Young Volunteers: A Randomized Controlled Single-Blind Pilot Study.

    PubMed

    Rajkumar, Hemalatha; Kumar, Manoj; Das, Nilita; Kumar, S Nishanth; Challa, Hanumanth R; Nagpal, Ravinder

    2015-05-01

    This study investigated the effect of 6-week supplementation of a probiotic strain Lactobacillus salivarius UBL S22 with or without prebiotic fructo-oligosaccharide (FOS) on serum lipid profiles, immune responses, insulin sensitivity, and gut lactobacilli in 45 healthy young individuals. The patients were divided into 3 groups (15/group), that is, placebo, probiotic, and synbiotic. After 6 weeks, a significant reduction (P < .05) in total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides and increase in high-density lipoprotein cholesterol was observed in the probiotic as well as in the synbiotic group when compared to placebo; however, the results of total cholesterol and LDL-cholesterol were more pronounced in the synbiotic group. Similarly, when compared to the placebo group, the serum concentrations of inflammatory markers such as high sensitivity C-reactive protein, interleukin (IL) 6, IL-1β, and tumor necrosis factor α were significantly (P < .05) reduced in both the experimental groups, but again the reduction in the synbiotic group was more pronounced. Also, an increase (P < .05) in the fecal counts of total lactobacilli and a decrease (P < .05) in coliforms and Escherichia coli was observed in both the experimental groups after 6 weeks of ingestion. Overall, the combination of L salivarius with FOS was observed to be more beneficial than L salivarius alone, thereby advocating that such synbiotic combinations could be therapeutically exploited for improved health and quality of life. PMID:25331262

  6. Galactoglucomannan Oligosaccharide Supplementation Affects Nutrient Digestibility, Fermentation End-Product Production, and Large Bowel Microbiota of the Dog

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A galactoglucomannan oligosaccharide (GGMO) obtained from fiberboard production was evaluated as a dietary supplement for dogs. The GGMO substrate contained high concentrations of oligosaccharides containing mannose, xylose, and glucose, with the mannose component accounting for 35% of dry matter. ...

  7. Enzymatic production of pectic oligosaccharides from onion skins.

    PubMed

    Babbar, Neha; Baldassarre, Stefania; Maesen, Miranda; Prandi, Barbara; Dejonghe, Winnie; Sforza, Stefano; Elst, Kathy

    2016-08-01

    Onion skins are evaluated as a new raw material for the enzymatic production of pectic oligosaccharides (POS) with a targeted degree of polymerization (DP). The process is based on a two-stage process consisting of a chelator-based crude pectin extraction followed by a controlled enzymatic hydrolysis. Treatment of the extracted crude onion skin's pectin with various enzymes (EPG-M2, Viscozyme and Pectinase) shows that EPG-M2 is the most appropriate enzyme for tailored POS production. The experiments reveal that the highest amount of DP2 and DP3 is obtained at a time scale of 75-90min with an EPG-M2 concentration of 26IU/mL. At these conditions the production amounts 2.5-3.0% (w/w) d.m for DP2 and 5.5-5.6% (w/w) d.m for DP3 respectively. In contrast, maximum DP4 production of 5.2-5.5% (w/w) d.m. is obtained with 5.2IU/mL at a time scale of 15-30min. Detailed LC-MS analysis reveals the presence of more methylated oligomers compared to acetylated forms in the digests. PMID:27112872

  8. Galactoglucomannan oligosaccharide Supplementation affects Nutrient Digestibility, Fermentation End-product Production, and Large Bowel Microbiota of the Dog

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A galactoglucomannan oligosaccharide (GGMO) obtained from fiberboard production was evaluated as a dietary supplement for dogs. The GGMO substrate contained high concentrations of mannose, xylose, and glucose oligosaccharides. Adult dogs assigned to a 6x6 Latin square design were fed six diets, ea...

  9. Pectic oligosaccharides from agricultural by-products: production, characterization and health benefits.

    PubMed

    Babbar, Neha; Dejonghe, Winnie; Gatti, Monica; Sforza, Stefano; Elst, Kathy

    2016-08-01

    Pectin containing agricultural by-products are potential sources of a new class of prebiotics known as pectic oligosaccharides (POS). In general, pectin is made up of homogalacturonan (HG, α-1,4-linked galacturonic acid monomers) and rhamnogalacturonan (RG, alternate galacturonic acid and rhamnose backbone with neutral side chains). Controlled hydrolysis of pectin containing agricultural by-products like sugar beet, apple, olive and citrus by chemical, enzymatic and hydrothermal can be used to produce oligo-galacturonides (GalpOS), galacto-oligosaccharides (GalOS), rhamnogalacturonan-oligosaccharides (RGOS), etc. However, extensive research is needed to establish the role of POS, both as a prebiotic as well as therapeutic agent. This review comprehensively covers different facets of POS, including the nature and chemistry of pectin and POS, potential agricultural residual sources of pectin, pre-treatment methods for facilitating selective extraction of pectin, identification and characterization of POS, health benefits and important applications of POS in food and feed. This review has been compiled to establish a platform for future research in the purification and characterization of POS and for in vivo and in vitro studies of important POS, so that they could be commercially exploited. PMID:25641325

  10. Oligosaccharides and monomeric carbohydrates production from olive tree pruning biomass.

    PubMed

    Mateo, Soledad; Puentes, Juan G; Sánchez, Sebastián; Moya, Alberto J

    2013-04-01

    Using the severity factor, it has been possible to study cellulose and hemicellulose fractional conversion, sugar yields change and oligosaccharides variation through olive tree pruning biomass pretreatments with acid or liquid hot water under pressure. The temperatures tested were in the range 180-230°C, operation time varying between 0 and 30min and acid concentration used did not exceed 0.05M. Complete hemicellulose solubilization in autohydrolysis was achieved using severity factors (logR0) close to 3.9 (most sugars are like oligomers), while if sulfuric acid 0.025M is employed, this parameter could be smaller (≥3.4). With these treatments, we have obtained cellulose conversions between 30 and 42% from liquid hot water experiments, 40-51% with sulfuric acid 0.025M and 42-57% when the acid concentration was 0.05M. The best results in terms of maximum yield in total sugars, d-glucose and d-xylose, with a low amount of acetic acid and hydroxymethylfurfural, was obtained at 200°C, 0min (what means that there is no time of temperature maintenance, only heating and cooling) and H2SO4 0.025M. PMID:23499077

  11. A combination of soy isoflavones and cello-oligosaccharides changes equol/O-desmethylangolensin production ratio and attenuates bone fragility in ovariectomized mice.

    PubMed

    Fujii, Shungo; Takahashi, Nobuyuki; Inoue, Hirofumi; Katsumata, Shin-Ichi; Kikkawa, Yuji; Machida, Makoto; Ishimi, Yoshiko; Uehara, Mariko

    2016-08-01

    We examined the cooperative effects of isoflavones and cello-oligosaccharides on daidzein metabolism and bone fragility in ovariectomized mice. Cello-oligosaccharides increased urinary equol and decreased O-desmethylangolensin. A combination of isoflavones and cello-oligosaccharides attenuated decreases in bone breaking force and stiffness caused by ovariectomy. Combination treatment with isofalvones and cello-oligosaccharides increases urinary equol/O-desmethylangolensin production ratio and prevents ovariectomy-induced abnormalities in bone strength. PMID:27191709

  12. In vitro evaluation of defined oligosaccharide fractions in an equine model of inflammation

    PubMed Central

    2013-01-01

    Background Dietary supplementation with oligosaccharides has been proven to be beneficial for health in several mammalian species. Next to prebiotic effects resulting in a modulation of gut micro biota, immunomodulatory effects of oligosaccharides have been documented in vivo. Supplementation with defined oligosaccharide fractions has been shown to attenuate allergic responses and enhance defensive immune responses. Despite the accumulating evidence for immunomodulatory effects, very limited information is available regarding the direct mechanism of action of oligosaccharides. This study aims to elucidate the effects of selected oligosaccharide fractions on the lipopolysaccharide (LPS) induced inflammatory response in equine peripheral blood mononuclear cells (PBMCs). We investigated three different products containing either galacto-oligosaccharides (GOS) alone, a combination of GOS with fructo-oligosaccharides (FOS), and a triple combination of GOS and FOS with acidic oligosaccharides (AOS), at different concentrations. These products have been used in an identical composition in various previously published in vivo experiments. As the selected oligosaccharide fractions were derived from natural products, the fractions contained defined amounts of mono- and disaccharides and minor amounts of endotoxin, which was taken into account in the design of the study and the analysis of data. Acquired data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses. Results Exposing cultured PBMCs to either GOS or GOS/FOS fractions resulted in a substantial dose-dependent increase of tumour necrosis factor-α (TNF-α) production in LPS challenged PBMCs. In contrast, incubation with GOS/FOS/AOS resulted in a dose-dependent reduction of both TNF-α and interleukin-10 production following LPS challenge. In addition, incubation with GOS/FOS/AOS significantly increased the apparent PBMC viability, indicating a protective or

  13. Evolved beta-galactosidases from Geobacillus stearothermophilus with improved transgalactosylation yield for galacto-oligosaccharide production.

    PubMed

    Placier, Gaël; Watzlawick, Hildegard; Rabiller, Claude; Mattes, Ralf

    2009-10-01

    A mutagenesis approach was applied to the beta-galactosidase BgaB from Geobacillus stearothermophilus KVE39 in order to improve its enzymatic transglycosylation of lactose into oligosaccharides. A simple screening strategy, which was based on the reduction of the hydrolysis of a potential transglycosylation product (lactosucrose), provided mutant enzymes possessing improved synthetic properties for the autocondensation product from nitrophenyl-galactoside and galacto-oligosaccharides (GOS) from lactose. The effects of the mutations on enzyme activity and kinetics were determined. An change of one arginine to lysine (R109K) increased the oligosaccharide yield compared to that for the wild-type BgaB. Subsequently, saturation mutagenesis at this position demonstrated that valine and tryptophan further increased the transglycosylation performance of BgaB. During the transglycosylation reaction with lactose of the evolved beta-galactosidases, a major trisaccharide was formed. Its structure was characterized as beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->4)-D-glucopyranoside (3'-galactosyl-lactose). At the lactose concentration of 18% (wt/vol), this trisaccharide was obtained in yields of 11.5% (wt/wt) with GP21 (BgaB R109K), 21% with GP637.2 (BgaB R109V), and only 2% with the wild-type BgaB enzyme. GP643.3 (BgaB R109W) was shown to be the most efficient mutant, with a 3'-galactosyl-lactose production of 23%. PMID:19666723

  14. Production process for high-quality pea-protein isolate with low content of oligosaccharides and phytate.

    PubMed

    Fredrikson, M; Biot, P; Alminger, M L; Carlsson, N G; Sandberg, A S

    2001-03-01

    A process for pea-protein isolate production, resulting in low content of phytate and oligosaccharides, has been developed. Oligosaccharides were removed from the protein fraction through ultrafiltration. Ultrafiltration of 50- and 100-kD molecular-weight cutoffs (MWCOs) were tested, and both effectively separated the oligosaccharides from the protein. Phytate degradation was achieved by incubation of the pea-protein solution by addition of exogenous phytase enzyme. An almost complete degradation of inositol hexa-, penta-, tetra-, and triphosphates was reached using an incubation time of 1 h. The reduced content of oligosaccharides and inositol phosphates is likely to result in reduced flatulence and improved mineral bioavailability. These qualities of the pea-protein isolate make it a suitable protein source for infant formula production. PMID:11312837

  15. Metabolism of Oligosaccharides and Starch in Lactobacilli: A Review

    PubMed Central

    Gänzle, Michael G.; Follador, Rainer

    2012-01-01

    Oligosaccharides, compounds that are composed of 2–10 monosaccharide residues, are major carbohydrate sources in habitats populated by lactobacilli. Moreover, oligosaccharide metabolism is essential for ecological fitness of lactobacilli. Disaccharide metabolism by lactobacilli is well understood; however, few data on the metabolism of higher oligosaccharides are available. Research on the ecology of intestinal microbiota as well as the commercial application of prebiotics has shifted the interest from (digestible) disaccharides to (indigestible) higher oligosaccharides. This review provides an overview on oligosaccharide metabolism in lactobacilli. Emphasis is placed on maltodextrins, isomalto-oligosaccharides, fructo-oligosaccharides, galacto-oligosaccharides, and raffinose-family oligosaccharides. Starch is also considered. Metabolism is discussed on the basis of metabolic studies related to oligosaccharide metabolism, information on the cellular location and substrate specificity of carbohydrate transport systems, glycosyl hydrolases and phosphorylases, and the presence of metabolic genes in genomes of 38 strains of lactobacilli. Metabolic pathways for disaccharide metabolism often also enable the metabolism of tri- and tetrasaccharides. However, with the exception of amylase and levansucrase, metabolic enzymes for oligosaccharide conversion are intracellular and oligosaccharide metabolism is limited by transport. This general restriction to intracellular glycosyl hydrolases differentiates lactobacilli from other bacteria that adapted to intestinal habitats, particularly Bifidobacterium spp. PMID:23055996

  16. Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells.

    PubMed

    Loreti, Nazareth; Ambao, Verónica; Andreone, Luz; Trigo, Romina; Bussmann, Ursula; Campo, Stella

    2013-02-01

    Granulosa cell (GC) inhibin A and B production is regulated by FSH and gonadal factors. This gonadotrophin is released as a mixture of glycoforms, which induce different biological responses in vivo and in vitro. Our aim was to determine the effect of recombinant human FSH (rhFSH) glycosylation variants on inhibin A and B production by rat GCs. Preparative isoelectro focusing was used to isolate more acidic/sialylated (pH <4.00) and less acidic/sialylated (pH >5.00) rhFSH charge analogues. Concanavalin A was used to isolate unbound and firmly bound rhFSH glycoforms on the basis of their oligosaccharide complexity. GCs, obtained from oestrogen-primed immature rats, were cultured with either native rhFSH or its glycosylation variants. Inhibin A and B were determined using specific ELISAs. Results were expressed as mean±s.e.m. Under basal conditions, inhibin A was the predominant dimer produced (inhibin A: 673±55; inhibin B: 80±4  pg/ml). More acidic/sialylated charge analogues stimulated inhibin B production when compared to inhibin A at all doses studied; by contrast, less acidic/sialylated charge analogues stimulated inhibin A production and elicited no effect on inhibin B. Glycoforms bearing complex oligosaccharides showed a potent stimulatory effect on inhibin B when compared to inhibin A production (i.e. dose 1  ng/ml: 4.9±0.5 vs 0.9±0.1-fold stimulation, P<0.001). Glycoforms bearing hybrid-type oligosaccharides favoured inhibin A production (i.e. dose 4  ng/ml 2.9±0.1 vs 1.6±0.1-fold stimulation, P<0.05). These results show that the sialylation degree as well as the complexity of oligosaccharides present in the rhFSH molecule may be considered additional factors that differentially regulate dimeric inhibin production by rat GCs. PMID:23166369

  17. Recyclable Strategy for the Production of High-Purity Galacto-oligosaccharides by Kluyveromyces lactis.

    PubMed

    Sun, Huaisheng; You, Shengping; Wang, Mengfan; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-07-20

    A recyclable strategy for the production of high-purity (>95%) galacto-oligosaccharides (GOS) was developed using Kluyveromyces lactis in both the synthesis and purification steps. For the synthesis of GOS, ethanol-permeabilized cells (p-cells) of K. lactis were used because the enhanced permeability facilitated the mass transfer of the substrate and the release of oligosaccharide products. For the purification of GOS, non-permeabilized K. lactis cells (np-cells) were preferred as a result of their intrinsic cell membrane barrier toward GOS, which led to the selective consumption of carbohydrate. In this way, undesired glucose, galactose, and lactose in the raw GOS solution can be completely removed. This strategy is recyclable not only because of the high stability and reusability of p-cells and np-cells but also because the ethanol, which is simultaneously generated during the purification, can be reused for the preparation of p-cells. The strategy proposed in this study is a promising candidate for the efficient production of high-purity GOS. PMID:27366924

  18. Galacto-oligosaccharides and Colorectal Cancer: Feeding our Intestinal Probiome

    PubMed Central

    Bruno-Barcena, Jose M.; Azcarate-Peril, M. Andrea

    2014-01-01

    Prebiotics are ingredients selectively fermented by the intestinal microbiota that promote changes in the microbial community structure and/or their metabolism, conferring health benefits to the host. Studies show that β (1–4) galacto-oligosaccharides [β (1–4) GOS], lactulose and fructo-oligosaccharides increase intestinal concentration of lactate and short chain fatty acids, and stool frequency and weight, and they decrease fecal concentration of secondary bile acids, fecal pH, and nitroreductase and β-glucuronidase activities suggesting a clear role in colorectal cancer (CRC) prevention. This review summarizes research on prebiotics bioassimilation, specifically β (1–4) GOS, and their potential role in CRC. We also evaluate research that show that the impact of prebiotics on host physiology can be direct or through modulation of the gut intestinal microbiome, specifically the probiome (autochtonous beneficial bacteria), we present studies on a potential role in CRC progression to finally describe the current state of β (1–4) GOS generation for industrial production. PMID:25584074

  19. Comparative structural characterization of 7 commercial galacto-oligosaccharide (GOS) products.

    PubMed

    van Leeuwen, Sander S; Kuipers, Bas J H; Dijkhuizen, Lubbert; Kamerling, Johannis P

    2016-04-29

    Many β-galactosidase enzymes convert lactose into a mixture of galacto-oligosaccharides (GOS) when incubated under the right conditions. Recently, the composition of commercial Vivinal GOS produced by Bacillus circulans β-galactosidase was studied in much detail in another study by van Leeuwen et al. As a spin-off of this study, we used the developed analytical strategy for the evaluation of 6 anonymous commercial GOS products, in comparison with Vivinal GOS. These GOS products were first subjected to HPLC-SEC, calibrated HPAEC-PAD profiling (glucose units in relation to a malto-oligosaccharide ladder), and 1D (1)H NMR spectroscopy. For a more detailed analysis and support of the conclusions based on the initial analysis, the GOS products were separated into DP-pure subpools on Bio-Gel P-2 (MALDI-TOF-MS analysis), which were subjected to calibrated HPAEC-PAD profiling and (1)H NMR analysis. Unidentified peaks from different GOS products, not present in Vivinal GOS, were isolated for detailed structural characterization. In this way, the differences between the various GOS products in terms of DP distribution and type of glycosidic linkages were established. A total of 13 new GOS structures were characterized, adding structural-reporter-group signals and HPAEC-PAD based glucose unit G.U. values to the analytical toolbox. The newly characterized products enhance the quality of the database with GOS structures up to DP4. The combined data provide a firm basis for the rapid profiling of the GOS products of microbial β-galactosidase enzymes. PMID:27035911

  20. Efficient free fatty acid production in engineered Escherichia coli strains using soybean oligosaccharides as feedstock.

    PubMed

    Wang, Dan; Wu, Hui; Thakker, Chandresh; Beyersdorf, Jared; Bennett, George N; San, Ka-Yiu

    2015-01-01

    To be competitive with current petrochemicals, microbial synthesis of free fatty acids can be made to rely on a variety of renewable resources rather than on food carbon sources, which increase its attraction for governments and companies. Industrial waste soybean meal is an inexpensive feedstock, which contains soluble sugars such as stachyose, raffinose, sucrose, glucose, galactose, and fructose. Free fatty acids were produced in this report by introducing an acyl-ACP carrier protein thioesterase and (3R)-hydroxyacyl-ACP dehydratase into E. coli. Plasmid pRU600 bearing genes involved in raffinose and sucrose metabolism was also transformed into engineered E. coli strains, which allowed more efficient utilization of these two kinds of specific oligosaccharide present in the soybean meal extract. Strain ML103 (pRU600, pXZ18Z) produced ~1.60 and 2.66 g/L of free fatty acids on sucrose and raffinose, respectively. A higher level of 2.92 g/L fatty acids was obtained on sugar mixture. The fatty acid production using hydrolysate obtained from acid or enzyme based hydrolysis was evaluated. Engineered strains just produced ~0.21 g/L of free fatty acids with soybean meal acid hydrolysate. However, a fatty acid production of 2.61 g/L with a high yield of 0.19 g/g total sugar was observed on an enzymatic hydrolysate. The results suggest that complex mixtures of oligosaccharides derived from soybean meal can serve as viable feedstock to produce free fatty acids. Enzymatic hydrolysis acts as a much more efficient treatment than acid hydrolysis to facilitate the transformation of industrial waste from soybean processing to high value added chemicals. PMID:25919701

  1. Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

    PubMed Central

    Lezyk, Mateusz; Jers, Carsten; Kjaerulff, Louise; Gotfredsen, Charlotte H.; Mikkelsen, Maria D.; Mikkelsen, Jørn D.

    2016-01-01

    This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides. PMID:26800369

  2. Purification, characterization, and prebiotic properties of pectic oligosaccharides from orange peel wastes.

    PubMed

    Gómez, Belén; Gullón, Beatriz; Remoroza, Connie; Schols, Henk A; Parajó, Juan C; Alonso, José L

    2014-10-01

    Pectic oligosaccharides (POS) were obtained by hydrothermal treatment of orange peel wastes (OPW) and purified by membrane filtration to yield a refined product containing 90 wt % of the target products. AraOS (DP 3-21), GalOS (DP 5-12), and OGalA (DP 2-12, with variable DM) were identified in POS mixtures, but long-chain products were also present. The prebiotic potential of the concentrate was assessed by in vitro fermentation using human fecal inocula. For comparative purposes, similar experiments were performed using orange pectin and commercial fructo-oligosaccharides (FOS) as substrates for fermentation. The dynamics of selected microbial populations was assessed by fluorescent in situ hybridization (FISH). Gas generation, pH, and short-chain fatty acid (SCFA) production were also measured. Under the tested conditions, all of the considered substrates were utilized by the microbiota, and fermentation resulted in increased numbers of all the bacterial groups, but the final profile of the microbial population depended on the considered carbon source. POS boosted particularly the numbers of bifidobacteria and lactobacilli, so that the ratio between the joint counts of both genera and the total cell number increased from 17% in the inocula to 27% upon fermentation. SCFA generation from POS fermentation was similar to that observed with FOS, but pectin fermentation resulted in reduced butyrate generation. PMID:25207862

  3. Recombinant expression of a chitosanase and its application in chitosan oligosaccharide production.

    PubMed

    Liu, Ya-Li; Jiang, Shu; Ke, Zu-Min; Wu, Hai-Shui; Chi, Cheng-Wu; Guo, Zhan-Yun

    2009-04-21

    Recently, considerable attention has been focused on chitosan oligosaccharides (COSs) due to their various biological activities. COSs can be prepared by enzymatic degradation of chitosan, which is the deacetylation product of chitin, one of the most abundant biopolymers in nature. In the current study, we recombinantly expressed a chitosanase and used it for COS preparation. A bacillus-derived GH8 family chitosanase with a 6xHis tag fused at its N-terminal was expressed in the Escherichia coli strain BL21(DE3) as a soluble and active form. Its expression level could be as high as 500 mg/L. Enzymatic activity could reach approximately 140,000 U/L under our assay conditions. The recombinant chitosanase could be purified essentially to homogeneity by immobilized metal-ion affinity chromatography. The enzyme could efficiently convert chitosan into monomer-free COS: 1g of enzyme could hydrolyze about 100 kg of chitosan. Our present work has provided a cheap chitosanase for large-scale COS production in industry. PMID:19254792

  4. Heat treatment of curdlan enhances the enzymatic production of biologically active β-(1,3)-glucan oligosaccharides.

    PubMed

    Kumagai, Yuya; Okuyama, Masayuki; Kimura, Atsuo

    2016-08-01

    Biologically active β-(1,3)-glucan oligosaccharides were prepared from curdlan using GH64 enzyme (KfGH64). KfGH64 showed low activity toward native curdlan; thereby pretreatment conditions of curdlan were evaluated. KfGH64 showed the highest activity toward curdlan with heat treatment. The most efficient pretreatment (90°C for 0.5h) converted approximately 60% of curdlan into soluble saccharides under the optimized enzyme reaction conditions (pH 5.5, 37°C, 100rpm mixing speed, 24h, and 10μg of KfGH64/1g of curdlan). The resulting products were predominantly laminaripentaose and a small amount of β-(1,3)-glucans with an average degree of polymerization (DP) of 13 and 130. The products did not contain small oligosaccharides (DP<5), indicating that the hydrolysis of heat-treated curdlan by KfGH64 is a suitable method for the production of biologically active β-(1,3)-glucan oligosaccharides. PMID:27112889

  5. Rapid Screening of Bovine Milk Oligosaccharides in a Whey Permeate Product and Domestic Animal Milks by Accurate Mass Database and Tandem Mass Spectral Library.

    PubMed

    Lee, Hyeyoung; Cuthbertson, Daniel J; Otter, Don E; Barile, Daniela

    2016-08-17

    A bovine milk oligosaccharide (BMO) library, prepared from cow colostrum, with 34 structures was generated and used to rapidly screen oligosaccharides in domestic animal milks and a whey permeate powder. The novel library was entered into a custom Personal Compound Database and Library (PCDL) and included accurate mass, retention time, and tandem mass spectra. Oligosaccharides in minute-sized samples were separated using nanoliquid chromatography (nanoLC) coupled to a high resolution and sensitive quadrupole-Time of Flight (Q-ToF) MS system. Using the PCDL, 18 oligosaccharides were found in a BMO-enriched product obtained from whey permeate processing. The usefulness of the analytical system and BMO library was further validated using milks from domestic sheep and buffaloes. Through BMO PCDL searching, 15 and 13 oligosaccharides in the BMO library were assigned in sheep and buffalo milks, respectively, thus demonstrating significant overlap between oligosaccharides in bovine (cow and buffalo) and ovine (sheep) milks. This method was shown to be an efficient, reliable, and rapid tool to identify oligosaccharide structures using automated spectral matching. PMID:27428379

  6. Oligosaccharides in Food and Agriculture

    NASA Astrophysics Data System (ADS)

    Collins, Michelle E.; Rastall, Robert A.

    Oligosaccharides are an integral part of the daily diet for humans and animals. They are primarily used for their nutritional properties, however they are currently receiving much attention due to their physiological effect on the microflora of the gastrointestinal tract. Galacto-oligosaccharides and the fructan-type oligosaccharides, namely FOS and inulin are well established as beneficial to the host and are classified as prebiotic based on data from clinical studies. These compounds dominate this sector of the market, although there are oligosaccharides emerging which have produced very interesting in vitro results in terms of prebiotic status and human trials are required to strengthen the claim. Such compounds include pectic oligosaccharides, gluco-oligosaccharides, gentio-oligosaccharides, kojio-oligosaccharides, and alternan oligosaccharides. The raw materials for production of these prebiotic compounds are derived from natural sources such as plants but also from by products of the food processing industry. In addition to being prebiotic these compounds can be incorporated into foodstuffs due to the physiochemical properties they possess.

  7. Production of lactulose oligosaccharides by isomerisation of transgalactosylated cheese whey permeate obtained by β-galactosidases from dairy Kluyveromyces.

    PubMed

    Padilla, Beatriz; Frau, Florencia; Ruiz-Matute, Ana Isabel; Montilla, Antonia; Belloch, Carmela; Manzanares, Paloma; Corzo, Nieves

    2015-08-01

    β-Galactosidases from Kluyveromyces lactis and Kluyveromyces marxianus isolated from artisanal ewes' milk cheeses, were used to transgalactosylate lactose from cheese whey permeate (WP). The content of galactooligosaccharides (GOS) obtained by transgalactosylation was comparable with that formed using pure lactose as substrate. In order to obtain a mixture with higher prebiotic oligosaccharide content, isomerisation of the transgalactosylated WP was carried out using sodium aluminate as catalyst. The transgalactosylated mixtures at 6 h of reaction contained amounts of prebiotic carbohydrates (tagatose, lactulose, GOS and oligosaccharides derived from lactulose, OsLu) close to 50 g/100 g of total carbohydrates for all the strains tested, corresponding to 322 g prebiotics/kg whey permeate. Thus, the suitability of this methodology to produce mixtures of dietary non-digestible carbohydrates with prebiotic properties from WP has been demonstrated, which is interesting for the food industry since it increases the value and the applicability of this by-product from cheese manufacture. PMID:26004434

  8. Engineering the glucansucrase GTFR enzyme reaction and glycosidic bond specificity: toward tailor-made polymer and oligosaccharide products.

    PubMed

    Hellmuth, Hendrik; Wittrock, Sabine; Kralj, Slavko; Dijkhuizen, Lubbert; Hofer, Bernd; Seibel, Jürgen

    2008-06-24

    Two long-standing questions about glucansucrases (EC 2.4.1.5) are how they control oligosaccharide versus polysaccharide synthesis and how they direct their glycosidic linkage specificity. This information is required for the production of tailor-made saccharides. Mutagenesis promises to be an effective tool for enzyme engineering approaches for altering the regioselectivity and acceptor substrate specificity. Therefore, we chose the most conserved motif around the transition state stabilizer in glucansucrases for a random mutagenesis of the glucansucrase GTFR of Streptococcus oralis, yielding different variants with altered reaction specificity. Modifications at position S628 achieved by saturation mutagenesis guided the reaction toward the synthesis of short chain oligosaccharides with a drastically increased yield of isomaltose (47%) or leucrose (64%). Alternatively, GTFR variant R624G/V630I/D717A exhibited a drastic switch in regioselectivity from a dextran type with mainly alpha-1,6-glucosidic linkages to a mutan type polymer with predominantly alpha-1,3-glucosidic linkages. Targeted modifications demonstrated that both mutations near the transition state stabilizer, R624G and V630I, are contributing to this alteration. It is thus shown that mutagenesis can guide the transglycosylation reaction of glucansucrase enzymes toward the synthesis of (a) various short chain oligosaccharides or (b) novel polymers with completely altered linkages, without compromising their high transglycosylation activity and efficiency. PMID:18512955

  9. In vitro digestion and fermentation properties of linear sugar-beet arabinan and its oligosaccharides.

    PubMed

    Moon, Jin Seok; Shin, So Yeon; Choi, Hye Sun; Joo, Wooha; Cho, Seung Kee; Li, Ling; Kang, Jung-Hyun; Kim, Tae-Jip; Han, Nam Soo

    2015-10-20

    This study was conducted to investigate the prebiotic effects of linear arabino-oligosaccharides (LAOS) and debranched (linear) sugar beet arabinan (LAR) for the development of new prebiotics. LAOS were prepared from LAR by enzymatic hydrolysis with endo-arabinanase from Bacillus licheniformis, followed by removal of the arabinose fraction by incubation with resting cells of Leuconostoc mesenteroides. The resulting LAOS contained DP2 (28.7%), DP3 (49.9%), DP4 (20.1%), and DP5 (1.16%). A standardized digestibility test showed that LAOS and LAR were not digestible. Individual cultures of 24 strains of gastrointestinal bacteria showed that LAOS and LAR stimulated growth of Lactobacillus brevis, Bifidobacterium longum, and Bacteroides fragilis. In vitro batch fermentation using human fecal samples showed that LAOS had higher bifidogenic properties than LAR; LAOS increased the population of bifidobacteria which produced short-chain fatty acids (SCFAs). LAOS was fermented slowly compared to fructo-oligosaccharides and this may permit SCFA production in the distal colon. This study demonstrates that LAOS prepared from LAR are promising dietary substrates for improvement of human intestinal health. PMID:26256159

  10. Production of Feruloyated Arabinoxylo-oligosaccharides from Maize (Zea mays) Bran by Microwave-Assisted Autohydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize bran was treated with microwave irradiation (160 – 200 °C for 2 – 20 min) to release feruolyated arabinoxylo-oligosaccharides (AXOS). Lower temperatures and shorter treatment times were consistent with low AXOS yields, while higher temperatures and longer reaction times also resulted in low y...

  11. Production and bioactivity of pectic oligosaccharides from fruit and vegetable biomass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pectin is abundant in various agro-industrial bio-resources such as citrus peel, apple pomace, cranberry pulp and sugar beet pulp. These materials can therefore be considered as a source of potential bioactive pectic oligosaccharides. This chapter reviews the various extraction and purification meth...

  12. Xylo-oligosaccharides production by autohydrolysis of corn fiber separated from DDGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylo-oligosaccharides (XOS) are reported to have beneficial health properties, and are considered to be functional food ingredients. XOS was produced using corn fiber separated from distillers dried grains with solubles (DDGS). Corn fiber was treated with deionized water in a Parr-reactor, at temper...

  13. The Characterisation of an Alkali-Stable Maltogenic Amylase from Bacillus lehensis G1 and Improved Malto-Oligosaccharide Production by Hydrolysis Suppression

    PubMed Central

    Abdul Manas, Nor Hasmaliana; Pachelles, Samson; Mahadi, Nor Muhammad; Illias, Rosli Md.

    2014-01-01

    A maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 was cloned, expressed in Escherichia coli, purified and characterised for its hydrolysis and transglycosylation properties. The enzyme exhibited high stability at pH values from 7.0 to 10.0. The hydrolysis of β-cyclodextrin (β-CD) produced malto-oligosaccharides of various lengths. In addition to hydrolysis, MAG1 also demonstrated transglycosylation activity for the synthesis of longer malto-oligosaccharides. The thermodynamic equilibrium of the multiple reactions was shifted towards synthesis when the reaction conditions were optimised and the water activity was suppressed, which resulted in a yield of 38% transglycosylation products consisting of malto-oligosaccharides of various lengths. Thin layer chromatography and high-performance liquid chromatography analyses revealed the presence of malto-oligosaccharides with a higher degree of polymerisation than maltoheptaose, which has never been reported for other maltogenic amylases. The addition of organic solvents into the reaction further suppressed the water activity. The increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15 and the increased specificity toward maltopentaose production demonstrated the enhanced synthetic property of the enzyme. The high transglycosylation activity of maltogenic amylase offers a great advantage for synthesising malto-oligosaccharides and rare carbohydrates. PMID:25221964

  14. Simultaneous analysis of C1 and C4 oxidized oligosaccharides, the products of lytic polysaccharide monooxygenases acting on cellulose.

    PubMed

    Westereng, Bjørge; Arntzen, Magnus Ø; Aachmann, Finn L; Várnai, Anikó; Eijsink, Vincent G H; Agger, Jane Wittrup

    2016-05-01

    Lytic polysaccharide monooxygenases play a pivotal role in enzymatic deconstruction of plant cell wall material due to their ability to catalyze oxidative cleavage of glycosidic bonds. LPMOs may release different products, often in small amounts, with various oxidation patterns (C1 or C4) and with varying stabilities, making accurate analysis of product profiles a major challenge. So far, HPAEC has been the method of choice but it has limitations with respect to analysis of C4-oxidized products. Here, we compare various HPLC methods and present procedures that allow efficient separation of intact C1- and C4-oxidized products. We demonstrate that both PGC and HILIC (in WAX-mode) can separate C1- and C4-oxidized products and that PGC gives superior chromatographic performance. In contrast to HPAEC, these methods are directly compatible with mass spectroscopy and charged aerosol detection (CAD), which enables online peak validation and quantification with LOD levels in the low ng range. While the novel methods show lower resolution than HPAEC, this is compensated by easy peak identification, allowing, for example, discrimination between chromatographically highly similar native and C4-oxidized cello-oligomers. HPAEC-MS studies revealed chemical oxidation of C4-geminal diol products, which implies that peaks commonly believed to be C4-oxidized cello-oligomers, in fact are on-column generated derivatives. Non-destructive separation of C4-oxidized cello-oligosaccharides on the PGC column allowed us, for the first time, to isolate C4-oxidized standards. HPAEC fractionation of a purified C4-oxidized tetramer revealed that on-column decomposition leads to formation of the native trimer, which may explain why product mixtures generated by C4-oxidizing LPMOs seem to be rich in native oligosaccharides when analyzed by HPAEC. The findings and methods described here will aid in future studies in the emerging LPMO field. PMID:27059395

  15. Cashew juice containing prebiotic oligosaccharides.

    PubMed

    da Silva, Isabel Moreira; Rabelo, Maria Cristiane; Rodrigues, Sueli

    2014-09-01

    The enzyme dextransucrase in a medium containing sucrose and an acceptor as substrate synthesizes prebiotics oligosaccharides. The cashew apple juice works as a source of acceptors because it is rich in glucose and fructose (enzyme acceptors). The use of cashew apple juice becomes interesting because it aims at harnessing the peduncle of the cashew that is wasted during the nut processing, which is the product of greater economic expression. The production of dextransucrase enzyme was done by fermentative process by inoculating the bacterium Leuconostoc mesenteroides NRRL B512F into a culture medium containing sucrose as the only carbon source. Thus, the aim of this work was the production of prebiotic oligosaccharides by enzymatic process with addition of the dextransucrase enzyme to the clarified cashew apple juice. Dextran yield was favored by the combination of low concentrations of sucrose and reducing sugars. The formation of oligosaccharides was favored by increasing the concentration of reducing sugars and by the combination of high concentrations of sucrose and reducing sugars, the highest concentration of oligosaccharides obtained was 104.73 g/L and the qualitative analysis showed that at concentrations of 25 g/L and 75 g/L of sucrose and reducing sugar, respectively, it is possible to obtain oligosaccharides of degree of polymerization up to 12. The juice containing prebiotic oligosaccharide is a potential new functional beverage. PMID:25190866

  16. Assessment of suitability of vine shoots for hemicellulosic oligosaccharides production through aqueous processing.

    PubMed

    Dávila, Izaskun; Gordobil, Oihana; Labidi, Jalel; Gullón, Patricia

    2016-07-01

    Vine shoots were subjected to non-isothermal aqueous processing. A range of severities (S0) from 3.20 to 4.65 was assayed and their effects in terms of solubilization, composition, molar mass distribution, structural characterization and thermal stability of the liquors were studied using HPLC, HPSEC, TGA and FTIR. The spent solids were characterized by HPLC and FTIR. When autohydrolysis was carried out at S0=4.01, the substrate solubilization achieved a 38.7% of the raw material and 83.1% of the initial xylan was converted into xylooligosaccharides (XOS). The amount of TOS (total oligosaccharides) in the hydrolysates was 28.4g/L while the other non volatile compounds (ONVC) were 0.08g/g NVC. The spent solid from the treatment at S0=4.01 was composed about 90% of cellulose and lignin. Therefore, it can be concluded that autohydrolysis is a suitable pretreatment of vine shoots such as a first stage of a biomass refinery. PMID:27054881

  17. Application of ultrasound for enhanced extraction of prebiotic oligosaccharides from selected fruits and vegetables.

    PubMed

    Jovanovic-Malinovska, Ruzica; Kuzmanova, Slobodanka; Winkelhausen, Eleonora

    2015-01-01

    Ultrasound assisted extraction (UAE) was used to extract oligosaccharides from selected fruits (blueberry, nectarine, raspberry, watermelon) and vegetables (garlic, Jerusalem artichoke, leek, scallion, spring garlic and white onion). The individual fractions of the oligosaccharides were analyzed: 1-kestose (GF2), nystose (GF3) and 1F-β-fructofuranosylnystose (GF4) from the fructo-oligosaccharides (FOS), and raffinose and stachyose from the raffinose family oligosaccharides (RFO). Extraction parameters including solvent concentration (35-85% v/v), extraction temperature (25-50°C) and sonication time (5-15min) were examined using response surface methodology (RSM). Ethanol concentration of 63% v/v, temperature of 40°C and extraction time of 10min gave maximal concentration of the extracted oligosaccharides. The experimental values under optimal conditions were consistent with the predicted values. UAE increased the concentration of extracted oligosaccharides in all fruits and vegetables from 2 to 4-fold compared to conventional extraction. The highest increase of total oligosaccharides extracted by UAE was detected in Jerusalem artichoke, 7.17±0.348g/100gFW, compared to 1.62±0.094g/100gFW with conventional method. PMID:25116595

  18. Hemicellulose isolation, characterization, and the production of xylo-oligosaccharides from the wastewater of a viscose fiber mill.

    PubMed

    Zhang, Yuedong; Yu, Guang; Li, Bin; Mu, Xindong; Peng, Hui; Wang, Haisong

    2016-05-01

    Viscose fiber mills generate a lot of wastewater enriched with hemicelluloses. The structure of the hemicellulose in the wastewater was characterized and the hemicellulose was isolated to produce xylo-oligosaccharides (XOS). It was confirmed that the hemicellulose was mainly 4-O-methylglucuronoxylan with a small amount of glucomannan and xyloglucan. The 4-O-methylglucuronoxylan was completely de-acetylated and linear with a few 4-O-methyl glucuronic acid attached. After purified by the acid precipitation and washing, the hemicellulose was pretreated by dilute acid, and then subjected to xylanase hydrolysis. After the dilute H2SO4 pretreatment at pH 2.6 and 150°C for 30min and the followed xylanase hydrolysis (65IU/g xylan), the total XOS yield was improved from 0.215 to 0.578g/g xylan. The percentage of XOS in the final sugar product was 68.9%. These results demonstrated the potential economical and environmental benefits of the process to utilize the byproducts from viscose fiber mills. PMID:26877018

  19. Nature and biosynthesis of galacto-oligosaccharides related to oligosaccharides in human breast milk

    PubMed Central

    Intanon, Montira; Arreola, Sheryl Lozel; Pham, Ngoc Hung; Kneifel, Wolfgang; Haltrich, Dietmar; Nguyen, Thu-Ha

    2014-01-01

    Human milk oligosaccharides (HMO) are prominent among the functional components of human breast milk. While HMO have potential applications in both infants and adults, this potential is limited by the difficulties in manufacturing these complex structures. Consequently, functional alternatives such as galacto-oligosaccharides are under investigation, and nowadays, infant formulae are supplemented with galacto-oligosaccharides to mimic the biological effects of HMO. Recently, approaches toward the production of defined human milk oligosaccharide structures using microbial, fermentative methods employing single, appropriately engineered microorganisms were introduced. Furthermore, galactose-containing hetero-oligosaccharides have attracted an increasing amount of attention because they are structurally more closely related to HMO. The synthesis of these novel oligosaccharides, which resemble the core of HMO, is of great interest for applications in the food industry. PMID:24571717

  20. Cellulosimicrobium cellulans strain E4-5 enzymatic hydrolysis of curdlan for production of (1 → 3)-linked β-D-glucan oligosaccharides.

    PubMed

    Fu, Yunbin; Cheng, Likun; Meng, Yanyu; Li, Shuguang; Zhao, Xiaoming; Du, Yuguang; Yin, Heng

    2015-12-10

    In order to find an efficient enzymatic tool for curdlan degradation to produce (1 → 3)-linked β-D-glucan oligosaccharides, strain E4-5 (registration number JN089883, Genbank) was isolated from seaside soil. The 16S rRNA gene sequencing classified it as Cellulosimicrobium cellulans. It was the first reported microorganism that succeeded in degrading high-set heated curdlan blocks. The ferments of strain E4-5 also showed good degradation effects on laminaran and alkali-neutralized curdlan. Due to the products with less amount of glucose, it was assumed that endo-1,3-β-glucanases of strain E4-5 had a greater hydrolyzing effect than exo-1,3-β-glucanases. This indicated that strain E4-5 was a promising microorganism to hydrolyze (1 → 3)-linked β-D-glucan. Moreover, alkali-neutralization pretreatment was effective for promoting a more diversified degree of polymerization (DP) of (1 → 3)-linked β-D-glucan oligosaccharides under enzymatic hydrolysis and will pave the way for making full use of curdlan for production of glucan oligosaccharides. PMID:26428180

  1. Production of high-purity isomalto-oligosaccharides syrup by the enzymatic conversion of transglucosidase and fermentation of yeast cells.

    PubMed

    Pan, Yen-Chung; Lee, Wen-Chien

    2005-03-30

    A method for the production of high-purity isomalto-oligosaccharides (IMO) involving the transglucosylation by transglucosidase and yeast fermentation was proposed. The starch of rice crumbs was enzymatically liquefied and saccharified, and then converted to low-purity IMO syrup by transglucosylation. The low-purity IMO produced either from rice crumbs or tapioca flour as the starch source could be effectively converted to high-purity IMO by yeast fermentation to remove the digestible sugars including glucose, maltose, and maltotriose. Both Saccharomyces carlsbergensis and Saccharomyces cerevisiae were able to ferment glucose in the IMO syrup. Cells of S. carlsbergensis harvested from the medium of malt juice were also able to ferment maltose and maltotriose. A combination of these two yeasts or S. carlsbergensis alone could be used to totally remove the digestible sugars in the IMO, coupled with the production of ethanol. The resultant high-purity IMO, including mainly isomaltose, panose, and isomaltotriose made up more than 98% w/w of the total sugars after a 3-day fermentation. When the low-purity IMO was produced from the starch of tapioca flour, 3-day fermentation under the same conditions resulted in IMO with purity lower than that from rice crumbs. For low-purity IMO from rice crumbs, fermentation with washed S. carlsbergensis cells harvested at log phase was the most effective. However, for the low-purity IMO from tapioca flour, incubation with S. cerevisiae for the first 24 h and then supplementing with an equal amount of S. carlsbergensis cells for further fermentation was the most effective approach for producing high-purity IMO. PMID:15672377

  2. Oligosaccharide-specific induction of interleukin 10 production by B220+ cells from schistosome-infected mice: a mechanism for regulation of CD4+ T-cell subsets.

    PubMed Central

    Velupillai, P; Harn, D A

    1994-01-01

    Defining the factors and/or mechanisms that lead to the predominance of a particular CD4+ T-cell subset (Th-1 vs. Th-2) is an area of intense investigation. In murine schistosomiasis, Th-2-type T cells become predominant after deposition of eggs. The most immunoreactive egg components are glycoproteins. Previously we identified two interesting oligosaccharides found on schistosome eggs and schistosomula. One, lacto-N-fucopentaose III (LNFP-III) contains the interesting trisaccharide Lewisx, which is a weak ligand for P-selectin and is a sugar also found on the alpha and beta chains of the integrin lymphocyte function-associated molecule 1, a ligand for intercellular adhesion molecule 1. Because of the correlation between schistosome egg glycoproteins and Th-2 dominance, the present study examined whether LNFP-III and structurally related oligosaccharides were lymphostimulatory and/or able to induce factors known to down-regulate Th-1 cells. We found that LNFP-III and related sugars did induce proliferation of splenic non-T cells, B220+,CD4-,CD8- cells (B cells) of schistosome-infected and naive mice. In contrast to proliferation, LNFP-III was the only oligosaccharide that induced spleen cells to produce large amounts of interleukin 10 and prostaglandin E2, two molecules known to down-regulate Th-1 cells. Further, only spleen cells from infected mice produced cytokines after oligosaccharide stimulation. Interestingly, LNFP-III stimulation did not induce production of interleukin 4. Thus, a specific carbohydrate ligand has been identified that stimulates B cells to proliferate and produce factors that down-regulate Th-1 T cells. Further, we suggest that identical or structurally related ligands may contribute to the known Th-1 down-regulation in other parasitic diseases and in chronic blood-vascular diseases such as human immunodeficiency virus infection and a number of metastatic carcinomas and that this effect may, therefore, be a general phenomenon. PMID:7904066

  3. Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

    PubMed Central

    van Hijum, S. A. F. T.; van Geel-Schutten, G. H.; Rahaoui, H.; van der Maarel, M. J. E. C.; Dijkhuizen, L.

    2002-01-01

    Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established. PMID:12200292

  4. Sucrose and Related Oligosaccharides

    NASA Astrophysics Data System (ADS)

    Eggleston, Gillian

    Sucrose (α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside) is the most common low-molecular-weight sugar found in the plant kingdom. It is ubiquitously known as common table sugar and primarily produced industrially from sugarcane (Saccharum officinarum) and sugar beet (Beta vulgaris); the basics of the industrial manufacture of sucrose are outlined in this chapter. Commercial sucrose has a very high purity (> 99.9%) making it one of the purest organic substances produced on an industrial scale. Value-addition to sucrose via chemical and biotechnological reactions is becoming more important for the diversification of the sugar industry to maintain the industries' competitiveness in a world increasingly turning to a bio-based economy. The basis for the chemical reactivity of sucrose is the eight hydroxyl groups present on the molecule, although, sucrose chemical reactivity is regarded as difficult. Increasing use of enzymatic biotechnological techniques to derivatize sucrose is expected, to add special functionalities to sucrose products like biodegradability, biocompatibility, and non-toxicity. Analysis of sucrose by colorimetric, enzymatic, oxidation-reduction and chromatography methods are discussed. Oligosaccharides related to sucrose are outlined in detail and include sucrose-based plant, honey and in vitro oligosaccharides.

  5. Variations in prebiotic oligosaccharide fermentation by intestinal lactic acid bacteria.

    PubMed

    Endo, Akihito; Nakamura, Saki; Konishi, Kenta; Nakagawa, Junichi; Tochio, Takumi

    2016-01-01

    Prebiotic oligosaccharides confer health benefits on the host by modulating the gut microbiota. Intestinal lactic acid bacteria (LAB) are potential targets of prebiotics; however, the metabolism of oligosaccharides by LAB has not been fully characterized. Here, we studied the metabolism of eight oligosaccharides by 19 strains of intestinal LAB. Among the eight oligosaccharides used, 1-kestose, lactosucrose and galactooligosaccharides (GOSs) led to the greatest increases in the numbers of the strains tested. However, mono- and disaccharides accounted for more than half of the GOSs used, and several strains only metabolized the mono- and di-saccharides in GOSs. End product profiles indicated that the amounts of lactate produced were generally consistent with the bacterial growth recorded. Oligosaccharide profiling revealed the interesting metabolic manner in Lactobacillus paracasei strains, which metabolized all oligosaccharides, but left sucrose when cultured with fructooligosaccharides. The present study clearly indicated that the prebiotic potential of each oligosaccharide differs. PMID:26888650

  6. Production of lactose-free galacto-oligosaccharide mixtures: comparison of two cellobiose dehydrogenases for the selective oxidation of lactose to lactobionic acid.

    PubMed

    Maischberger, Thomas; Nguyen, Thu-Ha; Sukyai, Prakit; Kittl, Roman; Riva, Sergio; Ludwig, Roland; Haltrich, Dietmar

    2008-08-11

    Galacto-oligosaccharides, complex mixtures of various sugars, are produced by transgalactosylation from lactose using beta-galactosidase and are of great interest for food and feed applications because of their prebiotic properties. Most galacto-oligosaccharide preparations currently available in the market contain a significant amount of monosaccharides and lactose. The mixture of galacto-oligosaccharides (GalOS) in this study produced from lactose using recombinant beta-galactosidase from Lactobacillus reuteri contains 48% monosaccharides, 26.5% lactose and 25.5% GalOS. To remove efficiently both monosaccharides and lactose from this GalOS mixture containing significant amounts of prebiotic non-lactose disaccharides, a biocatalytic approach coupled with subsequent chromatographic steps was used. Lactose was first oxidised to lactobionic acid using fungal cellobiose dehydrogenases, and then lactobionic acid and monosaccharides were removed by ion-exchange and size-exclusion chromatography. Two different cellobiose dehydrogenases (CDH), originating from Sclerotium rolfsii and Myriococcum thermophilum, were compared with respect to their applicability for this process. CDH from S. rolfsii showed higher specificity for the substrate lactose, and only few other components of the GalOS mixture were oxidised during prolonged incubation. Since these sugars were only converted once lactose oxidation was almost complete, careful control of the CDH-catalysed reaction will significantly reduce the undesired oxidation, and hence subsequent removal, of any GalOS components. Removal of ions and monosaccharides by the chromatographic steps gave an essentially pure GalOS product, containing less than 0.3% lactose and monosaccharides, in a yield of 60.3%. PMID:18353295

  7. Non-digestible oligosaccharides used as prebiotic agents: mode of production and beneficial effects on animal and human health.

    PubMed

    Grizard, D; Barthomeuf, C

    1999-01-01

    Prebiotic agents are food ingredients that are potentially beneficial to the health of consumers. The main commercial prebiotic agents consist of oligosaccharides and dietary fibres (mainly inulin). They are essentially obtained by one of three processes: 1) the direct extraction of natural polysaccharides from plants; 2) the controlled hydrolysis of such natural polysaccharides; 3) enzymatic synthesis, using hydrolases and/or glycosyl transferases. Both of these enzyme types catalyse transglycosylation reactions, allowing synthesis of small molecular weight synthetic oligosaccharides from mono- and disaccharides. Presently, in Europe, inulin-type fructans, characterised by the presence of fructosyl units bound to the beta-2,1 position of sucrose, are considered as one of the carbohydrate prebiotic references. Prebiotics escape enzymatic digestion in the upper gastrointestinal tract and enter the caecum without change to their structure. None are excreted in the stools, indicating that they are fermented by colonic flora so as to give a mixture of short-chain fatty acids (acetate, propionate and butyrate), L-lactate, carbon dioxide and hydrogen. By stimulating bifidobacteria, they may have the following implications for health: 1) potential protective effects against colorectal cancer and infectious bowel diseases by inhibiting putrefactive bacteria (Clostridium perfringens ) and pathogen bacteria (Escherichia coli, Salmonella, Listeria and Shigella ), respectively; 2) improvement of glucid and lipid metabolisms; 3) fibre-like properties by decreasing the renal nitrogen excretion; 4) improvement in the bioavailability of essential minerals; and 5) low cariogenic factor. These potential beneficial effects have been largely studied in animals but have not really been proven in humans. The development of a second generation of oligosaccharides and the putative implication of a complex bacterial trophic chain in the intestinal prebiotic fermentation process are also

  8. Characterization of an antigenic oligosaccharide from Leptospira interrogans serovar pomona and its role in immunity.

    PubMed Central

    Midwinter, A; Vinh, T; Faine, S; Adler, B

    1994-01-01

    An antigenic oligosaccharide fraction derived from the lipopolysaccharide of Leptospira interrogans serovar pomona was isolated by endo-glycosidase H digestion and column chromatography. The oligosaccharide contained rhamnose, ribose, glucose, and glucosamine and inhibited the binding of opsonic, protective monoclonal antibodies directed against the lipopolysaccharide. When conjugated to diphtheria toxoid, the oligosaccharide elicited the production of agglutinating, opsonic antibodies. Images PMID:7960129

  9. Quality and nutritional properties of pasta products enriched with immature wheat grain.

    PubMed

    Casiraghi, Maria Cristina; Pagani, Maria Ambrogina; Erba, Daniela; Marti, Alessandra; Cecchini, Cristina; D'Egidio, Maria Grazia

    2013-08-01

    In this study, nutritional and sensory properties of pasta enriched with 30% immature wheat grain (IWG), a natural source of fructo-oligosaccharides (FOS), are evaluated. Colour and cooking quality, nutritional value and glycaemic index (GI) of pasta were assessed in comparison with commercially enriched inulin and 100% wholewheat pastas. IWG integration induced deep changes in colour, without negatively affecting the cooking quality of pasta, and promoted nutritional quality by increasing the fibre content; IWG pasta presented a remarkable leaching of FOS in cooking water, thus providing only 1 g of FOS per serving. IWG pastas showed a GI of 67 (dried) and 79 (fresh), not significantly different from commercial pasta products. IWG can be considered an interesting ingredient to obtain functional products 'naturally enriched' in FOS and fibre. Results about FOS leaching suggest that, in dealing with functional effects, the actual prebiotic content should be carefully considered on food 'as eaten'. PMID:23373796

  10. Inulin and levan synthesis by probiotic Lactobacillus gasseri strains: characterization of three novel fructansucrase enzymes and their fructan products.

    PubMed

    Anwar, Munir A; Kralj, Slavko; Piqué, Anna Villar; Leemhuis, Hans; van der Maarel, Marc J E C; Dijkhuizen, Lubbert

    2010-04-01

    Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. Here, we report an evaluation of fructan synthesis in three Lactobacillus gasseri strains, identification of the fructansucrase-encoding genes and characterization of the recombinant proteins and fructan (oligosaccharide) products. High-performance anion-exchange chromatography and nuclear magnetic resonance analysis of the fructo-oligosaccharides (FOS) and polymers produced by the L. gasseri strains and the recombinant enzymes revealed that, in situ, L. gasseri strains DSM 20604 and 20077 synthesize inulin (and oligosaccharides) and levan products, respectively. L. gasseri DSM 20604 is only the second Lactobacillus strain shown to produce inulin polymer and FOS in situ, and is unique in its distribution of FOS synthesized, ranging from DP2 to DP13. The probiotic bacterium L. gasseri DSM 20243 did not produce any fructan, although we identified a fructansucrase-encoding gene in its genome sequence. Further studies showed that this L. gasseri DSM 20243 gene was prematurely terminated by a stop codon. Exchanging the stop codon for a glutamine codon resulted in a recombinant enzyme producing inulin and FOS. The three recombinant fructansucrase enzymes characterized from three different L. gasseri strains have very similar primary protein structures, yet synthesize different fructan products. An interesting feature of the L. gasseri strains is that they were unable to ferment raffinose, whereas their respective recombinant enzymes converted raffinose into fructan and FOS. PMID:20075040

  11. Purification, characterization and biological activities of a garlic oligosaccharide.

    PubMed

    Tsukamoto, Sadaji; Okamoto, Kouji; Inanaga, Junji; Karasaki, Yuji

    2008-06-01

    A novel oligosaccharide was purified from garlic (Allium sativum L.) bulbs via hot water extraction, ammonium sulfate precipitation, gel filtration and ion exchange chromatography. The molecular weight of the oligosaccharide was determined to be 1800. A nuclear magnetic resonance (NMR) study showed that ten fructose molecules were connected by beta1-2 linkage to a terminal glucose. The oligosaccharide had cytotoxic activities against human malignant lymphoma cells (U937) and colon adenocarcinoma cells (WiDr) in vitro. Furthermore, this oligosaccharide significantly suppressed the growth of murine colon adenocarcinoma cells (colon 26) in vivo. The oligosaccharide also stimulated interferon-gamma production by human peripheral blood lymphocyte in vitro, indicating that it may activate the immunological pathways and suppress the growth of tumors in vivo. PMID:18655544

  12. Elucidating the binding efficacy of β-galactosidase on graphene by docking approach and its potential application in galacto-oligosaccharide production.

    PubMed

    Satar, Rukhsana; Ismail, Syed Ahmed; Rehan, Mohd; Ansari, Shakeel Ahmed

    2016-05-01

    Herein, we propose the synthesis and characterization of graphene for the immobilization of β-galactosidase for improved galacto-oligosaccharide (GOS) production. The size of synthesized graphene was observed to be 25 nm by TEM analysis while interaction of enzyme with the nanosupport was observed by FTIR spectroscopy. Docking was obtained using molecular docking program Dock v.6.5 while the visual analyses and illustration of protein-ligand complex were investigated by utilizing chimera v.1.6.2 and PyMOL v.1.3 softwares. Immobilized β-galactosidase (IβG) showed improved stability against various physical and chemical denaturants. K m of IβG was increased to 6.41 mM as compared to 2.38 mM of soluble enzyme without bringing significant change in V max value. Maximum GOS content also registered an increase in lactose conversion. The maximum GOS production was achieved by immobilized enzyme at specific temperature and time. Hence, the developed nanosupport can be further exploited for developing a biosensor involving β-galactosidase or for immobilization of other industrially/therapeutically important enzymes. PMID:26861556

  13. Cellobiohydrolases Produce Different Oligosaccharides from Chitosan.

    PubMed

    Tegl, Gregor; Öhlknecht, Christoph; Vielnascher, Robert; Rollett, Alexandra; Hofinger-Horvath, Andreas; Kosma, Paul; Guebitz, Georg M

    2016-06-13

    Chito-oligosaccharides (COSs) are bioactive molecules with interesting characteristics; however, their exploitation is still restricted due to limited amounts accessible with current production strategies. Here we present a strategy for the production of COSs based on hydrolysis of chitosan by using readily available glycosidases. Cellobiohydrolases (EC 3.2.1.91) were compared with chitosanases (EC 3.2.1.132) regarding their ability for COS production, and the resulting fractions were analyzed by MS and NMR. The oligosaccharides had a degree of polymerization between three and six units, and the degree of acetylation (DA) varied depending on the applied enzyme. Different cellobiohydrolases produced COSs with varying DA, and based on comprehensive NMR analysis the preferred cleavage sites of the respective enzymes that show chitosanase and chitinase activity were elucidated. The study reveals the high potential of readily available cellulolytic enzymes besides chitosanases for the production of COSs with distinct structure facilitating access to this bioactive compound class. PMID:27214513

  14. The relevance of tick bites to the production of IgE antibodies to the mammalian oligosaccharide galactose-α-1,3-galactose

    PubMed Central

    Commins, Scott P.; James, Hayley R.; Kelly, Elizabeth A.; Pochan, Shawna L.; Workman, Lisa J.; Perzanowski, Matthew S.; Kocan, Katherine M.; Fahy, John V.; Nganga, Lucy W.; Ronmark, Eva; Cooper, Philip J.; Platts-Mills, Thomas A. E.

    2011-01-01

    Background In 2009, we reported a novel form of delayed anaphylaxis to red meat, which is related to serum IgE antibodies to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal). Most of these patients had tolerated meat for many years previously. The implication is that some exposure in adult life had stimulated the production of these IgE antibodies. Objectives To investigate possible causes of this IgE antibody response, focusing on evidence related to tick bites, which are common in the region where these reactions occur. Methods Serum assays were carried out using biotinylated proteins and extracts bound to a streptavidin ImmunoCAP. Results Prospective studies on IgE antibodies in three subjects following tick bites showed an increase in IgE to alpha-gal of twenty-fold or greater. Other evidence included i) a strong correlation between histories of tick bites and IgE to alpha-gal (χ2=26.8, p<0.001), ii) evidence that these IgE antibodies are common in areas where the tick Amblyomma americanum is common, and iii) a significant correlation between IgE antibodies to alpha-gal and IgE antibodies to proteins derived from A. americanum (rs=0.75, p<0.001). Conclusion The results presented here provide evidence that tick bites are a cause, or possibly the only cause, of IgE specific for alpha-gal in this area of the United States. Both the number of subjects becoming sensitized and the titer of IgE antibodies to alpha-gal are striking. Here we report the first example of a response to an ectoparasite giving rise to an important form of food allergy. PMID:21453959

  15. Trends in dairy and non-dairy probiotic products - a review.

    PubMed

    Vijaya Kumar, Bathal; Vijayendra, Sistla Venkata Naga; Reddy, Obulam Vijaya Sarathi

    2015-10-01

    Health awareness has grown to a greater extent among consumers and they are looking for healthy probiotic counterparts. Keeping in this view, the present review focuses recent developments in dairy and non-dairy probiotic products. All over the world, dairy probiotics are being commercialized in many different forms. However, the allergy and lactose intolerance are the major set-backs to dairy probiotics. Whereas, flavor and refreshing nature are the major advantages of non-dairy drinks, especially fruit juices. Phenotypic and genotypic similarities between dairy and non-dairy probiotics along with the matrix dependency of cell viability and cell functionality are reviewed. The heterogeneous food matrices of non-dairy food carriers are the major constraints for the survival of the probiotics, while the probiotic strains from non-dairy sources are satisfactory. Technological and functional properties, besides the viability of the probiotics used in fermented products of non-dairy origin are extremely important to get a competitive advantage in the world market. The functional attributes of dairy and non-dairy probiotic products are further enhanced by adding prebiotics such as galacto-oligosaccharide, fructo-oligosaccharide and inulin. PMID:26396359

  16. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases

    NASA Astrophysics Data System (ADS)

    Hamer, Stefanie Nicole; Cord-Landwehr, Stefan; Biarnés, Xevi; Planas, Antoni; Waegeman, Hendrik; Moerschbacher, Bruno Maria; Kolkenbrock, Stephan

    2015-03-01

    Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture.

  17. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases

    PubMed Central

    Hamer, Stefanie Nicole; Cord-Landwehr, Stefan; Biarnés, Xevi; Planas, Antoni; Waegeman, Hendrik; Moerschbacher, Bruno Maria; Kolkenbrock, Stephan

    2015-01-01

    Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture. PMID:25732514

  18. Oligosaccharide formation during commercial pear juice processing.

    PubMed

    Willems, Jamie L; Low, Nicholas H

    2016-08-01

    The effect of enzyme treatment and processing on the oligosaccharide profile of commercial pear juice samples was examined by high performance anion exchange chromatography with pulsed amperometric detection and capillary gas chromatography with flame ionization detection. Industrial samples representing the major stages of processing produced with various commercial enzyme preparations were studied. Through the use of commercially available standards and laboratory scale enzymatic hydrolysis of pectin, starch and xyloglucan; galacturonic acid oligomers, glucose oligomers (e.g., maltose and cellotriose) and isoprimeverose were identified as being formed during pear juice production. It was found that the majority of polysaccharide hydrolysis and oligosaccharide formation occurred during enzymatic treatment at the pear mashing stage and that the remaining processing steps had minimal impact on the carbohydrate-based chromatographic profile of pear juice. Also, all commercial enzyme preparations and conditions (time and temperature) studied produced similar carbohydrate-based chromatographic profiles. PMID:26988479

  19. Immobilization of xylanase on poly (ethylene glycol) methyl ether 5000 and its self-extractive bioconversion for the production of xylo-oligosaccharides.

    PubMed

    Li, Xin; Shan, Zongxing; Song, Xiangyang; Ouyang, Jia; Xu, Yong; Yong, Qiang; Yu, Shiyuan

    2014-02-01

    Endo-β-1,4-xylanase derived from Trichoderma reesei was covalently immobilized on poly (ethylene glycol) methyl ether 5000 (mPEG5000), and the resulting immobilized enzyme had a residual activity of 72.4 % with 82.9 % of PEGylated amino groups. Compared with the free enzyme, the immobilized xylanase was stable at pH values in the range of 4.0-6.0 and temperatures in the range of 50-65 °C. A self-extractive bioconversion system composed of immobilized xylanase, mPEG5000, and sodium citrate was used to produce xylo-oligosaccharides and provided a better distribution of the xylo-oligosaccharides than the free enzyme. Furthermore, the immobilized xylanase could be effectively recovered in situ following the hydrolysis reaction. PMID:24326682

  20. Complete genome sequence of a xanthan-degrading Microbacterium sp. strain XT11 with the potential for xantho-oligosaccharides production.

    PubMed

    Yang, Fan; Li, Lili; Si, Yang; Yang, Ming; Guo, Xiaoyu; Hou, Yingmin; Chen, Xiaoyi; Li, Xianzhen

    2016-03-20

    Here, we reported a high-quality complete genome sequence of a xanthan-degrading bacterium Microbacterium sp. XT11, newly isolated from garden soil, China. Several genes probably involved in xanthan degradation pathway were detected as a gene cluster in the genome. The genome information of strain XT11 will be valuable for clarifying the molecular mechanism for xanthan degradation and producing xantho-oligosaccharides. PMID:26853476

  1. Dietary Mannan Oligosaccharides: Counteracting the Side Effects of Soybean Meal Oil Inclusion on European Sea Bass (Dicentrarchus labrax) Gut Health and Skin Mucosa Mucus Production?

    PubMed Central

    Torrecillas, Silvia; Montero, Daniel; Caballero, Maria José; Pittman, Karin A.; Custódio, Marco; Campo, Aurora; Sweetman, John; Izquierdo, Marisol

    2015-01-01

    The main objective of this study was to assess the effects of 4 g kg−1 dietary mannan oligosaccharides (MOS) inclusion in soybean oil (SBO)- and fish oil (FO)-based diets on the gut health and skin mucosa mucus production of European sea bass juveniles after 8 weeks of feeding. Dietary MOS, regardless of the oil source, promoted growth. The intestinal somatic index was not affected, however dietary SBO reduced the intestinal fold length, while dietary MOS increased it. The dietary oil source fed produced changes on the posterior intestine fatty acid profiles irrespective of MOS dietary supplementation. SBO down-regulated the gene expression of TCRβ, COX2, IL-1β, TNFα, IL-8, IL-6, IL-10, TGFβ, and Ig and up-regulated MHCII. MOS supplementation up-regulated the expression of MHCI, CD4, COX2, TNFα, and Ig when included in FO-based diets. However, there was a minor up-regulating effect on these genes when MOS was supplemented in the SBO-based diet. Both dietary oil sources and MOS affected mean mucous cell areas within the posterior gut, however the addition of MOS to a SBO diet increased the mucous cell size over the values shown in FO fed fish. Dietary SBO also trends to reduce mucous cell density in the anterior gut relative to FO, suggesting a lower overall mucosal secretion. There are no effects of dietary oil or MOS in the skin mucosal patterns. Complete replacement of FO by SBO, modified the gut fatty acid profile, altered posterior gut-associated immune system (GALT)-related gene expression and gut mucous cells patterns, induced shorter intestinal folds and tended to reduce European sea bass growth. However, when combined with MOS, the harmful effects of SBO appear to be partially balanced by moderating the down-regulation of certain GALT-related genes involved in the functioning of gut mucous barrier and increasing posterior gut mucous cell diffusion rates, thus helping to preserve immune homeostasis. This denotes the importance of a balanced

  2. A novel thermostable GH5_7 β-mannanase from Bacillus pumilus GBSW19 and its application in manno-oligosaccharides (MOS) production.

    PubMed

    Zang, Haoyu; Xie, Shanshan; Wu, Huijun; Wang, Weiduo; Shao, Xiankun; Wu, Liming; Rajer, Faheem Uddin; Gao, Xuewen

    2015-10-01

    A novel thermostable mannanase from a newly isolated Bacillus pumilus GBSW19 has been identified, expressed, purified and characterized. The enzyme shows a structure comprising a 28 amino acid signal peptide, a glycoside hydrolase family 5 (GH5) catalytic domain and no carbohydrate-binding module. The recombinant mannanase has molecular weight of 45 kDa with an optimal pH around 6.5 and is stable in the range from pH 5-11. Meanwhile, the optimal temperature is around 65 °C, and it retains 50% relative activity at 60 °C for 12h. In addition, the purified enzyme can be activated by several ions and organic solvents and is resistant to detergents. Bpman5 can efficiently convert locus bean gum to mainly M2, M3 and M5, and hydrolyze manno-oligosaccharides with a minimum DP of 3. Further exploration of the optimum condition using HPLC to prepare oligosaccharides from locust bean gum was obtained as 10mg/ml locust bean gum incubated with 10 U/mg enzyme at 50 °C for 24h. By using this enzyme, locust bean gum can be utilized to generate high value-added oligosaccharides with a DP of 2-6. PMID:26215338

  3. Inulin Potential for Enzymatic Obtaining of Prebiotic Oligosaccharides.

    PubMed

    Flores, Adriana C; Morlett, Jesús A; Rodríguez, Raúl

    2016-08-17

    Oligosaccharides have been marketed since the 80s as low-calorie agents and recently have gained interest in the pharmaceutical and food industry as functional sweeteners and prebiotic enriching population of Bifidobacteria. Currently, they have an approximated value of $200 per kg and recently, inulin has been proposed as a feedstock for production of oligosaccharides through selective hydrolysis by action of endoinulinase. High optimum temperature (60°C) and thermostability are two important criteria that determine suitability of this enzyme for industrial applications as well as enzyme cost, a major limiting factor. Significant reduction in cost can be achieved by employing low-value and abundant inulin-rich plants as Jerusalem artichoke, dahlia, yacon, garlic, and onion, among others. In general, the early harvested tubers of these plants contain a greater amount of highly polymerized sugar fractions, which offer more industrial value than late-harvested tubers or those after storage. Also, development of recombinant microorganisms could be useful to reduce the cost of enzyme technology for large-scale production of oligosaccharides. In the case of fungal inulinases, several studies of cloning and modification have been made to achieve greater efficiency. The present paper reviews inulin from vegetable sources as feedstock for oligosaccharides production through the action of inulinases, the impact of polymerization degree of inulin and its availability, and some strategies to increase oligosaccharide production. PMID:25746219

  4. Structural characterization of (1→2)-β-xylose-(1→3)-α-arabinose-containing oligosaccharide products of extracted switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with α-arabinofuranosidase and β-endo-xylanase.

    PubMed

    Bowman, Michael J; Dien, Bruce S; Vermillion, Karl E; Mertens, Jeffrey A

    2014-10-29

    Switchgrass (Panicum virgatum, L.) is a potential dedicated biomass crop for use in biocatalytic conversion systems to biofuels. Nearly 30% of switchgrass cell wall material is xylan. The complete depolymerization of xylan is desirable both as an additional carbon source for microbial fermentation and to reduce inhibitory effects xylooligomers may have on cellulolytic glycoside hydrolase enzymes. To identify structural features of switchgrass xylan that are not distinguishable by mass spectrometry alone, a α-arabinofuranosidase enzyme was used to remove the arabinose side chains from alkali-extracted switchgrass xylan from three cultivars with simultaneous hydrolysis by β-endo-xylanase to enrich for oligosaccharide products with extended branching. The two most abundant enzymatic digestion products were separated and characterized by LC-MS(n), linkage analysis, and NMR. These two oligosaccharides were present in all three switchgrass cultivars and found to contain (1→2)-β-xylose-(1→3)-α-arabinose side chains, a linkage not previously reported in switchgrass. PMID:25240184

  5. Optimization of Oligosaccharide Synthesis from Cellobiose by Dextransucrase

    NASA Astrophysics Data System (ADS)

    Kim, Misook; Day, Donal F.

    There is a growing market for oligosaccharides as sweeteners, prebiotics, anticariogenic compounds, and immunostimulating agents in both food and pharmaceutical industries. Interest in novel carbohydrate-based products has grown because of their reduced toxicity and low immune response. Cellobiose is potentially valuable as a nondigestible sugar. The reaction of cellobiose, as an acceptor with a sucrose as a donor, catalyzed by a dextransucrase from Leuconostoc mesenteroides B-512FMCM, produced a series of cellobio-oligosaccharides. This production system was optimized using a Box-Behnken experimental design for 289 mM of sucrose and 250 mM of cellobiose and 54 U of the enzyme at pH 5.2 and 30 °C, to produce maximum yields of oligosaccharide.

  6. N alpha-(1-deoxy-D-fructos-1-yl)-L-arginine, an antioxidant compound identified in aged garlic extract.

    PubMed

    Ryu, K; Ide, N; Matsuura, H; Itakura, Y

    2001-03-01

    Aged garlic extract (AGE) has been shown to have antioxidant activity. The organosulfur compounds, S-allyl-L-cysteine and S-allylmercapto-L-cysteine, are responsible, at least in part, for the antioxidant activity of AGE. To identify major active components, we fractionated AGE, using hydrogen peroxide scavenging activity as an antioxidative index. Strong activity in the amino acid fraction was found and the major active compound was identified as N alpha-(1-deoxy-D-fructos-1-yl)-L-arginine (Fru-Arg). Antioxidant activity of Fru-Arg was comparable to that of ascorbic acid, scavenging hydrogen peroxide completely at 50 micromol/L and 37% at 10 micromol/L. Quantitative analysis using the established HPLC system revealed that AGE contained 2.1-2.4 mmol/L of Fru-Arg, but none was detected in either raw or heated garlic juice. Furthermore, it was shown that a minimum of 4 mo aging incubation was required for Fru-Arg to be generated. These findings indicate that the aging process is critical for the production of the antioxidant compound, Fru-Arg. These results may explain some of the variation in benefits among different commercially available garlic preparations. PMID:11238799

  7. Structural confirmation of oligosaccharides newly isolated from sugar beet molasses

    PubMed Central

    2012-01-01

    Background Sugar beet molasses is a viscous by-product of the processing of sugar beets into sugar. The molasses is known to contain sucrose and raffinose, a typical trisaccharide, with a well-established structure. Although sugar beet molasses contains various other oligosaccharides as well, the structures of those oligosaccharides have not been examined in detail. The purpose of this study was isolation and structural confirmation of these other oligosaccharides found in sugar beet molasses. Results Four oligosaccharides were newly isolated from sugar beet molasses using high-performance liquid chromatography (HPLC) and carbon-Celite column chromatography. Structural confirmation of the saccharides was provided by methylation analysis, matrix-assisted laser desorption/ionaization time of flight mass spectrometry (MALDI-TOF-MS), and nuclear magnetic resonance (NMR) measurements. Conclusion The following oligosaccharides were identified in sugar beet molasses: β-D-galactopyranosyl-(1- > 6)-β-D-fructofuranosyl-(2 <-> 1)-α-D-glucopyranoside (named β-planteose), α-D-galactopyranosyl-(1- > 1)-β-D-fructofuranosyl-(2 <-> 1)-α-D-glucopyranoside (named1-planteose), α-D-glucopyranosyl-(1- > 6)-α-D-glucopyranosyl-(1 <-> 2)-β-D-fructofuranoside (theanderose), and β-D-glucopyranosyl-(1- > 3)-α-D-glucopyranosyl-(1 <-> 2)-β-D-fructofuranoside (laminaribiofructose). 1-planteose and laminaribiofructose were isolated from natural sources for the first time. PMID:22925105

  8. Novel High-Molecular Weight Fucosylated Milk Oligosaccharides Identified in Dairy Streams

    PubMed Central

    Mehra, Raj; Barile, Daniela; Marotta, Mariarosaria; Lebrilla, Carlito B.; Chu, Caroline; German, J. Bruce

    2014-01-01

    Oligosaccharides are the third largest component in human milk. This abundance is remarkable because oligosaccharides are not digestible by the newborn, and yet they have been conserved and amplified during evolution. In addition to encouraging the growth of a protective microbiota dominated by bifidobacteria, oligosaccharides have anti-infective activity, preventing pathogens from binding to intestinal cells. Although it would be advantageous adding these valuable molecules to infant milk formula, the technologies to reproduce the variety and complexity of human milk oligosaccharides by enzymatic/organic synthesis are not yet mature. Consequently, there is an enormous interest in alternative sources of these valuable oligosaccharides. Recent research has demonstrated that bovine milk and whey permeate also contain oligosaccharides. Thus, a thorough characterization of oligosaccharides in bovine dairy streams is an important step towards fully assessing their specific functionalities. In this study, bovine milk oligosaccharides (BMOs) were concentrated by membrane filtration from a readily available dairy stream called “mother liquor”, and analyzed by high accuracy MALDI FT-ICR mass spectrometry. The combination of HPLC and accurate mass spectrometry allowed the identification of ideal processing conditions leading to the production of Kg amount of BMO enriched powders. Among the BMOs identified, 18 have high-molecular weight and corresponded in size to the most abundant oligosaccharides present in human milk. Notably 6 oligosaccharides contained fucose, a sugar monomer that is highly abundant in human milk, but is rarely observed in bovine milk. This work shows that dairy streams represent a potential source of complex milk oligosaccharides for commercial development of unique dairy ingredients in functional foods that reproduce the benefits of human milk. PMID:24810963

  9. Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine and melibiose using Aspergillus niger α-galactosidase, and properties of the products.

    PubMed

    Sakaki, Yohei; Tashiro, Mitsuru; Katou, Moe; Sakuma, Chiseko; Hirano, Takako; Hakamata, Wataru; Nishio, Toshiyuki

    2016-09-01

    Two kinds of oligosaccharides, N-acetylraffinosamine (RafNAc) and N-acetylplanteosamine (PlaNAc), were synthesized from N-acetylsucrosamine and melibiose using the transgalactosylation activity of Aspergillus niger α-galactosidase. RafNAc and PlaNAc are novel trisaccharides in which d-glucopyranose residues in raffinose (Raf) and planteose are replaced with N-acetyl-d-glucosamine. These trisaccharides were more stable in acidic solution than Raf. RafNAc was hydrolyzed more rapidly than Raf by α-galactosidase of green coffee bean. In contrast, RafNAc was not hydrolyzed by Saccharomyces cerevisiae invertase, although Raf was hydrolyzed well by this enzyme. These results indicate that the physicochemical properties and steric structure of RafNAc differ considerably from those of Raf. PMID:27254139

  10. Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis

    DOE PAGESBeta

    Xue, Saisi; Uppugundla, Nirmal; Bowman, Michael J.; Cavalier, David; Da Costa Sousa, Leonardo; Dale, Bruce E.; Balan, Venkatesh

    2015-11-26

    Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18–25 % of the total soluble sugars in the hydrolysate and 12–18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7–9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production frommore » cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature. Results indicate that, oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEXcorn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the

  11. Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis

    SciTech Connect

    Xue, Saisi; Uppugundla, Nirmal; Bowman, Michael J.; Cavalier, David; Da Costa Sousa, Leonardo; Dale, Bruce E.; Balan, Venkatesh

    2015-11-26

    Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18–25 % of the total soluble sugars in the hydrolysate and 12–18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7–9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production from cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature. Results indicate that, oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEXcorn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the

  12. Chromatographic Separations of Enantiomers and Underivatized Oligosaccharides

    SciTech Connect

    Ying Liu

    2004-12-19

    resolution, high column stability, and high sensitivity. In addition, this method showed potential usefulness for the sensitive and quick analysis of hydrolysis products of polysaccharides, and for trace level analysis of individual oligosaccharides or oligosaccharide isomers from biological systems.

  13. Conversion of biomass-derived oligosaccharides into lipids

    PubMed Central

    2014-01-01

    Background Oligocelluloses and oligoxyloses are partially hydrolyzed products from lignocellulosic biomass hydrolysis. Biomass hydrolysates usually contain monosaccharides as well as various amounts of oligosaccharides. To utilize biomass hydrolysates more efficiently, it is important to identify microorganisms capable of converting biomass-derived oligosaccharides into biofuels or biochemicals. Results We have demonstrated that the oleaginous yeast Cryptococcus curvatus can utilize either oligocelluloses or oligoxyloses as sole carbon sources for microbial lipid production. When oligocelluloses were used, lipid content and lipid coefficient were 35.9% and 0.20 g/g consumed sugar, respectively. When oligoxyloses were used, lipid coefficient was 0.17 g/g consumed sugar. Ion chromatography analysis showed oligocelluloses with a degree of polymerization from 2 to 9 were assimilated. Our data suggested that these oligosaccharides were transported into cells and then hydrolyzed by cytoplasmic enzymes. Further analysis indicated that these enzymes were inducible by oligocelluloses. Lipid production on cellulose by C. curvatus using the simultaneous saccharification and lipid production process in the absence of cellobiase achieved essentially identical results to that in the presence of cellobiase, suggesting that oligocelluloses generated in situ were utilized with high efficiency. This study has provided inspiring information for oligosaccharides utilization, which should facilitate biorefinery based on lignocellulosic biomass. Conclusions C. curvatus can directly utilize biomass-derived oligosaccharides. Oligocelluloses are transported into the cells and then hydrolyzed by cytoplasmic enzymes. A simultaneous saccharification and lipid production process can be conducted without oligocelluloses accumulation in the absence of cellobiase by C. curvatus, which could reduce the enzyme costs. PMID:24472330

  14. Intestinal infections and prebiotics: the roles of oligosaccharides in promoting health

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prebiotic oligosaccharides exert activity against pathogens partly by stimulating the growth and/or activity of commensal bacteria that provide health benefits (lower pH, bacteriocin production, immune system modulation, competitive exclusion). This review describes alternative mechanisms of action...

  15. The Human Milk Metabolome Reveals Diverse Oligosaccharide Profiles123

    PubMed Central

    Smilowitz, Jennifer T.; O’Sullivan, Aifric; Barile, Daniela; German, J. Bruce; Lönnerdal, Bo; Slupsky, Carolyn M.

    2013-01-01

    Breast milk delivers nutrition and protection to the developing infant. There has been considerable research on the high-molecular-weight milk components; however, low-molecular-weight metabolites have received less attention. To determine the effect of maternal phenotype and diet on the human milk metabolome, milk collected at day 90 postpartum from 52 healthy women was analyzed by using proton nuclear magnetic resonance spectroscopy. Sixty-five milk metabolites were quantified (mono-, di-, and oligosaccharides; amino acids and derivatives; energy metabolites; fatty acids and associated metabolites; vitamins, nucleotides, and derivatives; and others). The biological variation, represented as the percentage CV of each metabolite, varied widely (4–120%), with several metabolites having low variation (<20%), including lactose, urea, glutamate, myo-inositol, and creatinine. Principal components analysis identified 2 clear groups of participants who were differentiable on the basis of milk oligosaccharide concentration and who were classified as secretors or nonsecretors of fucosyltransferase 2 (FUT2) gene products according to the concentration of 2′-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose I. Exploration of the interrelations between the milk sugars by using Spearman rank correlations revealed significant positive and negative associations, including positive correlations between fucose and products of the FUT2 gene and negative correlations between fucose and products of the fucosyltransferase 3 (FUT3) gene. The total concentration of milk oligosaccharides was conserved among participants (%CV = 18%), suggesting tight regulation of total oligosaccharide production; however, concentrations of specific oligosaccharides varied widely between participants (%CV = 30.4–84.3%). The variability in certain milk metabolites suggests possible roles in infant or infant gut microbial development. This trial was registered at clinicaltrials.gov as NCT

  16. Optimization of hydrolysis conditions for the production of glucomanno-oligosaccharides from konjac using β-mannanase by response surface methodology.

    PubMed

    Chen, Junfan; Liu, Desheng; Shi, Bo; Wang, Hai; Cheng, Yongqiang; Zhang, Wenjing

    2013-03-01

    Glucomanno-oligosaccharides (GMO), usually produced from hydrolysis of konjac tubers with a high content of glucomannan, have a positive effect on Bifidobacterium as well as a variety of other physiological activities. Response surface methodology (RSM) was employed to optimize the hydrolysis time, hydrolysis temperature, pH and enzyme to substrate ratio (E/S) to obtain a high GMO yield from konjac tubers. From the signal-factor experiments, it was concluded that the change in the direct reducing sugar (DRS) is consistent with total reducing sugar (TRS) but contrary to the degree of polymerization (DP). DRS was used as an indicator of the content of GMO in the RSM study. The optimum RSM operating conditions were: reaction time of 3.4 h, reaction temperature of 41.0°C, pH of 7.1 and E/S of 0.49. The results suggested that the enzymatic hydrolysis was enhanced by temperature, pH and incubation time. Model validation showed good agreement between experimental results and the predicted responses. PMID:23465904

  17. Xylo- and cello-oligosaccharide oxidation by gluco-oligosaccharide oxidase from Sarocladium strictum and variants with reduced substrate inhibition

    PubMed Central

    2013-01-01

    Background The oxidation of carbohydrates from lignocellulose can facilitate the synthesis of new biopolymers and biochemicals, and also reduce sugar metabolism by lignocellulolytic microorganisms, reserving aldonates for fermentation to biofuels. Although oxidoreductases that oxidize cellulosic hydrolysates have been well characterized, none have been reported to oxidize substituted or branched xylo-oligosaccharides. Moreover, this is the first report that identifies amino acid substitutions leading to GOOX variants with reduced substrate inhibition. Results The recombinant wild type gluco-oligosaccharide oxidase (GOOX) from the fungus Sarocladium strictum, along with variants that were generated by site-directed mutagenesis, retained the FAD cofactor, and showed high activity on cello-oligosaccharide and xylo-oligosaccharides, including substituted and branched xylo-oligosaccharides. Mass spectrometric analyses confirmed that GOOX introduces one oxygen atom to oxidized products, and 1H NMR and tandem mass spectrometry analysis confirmed that oxidation was restricted to the anomeric carbon. The A38V mutation, which is close to a predicted divalent ion-binding site in the FAD-binding domain of GOOX but 30 Å away from the active site, significantly increased the kcat and catalytic efficiency of the enzyme on all oligosaccharides. Eight amino acid substitutions were separately introduced to the substrate-binding domain of GOOX-VN (at positions Y72, E247, W351, Q353 and Q384). In all cases, the Km of the enzyme variant was higher than that of GOOX, supporting the role of corresponding residues in substrate binding. Most notably, W351A increased Km values by up to two orders of magnitude while also increasing kcat up to 3-fold on cello- and xylo-oligosaccharides and showing no substrate inhibition. Conclusions This study provides further evidence that S. strictum GOOX has broader substrate specificity than the enzyme name implies, and that substrate inhibition can be

  18. Effects of a Formula Containing Two Types of Prebiotics, Bifidogenic Growth Stimulator and Galacto-oligosaccharide, and Fermented Milk Products on Intestinal Microbiota and Antibody Response to Influenza Vaccine in Elderly Patients: A Randomized Controlled Trial

    PubMed Central

    Nagafuchi, Shinya; Yamaji, Taketo; Kawashima, Akihiro; Saito, Yukiko; Takahashi, Takeshi; Yamamoto, Takayuki; Maruyama, Mitsuo; Akatsu, Hiroyasu

    2015-01-01

    We investigated the effect of a formula containing two different prebiotics (bifidogenic growth stimulator and galacto-oligosaccharide) and fermented milk products on intestinal microbiota and antibody responses to an influenza vaccine in enterally fed elderly in-patients. Patients were administered either formula containing prebiotics and fermented milk products (group F: n = 12, 79.9 ± 9.5 years old) or standard formula (group C: n = 12, 80.7 ± 10.1 years old) via percutaneous endoscopic gastrostomy during a 14-week intervention period. Subjects were immunized with an influenza vaccine (A/H1N1, A/H3N2, and B) at week 4 of the intervention. Blood biochemical indices, intestinal bacteria populations and antibody titers were analyzed. Bifidobacterium counts increased significantly in group F compared with group C. The enhanced antibody titers against A/H1N1 were maintained in group F for a longer period compared with group C. The titers against A/H3N2 were unchanged between both groups, and those against B were significantly lower in group F than in group C, although few subjects had seroprotective titers against A/H3N2 and B. These results suggest that administration of the formula containing prebiotics and fermented milk products may maintain antibody titers for longer periods through the improvement of intestinal microbiota. PMID:26096655

  19. Profiling pneumococcal type 3-derived oligosaccharides by high resolution liquid chromatography-tandem mass spectrometry

    PubMed Central

    Li, Guoyun; Li, Lingyun; Xue, Changhu; Middleton, Dustin; Linhardt, Robert J.; Avci, Fikri Y.

    2015-01-01

    Pneumococcal type-3 polysaccharide (Pn3P) is considered a major target for the development of a human vaccine to protect against Streptococcus pneumonia infection. Thus, it is critical to develop methods for the preparation and analysis of Pn3P-derived oligosaccharides to better understand its immunological properties. In this paper, we profile oligosaccharides, generated by the free radical depolymerization of Pn3P, using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Hydrophilic liquid interaction chromatography (HILIC)-mass spectrometry (MS) revealed a series of oligosaccharides with an even- and odd-number of saccharide residues, ranging from monosaccharide, degree of polymerization (dp1) to large oligosaccharides up to dp 20, generated by free radical depolymerization. Isomers of oligosaccharides with an even number of sugar residues were easily separated on a HILIC column, and their sequences could be distinguished by comparing MS/MS of these oligosaccharides and their reduced alditols. Fluorescent labeling with 2-aminoacridone (AMAC) followed by reversed phase (RP)-LC-MS/MS was applied to analyze and sequence poorly separated product mixtures, as RP-LC affords higher resolution of AMAC-labeled oligosaccharides than does HILIC-based separation. The present methodology can be potentially applied to profiling other capsular polysaccharides. PMID:25913329

  20. Bovine Milk as a Source of Functional Oligosaccharides for Improving Human Health12

    PubMed Central

    Zivkovic, Angela M.; Barile, Daniela

    2011-01-01

    Human milk oligosaccharides are complex sugars that function as selective growth substrates for specific beneficial bacteria in the gastrointestinal system. Bovine milk is a potentially excellent source of commercially viable analogs of these unique molecules. However, bovine milk has a much lower concentration of these oligosaccharides than human milk, and the majority of the molecules are simpler in structure than those found in human milk. Specific structural characteristics of milk-derived oligosaccharides are crucial to their ability to selectively enrich beneficial bacteria while inhibiting or being less than ideal substrates for undesirable and pathogenic bacteria. Thus, if bovine milk products are to provide human milk–like benefits, it is important to identify specific dairy streams that can be processed commercially and cost-effectively and that can yield specific oligosaccharide compositions that will be beneficial as new food ingredients or supplements to improve human health. Whey streams have the potential to be commercially viable sources of complex oligosaccharides that have the structural resemblance and diversity of the bioactive oligosaccharides in human milk. With further refinements to dairy stream processing techniques and functional testing to identify streams that are particularly suitable for enriching beneficial intestinal bacteria, the future of oligosaccharides isolated from dairy streams as a food category with substantiated health claims is promising. PMID:22332060

  1. Oligosaccharide mapping of heparan sulphate by polyacrylamide-gradient-gel electrophoresis and electrotransfer to nylon membrane.

    PubMed Central

    Turnbull, J E; Gallagher, J T

    1988-01-01

    A new method that we have called 'oligosaccharide mapping' is described for the analysis of radiolabelled heparan sulphate and other glycosaminoglycans. The method involves specific enzymic or chemical scission of polysaccharide chains followed by high-resolution separation of the degradation products by polyacrylamide-gradient-gel electrophoresis. The separated oligosaccharides are immobilized on charged nylon membranes by electrotransfer and detected by fluorography. A complex pattern of discrete bands is observed covering an oligosaccharide size range from degree of polymerization (d.p.) 2 (disaccharide) to approximately d.p. 40. Separation is due principally to differences in Mr, though the method also seems to detect variations in conformation of oligosaccharide isomers. Resolution of oligosaccharides is superior to that obtained with isocratic polyacrylamide-gel-electrophoresis systems or gel chromatography, and reveals structural details that are not accessible by other methods. For example, in this paper we demonstrate a distinctive repeating doublet pattern of iduronate-rich oligosaccharides in heparitinase digests of mouse fibroblast heparan sulphate. This pattern may be a general feature of mammalian heparan sulphates. Oligosaccharide mapping should be a valuable method for the analysis of fine structure and sequence of heparan sulphate and other complex polysaccharides, and for making rapid assessments of the molecular distinctions between heparan sulphates from different sources. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:2969727

  2. Pet food and feed applications of inulin, oligofructose and other oligosaccharides.

    PubMed

    Flickinger, E A; Fahey, G C

    2002-05-01

    Prebiotics may be considered as functional food ingredients. They are attracting considerable interest from pet owners, pet food manufacturers, livestock producers and feed manufacturers. The most common forms of prebiotics are nondigestible oligosaccharides (NDO), including inulin, oligofructose mannanoligosaccharides, gluco-oligosaccharides, and galacto-oligosaccharides. These NDO are nondigestible by enzymes present in the mammalian small intestine, but are fermented by bacteria present in the hindgut of nonruminants. Inulin and oligofructose are present in measurable quantities in feed ingredients like wheat, wheat by-products, barley, and peanut hulls. Consumption of prebiotic oligosaccharides elicits several purported health benefits. In companion animals, prebiotics have been shown to improve gut microbial ecology and enhance stool quality. In production livestock and poultry, prebiotics are employed to control pathogenic bacteria, reduce faecal odour, and enhance growth performance. Research to date indicates positive effects of prebiotics on health status and performance of companion animals, livestock, and poultry. PMID:12088533

  3. Analysis of Prebiotic Oligosaccharides

    NASA Astrophysics Data System (ADS)

    Sanz, M. L.; Ruiz-Matute, A. I.; Corzo, N.; Martínez-Castro, I.

    Carbohydrates and more specifically prebiotics, are complex mixtures of isomers with different degrees of polymerization (DP), monosaccharide units and/or glycosidic linkages. Many efforts are focused on the search for new products and the determination of their biological activity. However, the study of their chemical structure is fundamental to both acquire a basic knowledge of the carbohydrate and to increase the understanding of the mechanisms for their metabolic effect.

  4. A combined metabolomic and phylogenetic study reveals putatively prebiotic effects of high molecular weight arabino-oligosaccharides when assessed by in vitro fermentation in bacterial communities derived from humans.

    PubMed

    Sulek, Karolina; Vigsnaes, Louise Kristine; Schmidt, Line Rieck; Holck, Jesper; Frandsen, Henrik Lauritz; Smedsgaard, Jørn; Skov, Thomas Hjort; Meyer, Anne S; Licht, Tine Rask

    2014-08-01

    Prebiotic oligosaccharides are defined by their selective stimulation of growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial for health. However, apart from the short chain fatty acids, little is known about bacterial metabolites created by fermentation of prebiotics, and the significance of the size of the oligosaccharides remains largely unstudied. By in vitro fermentations in human fecal microbial communities (derived from six different individuals), we studied the effects of high-mass (HA, >1 kDa), low-mass (LA, <1 kDa) and mixed (BA) sugar beet arabino-oligosaccharides (AOS) as carbohydrate sources. Fructo-oligosaccharides (FOS) were included as reference. The changes in bacterial communities and the metabolites produced in response to incubation with the different carbohydrates were analyzed by quantitative PCR (qPCR) and Liquid Chromatography-Mass Spectrometry (LC-MS), respectively. All tested carbohydrate sources resulted in a significant increase of Bifidobacterium spp. between 1.79 fold (HA) and 1.64 fold (FOS) in the microbial populations after fermentation, and LC-MS analysis suggested that the bifidobacteria contributed to decomposition of the arabino-oligosaccharide structures, most pronounced in the HA fraction, resulting in release of the essential amino acid phenylalanine. Abundance of Lactobacillus spp. correlated with the presence of a compound, most likely a flavonoid, indicating that lactobacilli contribute to release of such health-promoting substances from plant structures. Additionally, the combination of qPCR and LC-MS revealed a number of other putative interactions between intestinal microbes and the oligosaccharides, which contributes to the understanding of the mechanisms behind prebiotic impact on human health. PMID:24905430

  5. Galacto-Oligosaccharide Prebiotics

    NASA Astrophysics Data System (ADS)

    Tzortzis, George; Vulevic, Jelena

    The wide recognition of bifidobacteria as health promoting bacteria (Boesten and de Vos, 2008) has attracted a lot of interest in identifying substances that can selectively promote their growth. Many studies using conventional culture and molecular techniques for bacterial identification have shown that breast-fed infants are characterized by an intestinal microbiota that is dominated by bifidobacteria (Benno et al., 1984), which is different from that of infants fed on cow's milk in that their microbiotas are characterized by lower counts of bifidobacteria, with greater numbers of more potentially harmful organisms such as clostridia and enterococci (Lunderquist et al., 1985). As a result of this difference in the microbiota composition, higher levels of ammonia, amines and phenols and other potentially harmful substances have also been found in infants fed cow's milk products (Lunderquist et al., 1985).

  6. Profiling of soluble neutral oligosaccharides from treated biomass using solid phase extraction and LC-TOF MS.

    PubMed

    Vismeh, Ramin; Humpula, James F; Chundawat, Shishir P S; Balan, Venkatesh; Dale, Bruce E; Jones, A Daniel

    2013-05-15

    Thermochemical pretreatments of cellulosic biomass are known to improve cell wall enzymatic digestibility, while simultaneously releasing substantial amounts of soluble oligosaccharides. Profiling of oligosaccharides released during pretreatment yields information essential for choosing glycosyl hydrolases necessary for cost-effective conversion of cellulosic biomass to desired biofuel/biochemical end-products. In this report we present a methodology for profiling of soluble neutral oligosaccharides released from ammonia fiber expansion (AFEX™)-pretreated corn stover. Our methodology employs solid phase extraction (SPE) enrichment of oligosaccharides using porous graphitized carbon (PGC), followed by high performance liquid chromatography (HPLC) separation using a polymeric amine based column and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). For structural elucidation on the chromatographic time scale, nonselective multiplexed collision-induced dissociation was performed for quasi-simultaneous acquisition of oligosaccharide molecular and fragment masses in a single analysis. These analyses revealed glucans up to degree of polymerization (DP) 22 without modifications. Additionally, arabinoxylans up to DP=6 were detected in pretreated biomass extracts (post-enzymatic digestion). Cross-ring fragment ion abundances were consistent with assignment of linkages between sugar units in glucans and also xylose backbone in arabinoxylans as 1-4 linkages. Comprehensive profiling of soluble oligosaccharides also demonstrated decreases in levels of acetate esters of arabinoxylan oligosaccharides with concomitant increases in nonacetylated oligosaccharides that were consistent with earlier observations of 85% release of acetate esters by AFEX™ pretreatment. PMID:23544634

  7. Effect of a Semi-Purified Oligosaccharide-Enriched Fraction from Caprine Milk on Barrier Integrity and Mucin Production of Co-Culture Models of the Small and Large Intestinal Epithelium.

    PubMed

    Barnett, Alicia M; Roy, Nicole C; McNabb, Warren C; Cookson, Adrian L

    2016-01-01

    Caprine milk contains the highest amount of oligosaccharides among domestic animals, which are structurally similar to human milk oligosaccharides (HMOs). This suggests caprine milk oligosaccharides may offer similar protective and developmental effects to that of HMOs. However, to date, studies using oligosaccharides from caprine milk have been limited. Thus, this study aimed to examine the impact of a caprine milk oligosaccharide-enriched fraction (CMOF) on barrier function of epithelial cell co-cultures of absorptive enterocytes (Caco-2 cells) and mucus-secreting goblet cells (HT29-MTX cells), that more closely simulate the cell proportions found in the small (90:10) and large intestine (75:25). Treatment of epithelial co-cultures with 0.4, 1.0, 2.0 and 4.0 mg/mL of CMOF was shown to have no effect on metabolic activity but did enhance cell epithelial barrier integrity as measured by trans-epithelial electrical resistance (TEER), in a dose-dependent manner. The CMOF at the maximum concentration tested (4.0 mg/mL) enhanced TEER, mucin gene expression and mucin protein abundance of epithelial co-cultures, all of which are essential components of intestinal barrier function. PMID:27164134

  8. Effect of a Semi-Purified Oligosaccharide-Enriched Fraction from Caprine Milk on Barrier Integrity and Mucin Production of Co-Culture Models of the Small and Large Intestinal Epithelium

    PubMed Central

    Barnett, Alicia M.; Roy, Nicole C.; McNabb, Warren C.; Cookson, Adrian L.

    2016-01-01

    Caprine milk contains the highest amount of oligosaccharides among domestic animals, which are structurally similar to human milk oligosaccharides (HMOs). This suggests caprine milk oligosaccharides may offer similar protective and developmental effects to that of HMOs. However, to date, studies using oligosaccharides from caprine milk have been limited. Thus, this study aimed to examine the impact of a caprine milk oligosaccharide-enriched fraction (CMOF) on barrier function of epithelial cell co-cultures of absorptive enterocytes (Caco-2 cells) and mucus-secreting goblet cells (HT29-MTX cells), that more closely simulate the cell proportions found in the small (90:10) and large intestine (75:25). Treatment of epithelial co-cultures with 0.4, 1.0, 2.0 and 4.0 mg/mL of CMOF was shown to have no effect on metabolic activity but did enhance cell epithelial barrier integrity as measured by trans-epithelial electrical resistance (TEER), in a dose-dependent manner. The CMOF at the maximum concentration tested (4.0 mg/mL) enhanced TEER, mucin gene expression and mucin protein abundance of epithelial co-cultures, all of which are essential components of intestinal barrier function. PMID:27164134

  9. Preparation of oligosaccharides from Chinese yam and their antioxidant activity.

    PubMed

    Chen, Yi-Feng; Zhu, Qin; Wu, Shengjun

    2015-04-15

    In the present study, the oligosaccharides from Chinese yam were prepared by hydrolysis with hydrogen peroxide (H2O2), which can cleave the glycosidic bonds in polysaccharides. The hydrolysis conditions were optimised by using a central composite design (CCD) as follows: reaction time 4.02 h, temperature 84.35 °C, and H2O2 concentration 2.46%, under which the yield of Chinese yam derived oligosaccharides (CYOs) reached 11.73%, which was consistent with the predicted yield by analysis of the results of CCD (11.89%). The CYOs products were partially characterised by chemical component and Fourier transform infrared spectrum. The CYOs scavenged hydroxyl radical by 89.05% at the concentration of 100 μg/mL, indicating that the CYOs may be a viable option for use as a food antioxidant. PMID:25466131

  10. Powder lemon juice containing oligosaccharides obtained by dextransucrase acceptor reaction synthesis and dehydrated in sprouted bed.

    PubMed

    Coelho, Raquel Macedo Dantas; Araújo, Antônia Daiana Andrade; Fontes, Cláudia Patrícia Mourão Lima; da Silva, Ana Raquel Araujo; da Costa, José Maria Correia; Rodrigues, Sueli

    2015-09-01

    Oligosaccharides can be synthesized using the sugars present in the fruit juices through the dextransucrase acceptor reaction. In the present work, the effect of reducing sugar and sucrose concentration on oligosaccharide formation in lemon juice was evaluated through response surface methodology. The oligosaccharide formation in lemon juice was favored at high concentrations of sucrose (75 g/L) and reducing sugar (75 g/L). At this synthesis conditions, an oligosaccharide concentration of 94.81 g/L was obtained with a conversion of 63.21% of the initial sugars into the target product. Oligosaccharides with degree of polymerization up to 11 were obtained. The lemon juice was dehydrated in spouted bed using maltodextrin as drying adjuvant. The powder obtained at 60°C with 20 % maltodextrin presented low moisture (2.24 %), low water activity (Aw = 0.18) and the lowest reconstitution time (~46 s). The results showed that lemon juice is suitable for oligosaccharides enzyme synthesis and can be dehydrated in spouted bed. PMID:26345014

  11. Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation.

    PubMed

    Taguchi, Yuya; Fujinami, Daisuke; Kohda, Daisuke

    2016-05-20

    The glycosylation of asparagine residues is the predominant protein modification in all three domains of life. An oligosaccharide chain is preassembled on a lipid-phospho carrier and transferred onto asparagine residues by the action of a membrane-bound enzyme, oligosaccharyltransferase. The oligosaccharide donor for the oligosaccharyl transfer reaction is dolichol-diphosphate-oligosaccharide in Eukaryota and polyprenol-diphosphate-oligosaccharide in Eubacteria. The donor in some archaeal species was reportedly dolichol-monophosphate-oligosaccharide. Thus, the difference in the number of phosphate groups aroused interest in whether the use of the dolichol-monophosphate type donors is widespread in the domain Archaea. Currently, all of the archaeal species with identified oligosaccharide donors have belonged to the phylum Euryarchaeota. Here, we analyzed the donor structures of two species belonging to the phylum Crenarchaeota, Pyrobaculum calidifontis and Sulfolobus solfataricus, in addition to two species from the Euryarchaeota, Pyrococcus furiosus and Archaeoglobus fulgidus The electrospray ionization tandem mass spectrometry analyses confirmed that the two euryarchaeal oligosaccharide donors were the dolichol-monophosphate type and newly revealed that the two crenarchaeal oligosaccharide donors were the dolichol-diphosphate type. This novel finding is consistent with the hypothesis that the ancestor of Eukaryota is rooted within the TACK (Thaum-, Aig-, Cren-, and Korarchaeota) superphylum, which includes Crenarchaea. Our comprehensive study also revealed that one archaeal species could contain two distinct oligosaccharide donors for the oligosaccharyl transfer reaction. The A. fulgidus cells contained two oligosaccharide donors bearing oligosaccharide moieties with different backbone structures, and the S. solfataricus cells contained two oligosaccharide donors bearing stereochemically different dolichol chains. PMID:27015803

  12. Effects of orally administered fumonisin B₁ (FB₁), partially hydrolysed FB₁, hydrolysed FB₁ and N-(1-deoxy-D-fructos-1-yl) FB₁ on the sphingolipid metabolism in rats.

    PubMed

    Hahn, Irene; Nagl, Veronika; Schwartz-Zimmermann, Heidi Elisabeth; Varga, Elisabeth; Schwarz, Christiane; Slavik, Veronika; Reisinger, Nicole; Malachová, Alexandra; Cirlini, Martina; Generotti, Silvia; Dall'Asta, Chiara; Krska, Rudolf; Moll, Wulf-Dieter; Berthiller, Franz

    2015-02-01

    Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in maize-based food and feed. Alkaline processing like nixtamalisation of maize generates partially and fully hydrolysed FB1 (pHFB1 and HFB1) and thermal treatment in the presence of reducing sugars leads to formation of N-(1-deoxy-D-fructos-1-yl) fumonisin B1 (NDF). The toxicity of these metabolites, in particular their effect on the sphingolipid metabolism, is either unknown or discussed controversially. We produced high purity FB1, pHFB1a+b, HFB1 and NDF and fed them to male Sprague Dawley rats for three weeks. Once a week, urine and faeces samples were collected over 24 h and analysed for fumonisin metabolites as well as for the sphinganine (Sa) to sphingosine (So) ratio by validated LC-MS/MS based methods. While the latter was significantly increased in the FB1 positive control group, the Sa/So ratios of the partially and fully hydrolysed fumonisins were indifferent from the negative control group. Although NDF was partly cleaved during digestion, the liberated amounts of FB1 did not raise the Sa/So ratio. These results show that the investigated alkaline and thermal processing products of FB1 were, at the tested concentrations, non-toxic for rats, and suggest that according food processing can reduce fumonisin toxicity for humans. PMID:25475052

  13. Urine oligosaccharide pattern in patients with hyperprolactinaemia.

    PubMed

    Ekman, Bertil; Wahlberg, Jeanette; Landberg, Eva

    2015-11-01

    Free milk-type oligosaccharides are produced during pregnancy and lactation and may have an impact on several cells in the immune system. Our aim was to investigate if patients with isolated hyperprolactinaemia, not related to pregnancy, also have increased synthesis and urinary excretion of milk-type oligosaccharides and to compare the excretion pattern with that found during pregnancy. Urine samples were collected as morning sample from 18 patients with hyperprolactinaemia, 13 healthy controls with normal prolactin levels and four pregnant women. After purification, lactose and free oligosaccharides were analysed and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection. The identity of peaks was confirmed by exoglycosidase treatment and comparison with oligosaccharide standards. Prolactin was measured in serum collected between 09 and 11 a.m. by a standardized immunochemical method. Patients with hyperprolactinaemia had higher urinary excretion of lactose than normoprolactinemic controls and urinary lactose correlated positively to prolactin levels (r = 0.51, p < 0.05). Increased levels of the fucosylated oligosaccharides 2-fucosyl lactose and lacto-di-fucotetraose were found in urine from three and two patients, respectively. The acidic oligosaccharide 3-sialyl lactose was found in high amount in urine from two patients with prolactin of >10,000 mU/l. However, pregnant women in their third trimester had the highest concentration of all these oligosaccharides and excretion increased during pregnancy. This study is first to show that both lactose and certain fucosylated and sialylated milk-type oligosaccharides are increased in some patients with hyperprolactinaemia. It remains to elucidate the functional importance of these findings. PMID:26275984

  14. Metabolic engineering of microbes for oligosaccharide and polysaccharide synthesis

    PubMed Central

    Ruffing, Anne; Chen, Rachel Ruizhen

    2006-01-01

    Metabolic engineering has recently been embraced as an effective tool for developing whole-cell biocatalysts for oligosaccharide and polysaccharide synthesis. Microbial catalysts now provide a practical means to derive many valuable oligosaccharides, previously inaccessible through other methods, in sufficient quantities to support research and clinical applications. The synthesis process based upon these microbes is scalable as it avoids expensive starting materials. Most impressive is the high product concentrations (up to 188 g/L) achieved through microbe-catalyzed synthesis. The overall cost for selected molecules has been brought to a reasonable range (estimated $ 30–50/g). Microbial synthesis of oligosaccharides and polysaccharides is a carbon-intensive and energy-intensive process, presenting some unique challenges in metabolic engineering. Unlike nicotinamide cofactors, the required sugar nucleotides are products of multiple interacting pathways, adding significant complexity to the metabolic engineering effort. Besides the challenge of providing the necessary mammalian-originated glycosyltransferases in active form, an adequate uptake of sugar acceptors can be an issue when another sugar is necessary as a carbon and energy source. These challenges are analyzed, and various strategies used to overcome these difficulties are reviewed in this article. Despite the impressive success of the microbial coupling strategy, there is a need to develop a single strain that can achieve at least the same efficiency. Host selection and the manner with which the synthesis interacts with the central metabolism are two important factors in the design of microbial catalysts. Additionally, unlike in vitro enzymatic synthesis, product degradation and byproduct formation are challenges of whole-cell systems that require additional engineering. A systematic approach that accounts for various and often conflicting requirements of the synthesis holds the key to deriving an

  15. Characterisation of branched gluco-oligosaccharides to study the mode-of-action of a glucoamylase from Hypocrea jecorina.

    PubMed

    Jonathan, M C; van Brussel, M; Scheffers, M S; Kabel, M A

    2015-11-01

    In the conversion of starch to fermentable glucose for bioethanol production, hydrolysis of amylopectin by α-amylases and glucoamylases is the slowest step. In this process, α-1,6-branched gluco-oligosaccharides accumulate and are slowly degraded. Glucoamylases that are able to degrade such branched oligosaccharides faster are economically beneficial. This research aimed at the isolation and characterisation of branched gluco-oligosaccharides produced from amylopectin digestion by α-amylase, to be used as substrates for comparing their degradation by glucoamylases. Branched gluco-oligosaccharides with a DP between five and twelve were purified using size exclusion chromatography. These structures were characterised after labelling with 2-aminobenzamide using UHPLC-MS(n) analysis. Further, the purified oligosaccharides were used to evaluate the mode-of-action of a glucoamylase from Hypocrea jecorina. The enzyme cleaves the α-1,4-linkage adjacent to the α-1,6-linkage at a lower rate than that of α-1,4-linkages in linear oligosaccharides. Hence, the branched gluco-oligosaccharides are a suitable substrate to evaluate glucoamylase activity on branched structures. PMID:26256324

  16. Assessment of the bifidogenic effect of substituted xylo-oligosaccharides obtained from corn straw.

    PubMed

    Moniz, Patrícia; Ho, Ai Ling; Duarte, Luís C; Kolida, Sofia; Rastall, Robert A; Pereira, Helena; Carvalheiro, Florbela

    2016-01-20

    This work evaluates the bifidogenic potential of substituted xylo-oligosaccharides (XOS) obtained from a lignocellulosic feedstock (corn straw). Autohydrolysis was used to selectively hydrolyse the xylan-rich hemicellulosic fraction and the soluble oligosaccharides were purified by gel filtration chromatography. Selected oligosaccharides fractions within the target ranges of polymerization degree (4-6 and 9-21, samples S1 and S2, respectively) were characterized and their bifidogenic potential was investigated by in vitro fermentations using human fecal inocula. Bacterial growth was assessed by fluorescent in situ hybridization (FISH). XOS consumption and short-chain fatty acids (SCFA) production were evaluated and compared with commercial oligosaccharides. Under the tested conditions, all the substrates were utilized by the microbiota, and fermentation resulted in increased bifidobacteria populations. Samples S1 and S2 increased bifidobacteria populations and the production profile of SCFA was similar for XOS samples and commercial oligosaccharides although XOS samples displayed the highest concentration of SCFA on longer fermentation times. PMID:26572377

  17. Pathogen-Derived Oligosaccharides Improve Innate Immune Response to Intracellular Parasite Infection

    PubMed Central

    Osanya, Alex; Song, Eun-Ho; Metz, Kyle; Shimak, Raeann M.; Boggiatto, Paola Mercedes; Huffman, Elise; Johnson, Charles; Hostetter, Jesse M.; Pohl, Nicola L.B.; Petersen, Christine A.

    2011-01-01

    Pathogen glycolipids, including Leishmania spp. lipophosphoglycan (LPG) and Mycobacterium tuberculosis mannosylated lipoarabinomannan (ManLAM), modulate essential interactions with host phagocytic cells. Polysaccharide and lipid components promote immunomodulation. Owing to the stereochemistry required to synthesize oligosaccharides, the roles for oligosaccharides in the pathogenesis of infectious diseases have remained largely unknown. Recent advances in carbohydrate chemistry allowed us to synthesize pathogen surface oligosaccharides to discern their immune response–altering activities. Trimannose cap carbohydrates from ManLAM and LPG altered the production of proinflammatory cytokines via a toll-like receptor (TLR2)–mediated mechanism in vitro and in vivo. In vivo treatment with trimannose led to increased Th1-polarizing, IL-12p40–producing cells from the draining lymph nodes of treated Leishmania major–infected mice compared with cells from untreated infected mice. Trimannose treatment increased the production of other Th1 proinflammatory cytokines (ie, interferon-γ, IL-6, and tumor necrosis factor-α) critical for a productive immune response to either pathogen. This significant difference in cytokine production between trimannose cap sugar–treated and control groups was not observed in draining lymph node cells from TLR2−/− mice. Type of inflammation and rate of bead entry into macrophages and dendritic cells were different for trimannose-coated beads compared with control oligosaccharide-coated beads, indicating selective lectin receptor/oligosaccharide interactions mediating cell entry and cytokine production. These novel findings may prompt the development of targeted oligosaccharide adjuvants against chronic infections. PMID:21763266

  18. Efficient separation of oxidized cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases.

    PubMed

    Westereng, Bjørge; Agger, Jane Wittrup; Horn, Svein J; Vaaje-Kolstad, Gustav; Aachmann, Finn L; Stenstrøm, Yngve H; Eijsink, Vincent G H

    2013-01-01

    We present an evaluation of HPLC-based analytical tools for the simultaneous analysis of native and oxidized cello-oligosaccharides, which are products of enzymatic cellulose degradation. Whereas cello-oligosaccharides arise from cellulose depolymerization by glycoside hydrolases, oxidized cello-oligosaccharides are produced by cellobiose dehydrogenase and the recently identified copper dependent lytic polysaccharide monooxygenases (LPMOs) currently classified as CBM33 and GH61. The latter enzymes are wide-spread and expected to play crucial roles in further development of efficient enzyme technology for biomass conversion. Three HPLC approaches with well documented performance in the field of oligosaccharide analysis have been investigated: high-performance anion-exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC) and porous graphitized carbon liquid chromatography (PGC-LC). HPAEC with pulsed amperometric detection (PAD) was superior for analysis of oxidized oligosaccharides, combining the best separation with superior sensitivity for oligosaccharide species with a degree of polymerization (DP) ranging from 1 to 10. Furthermore, the HPAEC method can be optimized for operation in a high-throughput manner (run time 10 min). Both PGC-LC and HILIC allow reasonable run times (41 and 25 min, respectively), with acceptable separation, but suffer from poor sensitivity compared to HPAEC-PAD. On the other hand, PGC-LC and HILIC benefit from being fully compatible with online mass spectrometry. Using an LC-MS setup, these methods will deliver much better sensitivity than what can be obtained with conventional detectors such as ultraviolet-, charged aerosol-, or evaporative light scattering and may reach sensitivities similar to or even better than what is obtained in HPAEC-PAD. Pure oxidized cello-oligosaccharide standards, ranging from DP2 to DP5, were obtained by semi-preparative PGC and characterized by MS and NMR analysis. PMID:23246088

  19. Xyloglucan oligosaccharides promote growth and activate cellulase: Evidence for a role of cellulase in cell expansion. [Pisum sativum L

    SciTech Connect

    McDougall, G.J.; Fry, S.C. )

    1990-07-01

    Oligosaccharides produced by the action of fungal cellulase on xyloglucans promoted the elongation of etiolated pea (Pisum sativum L.) stem segments in a straight-growth bioassay designed for the determination of auxins. The oligosaccharides were most active at about 1 micromolar. We tested the relative growth-promoting activities of four HPLC-purified oligosaccharides which shared a common glucose{sub 4} {center dot} xylose{sub 3} (XG7) core. The substituted oligosaccharides XG8 (glucose{sub 4} {center dot} xylose{sub 3} {center dot} galactose) and XG9n (glucose{sub 4} {center dot} xylose{sub 3} {center dot} galactose{sub 2}) were more effective than XG7 itself and XG9 (glucose{sub 4} {center dot} xylose{sub 3} {center dot} galactose {center dot} fucose). The same oligosaccharides also promoted the degradation, assayed viscometrically, of xyloglucan by an acidic cellulase from bean (Phaseolus vulgaris L.) leaves. The oligosaccharides were highly active at 10{sup {minus}4} molar, causing up to a fourfold increase in activity, but the effect was still detectable at 1 micromolar. Those oligosaccharides (XG8 and XG9n) which best promoted growth, stimulated cellulase activity to the greatest extent. The oligosaccharides did not stimulate the action of the cellulase in an assay based on the conversion of ({sup 3}H)xyloglucan to ethanol-soluble fragments. This suggests that the oligosaccharides enhanced the midchain hydrolysis of xyloglucan molecules (which would rapidly reduce the viscosity of the solution), at the expense of cleavage near the termini (which would yield ethanol-soluble products).

  20. Effect of chito-oligosaccharides over human faecal microbiota during fermentation in batch cultures.

    PubMed

    Mateos-Aparicio, Inmaculada; Mengíbar, Marian; Heras, Angeles

    2016-02-10

    Chitosan with high number of deacetylated units, its reacetylated derivative and COS obtained through an enzymatic treatment with chitosanase were tested in pH controlled batch cultures to investigate the ability of the human faecal microbiota to utilise them. Chitosan derivatives with high number of deacetylated units decreased the bacterial populations: Bifidobacterium spp., Eubacterium rectale/Clostridium coccoides, C. Histolyticum and Bacteroides/Prevotella. On the other hand, chitosan derivatives with high content of acetylated residues maintained the tested bacterial groups and could increase Lactobacillus/Enterococcus. Regarding short chain fatty acids (SCFA), only low Mw COS increased the production in similar levels than fructo-oligossacharides (FOS). The acetylated chitosans and their COS do not appear as potential prebiotics but did not affect negatively the faecal microbiota, while derivatives with high number of deacetylated units could induce a colonic microbiota imbalance. PMID:26686171

  1. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  2. Grafting of oligosaccharides onto synthetic polymer colloids.

    PubMed

    Mange, Siyabonga; Dever, Cédric; De Bruyn, Hank; Gaborieau, Marianne; Castignolles, Patrice; Gilbert, Robert G

    2007-06-01

    A new method to form colloidally stable oligosaccharide-grafted synthetic polymer particles has been developed. The oligosaccharides, of weight-average degree of polymerization approximately 38, were obtained by enzymatic debranching of amylopectin. Through the use of a cerium(IV)-based redox initiation process, oligosaccharide chains are grafted onto a synthetic polymer colloid comprising electrostatically stabilized poly(methyl methacrylate) or polystyrene latex particles swollen with methyl methacrylate monomer. Ce(IV) creates a radical species on these oligosaccharides, which then propagates, initially with aqueous-phase monomer, then with the methyl methacrylate monomer inside the particles. Ultracentrifugation, NMR, and total starch analyses together prove that the grafting process has occurred, with at least 7.7 wt % starch grafted and a grafting efficiency of 33%. The surfactant used in latex preparation was removed by dialysis, resulting in particles colloidally stabilized with only linear starch as a steric stabilizer. The debranched starch that comprises these oligosaccharides is found to be a remarkably effective colloidal stabilizer, albeit at low electrolyte concentration, stabilizing particles with very sparse surface coverage. PMID:17497920

  3. Effects of an onion by-product on bioactivity and safety markers in healthy rats.

    PubMed

    Roldán-Marín, Eduvigis; Krath, Britta N; Poulsen, Morten; Binderup, Mona-Lise; Nielsen, Tom H; Hansen, Max; Barri, Thaer; Langkilde, Søren; Cano, M Pilar; Sánchez-Moreno, Concepción; Dragsted, Lars O

    2009-12-01

    Onions are excellent sources of bioactive compounds including fructo-oligosaccharides (FOS) and polyphenols. An onion by-product was characterised in order to be developed as a potentially bioactive food ingredient. Our main aim was to investigate whether the potential health and safety effects of this onion by-product were shared by either of two derived fractions, an extract containing the onion FOS and polyphenols and a residue fraction containing mainly cell wall materials. We report here on the effects of feeding these products on markers of potential toxicity, protective enzymes and gut environment in healthy rats. Rats were fed during 4 weeks with a diet containing the products or a control feed balanced in carbohydrate. The onion by-product and the extract caused anaemia as expected in rodents for Allium products. No other toxicity was observed, including genotoxicity. Glutathione reductase (GR) and glutathione peroxidase (GPx1) activities in erythrocytes increased when rats were fed with the onion extract. Hepatic gene expression of Gr, Gpx1, catalase, 5-aminolevulinate synthase and NAD(P)H:quinone oxidoreductase was not altered in any group of the onion fed rats. By contrast, gamma-glutamate cysteine ligase catalytic subunit gene expression was upregulated but only in rats given the onion residue. The onion by-products as well as the soluble and insoluble fractions had prebiotic effects as evidenced by decreased pH, increased butyrate production and altered gut microbiota enzyme activities. In conclusion, the onion by-products have no in vivo genotoxicity, may support in vivo antioxidative defence and alter the functionality of the rat gut microbiota. PMID:19682402

  4. Synthesis of Galacto-oligosaccharide in Two-phase System.

    PubMed

    Gui, Li-Qiong; Wei, Dong-Zhi; Cui, Yu-Min; Yu, Jun-Tang

    1999-01-01

    35 of the total products of galacto-oligosaccharide (GOS) could be obtained from the two-phase system with cyclohexane and ethyl acetate as bulk organic phases and 15% phosphate buffer as aqueous phase. The effects of temperature pH of buffer lactose concentration galactose and glucose and the immobilization of enzyme on the synthesis of GOS were studied. It was found that the reaction temperature and initial lactose concentration didn'thave obvious effects while the addition of glucose and galactose somewhat affected the GOS yield and the GOS yields could reach 64.78% with lactase immobilized on resin D345. PMID:12136210

  5. Structural confirmation of novel oligosaccharides isolated from sugar beet molasses.

    PubMed

    Abe, Tatsuya; Kikuchi, Hiroto; Aritsuka, Tsutomu; Takata, Yusuke; Fukushi, Eri; Fukushi, Yukiharu; Kawabata, Jun; Ueno, Keiji; Onodera, Shuichi; Shiomi, Norio

    2016-07-01

    Eleven oligosaccharides were isolated from sugar beet molasses using carbon-Celite column chromatography and HPLC. The constituent sugars and linkage positions were determined using methylation analysis, MALDI-TOF-MS, and NMR measurements. The configurations of isolated oligosaccharides were confirmed based on detailed NMR analysis. Based on our results, three of the 11 oligosaccharides were novel. PMID:26920296

  6. Cell-associated oligosaccharides of Bradyrhizobium spp.

    PubMed Central

    Miller, K J; Gore, R S; Johnson, R; Benesi, A J; Reinhold, V N

    1990-01-01

    We report the initial characterization of the cell-associated oligosaccharides produced by four Bradyrhizobium strains: Bradyrhizobium japonicum USDA 110, USDA 94, and ATCC 10324 and Bradyrhizobium sp. strain 32H1. The cell-associated oligosaccharides of these strains were found to be composed solely of glucose and were predominantly smaller than the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species. Linkage studies and nuclear magnetic resonance analyses demonstrated that the bradyrhizobial glucans are linked primarily by beta-1,6 and beta-1,3 glycosidic bonds. Thus, the bradyrhizobia appear to synthesize cell-associated oligosaccharides of structural character substantially different from that of the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species. PMID:2294083

  7. On-line separation and characterization of hyaluronan oligosaccharides derived from radical depolymerization

    PubMed Central

    Zhao, Xue; Yang, Bo; Li, Lingyun; Zhang, Fuming; Linhardt, Robert J.

    2013-01-01

    Hydroxyl radicals are widely implicated in the oxidation of carbohydrates in biological and industrial processes and are often responsible for their structural modification resulting in functional damage. In this study, the radical depolymerization of the polysaccharide hyaluronan was studied in a reaction with hydroxyl radicals generated by Fenton Chemistry. A simple method for isolation and identification of the resulting non-sulfated oligosaccharide products of oxidative depolymerization was established. Hyaluronan oligosaccharides were analyzed using ion-pairing reversed phase high performance liquid chromotography coupled with tandem electrospray mass spectrometry. The sequence of saturated hyaluronan oligosaccharides having even- and odd-numbers of saccharide units, afforded through oxidative depolymerization, were identified. This study represents a simple, effective ‘fingerprinting’ protocol for detecting the damage done to hyaluronan by oxidative radicals. This study should help reveal the potential biological outcome of reactive-oxygen radical-mediated depolymerization of hyaluronan. PMID:23768593

  8. Structural characterization and antioxidant activities of κ-carrageenan oligosaccharides degraded by different methods.

    PubMed

    Sun, Yujiao; Yang, Bingying; Wu, Yanmin; Liu, Yang; Gu, Xiao; Zhang, Hong; Wang, Chengjian; Cao, Hongzhi; Huang, Linjuan; Wang, Zhongfu

    2015-07-01

    In the present study, four kinds of κ-carrageenan oligosaccharides were obtained by the degradation of parent κ-carrageenan using free radical depolymerization, mild acid hydrolysis, κ-carrageenase digestion and partial reductive hydrolysis, respectively. Their structure types were accurately and comparatively elucidated by ESI-MS and CID MS/MS. The antioxidant activities of different degraded products were investigated by four different antioxidant assays, including superoxide radical scavenging activity, hydroxyl radical scavenging activity, reducing power and DPPH radical scavenging activity. The methods of depolymerization had an influence on the antioxidant activities of κ-carrageenan oligosaccharides obtained from κ-carrageenan. These results indicated that the antioxidant activities of κ-carrageenan oligosaccharides could be related to the degree of polymerization, the content of reducing sugar and sulfate groups, and the structure of reducing terminus. PMID:25704717

  9. Comparative study of fungal strains for thermostable inulinase production.

    PubMed

    Flores-Gallegos, Adriana C; Contreras-Esquivel, Juan C; Morlett-Chávez, Jesús A; Aguilar, Cristóbal N; Rodríguez-Herrera, Raúl

    2015-04-01

    Fructose and fructo-oligosaccharides (FOS) are important ingredients in the food industry. Fructose is considered an alternative sweetener to sucrose because it has higher sweetening capacity and increases iron absorption in children, and FOS's are a source of dietary fiber with a bifidogenic effect. Both compounds can be obtained by enzymatic hydrolysis of inulin. However, inulin presents limited solubility at room temperature, thus, fructose and FOS production is carried out at 60°C. Therefore, there is a growing interest to isolate and characterize thermostable inulinases. The aim of this work was to evaluate the capacity of different fungal strains to produce potential thermostable inulinases. A total of 27 fungal strains belonging to the genera Aspergillus, Penicillium, Rhizopus, Rhizomucor and Thermomyces were evaluated for production of inulinase under submerged culture using Czapek Dox medium with inulin as a sole carbon source. Strains were incubated at 37°C and 200 rpm for 96 h. Crude enzyme extract was obtained to evaluate inulinase and invertase activity. In order to select the fungal strain with the highest thermostable inulinase production, a selection criterion was established. It was possible to determine the highest inulinase activity for Rhizopus microsporus 13aIV (10.71 U/mL) at 36 h with an optimum temperature of inulinase of 70°C. After 6 h at 60°C, the enzyme did not show any significant loss of activity and retained about 87% activity, while it only retains 57% activity at 70°C. According to hydrolysis products, R. microsporus produced endo and exo-inulinase. PMID:25454696

  10. Versatile strategy for the divergent synthesis of linear oligosaccharide domain variants of Quillaja saponin vaccine adjuvants.

    PubMed

    Fernández-Tejada, Alberto; Tan, Derek S; Gin, David Y

    2015-09-21

    We describe a new, versatile synthetic approach to Quillaja saponin variants based on the natural product immunoadjuvant QS-21. This modular, divergent strategy provides efficient access to linear oligosaccharide domain variants with modified sugars and regiochemistries. This new synthetic approach opens the door to the rapid generation of diverse analogues to identify novel saponin adjuvants with improved synthetic accessibility. PMID:26243268

  11. Oligosaccharides of Cabernet Sauvignon, Syrah and Monastrell red wines.

    PubMed

    Apolinar-Valiente, Rafael; Romero-Cascales, Inmaculada; Williams, Pascale; Gómez-Plaza, Encarna; López-Roca, José María; Ros-García, José María; Doco, Thierry

    2015-07-15

    Wine oligosaccharides were recently characterized and their concentrations, their composition and their roles on different wines remain to be determined. The concentration and composition of oligosaccharides in Cabernet Sauvignon, Syrah and Monastrell wines was studied. Oligosaccharide fractions were isolated by high resolution size-exclusion chromatography. The neutral and acidic sugar composition was determined by gas chromatography. The MS spectra of the oligosaccharides were performed on an AccuTOF mass spectrometer. Molar-mass distributions were determined by coupling size exclusion chromatography with a multi-angle light scattering device (MALLS) and a differential refractive index detector. Results showed significant differences in the oligosaccharidic fraction from Cabernet Sauvignon, Syrah and Monastrell wines. This study shows the influence that the grape variety seems have on the quantity, composition and structure of oligosaccharides in the finished wine. To our knowledge, this is the first report to research the oligosaccharides composition of Cabernet Sauvignon, Syrah and Monastrell wines. PMID:25722170

  12. Delta inulin: a novel, immunologically active, stable packing structure comprising β-D-[2 -> 1] poly(fructo-furanosyl) α-D-glucose polymers.

    PubMed

    Cooper, Peter D; Petrovsky, Nikolai

    2011-05-01

    We report a novel isoform of β-D-[2 → 1] poly(fructo-furanosyl) α-D-glucose termed delta inulin (DI), comparing it with previously described alpha (AI), beta (BI) and gamma (GI) isoforms. In vitro, DI is the most immunologically active weight/weight in human complement activation and in binding to monocytes and regulating their chemokine production and cell surface protein expression. In vivo, this translates into potent immune adjuvant activity, enhancing humoral and cellular responses against co-administered antigens. As a biocompatible polysaccharide particle, DI is safe and well tolerated by subcutaneous or intramuscular injection. Physico-chemically, DI forms as an insoluble precipitate from an aqueous solution of suitable AI, BI or GI held at 37-48°C, whereas the precipitate from the same solution at lower temperatures has the properties of AI or GI. DI can also be produced by heat conversion of GI suspensions at 56°C, whereas GI is converted from AI at 45°C. DI is distinguished from GI by its higher temperature of solution in dilute aqueous suspension and by its lower solubility in dimethyl sulfoxide, both consistent with greater hydrogen bonding in DI's polymer packing structure. DI suspensions can be dissolved by heat, re-precipitated by cooling as AI and finally re-converted back to DI by repeated heat treatment. Thus, DI, like the previously described inulin isoforms, reflects the formation of a distinct polymer aggregate packing structure via reversible noncovalent bonding. DI forms the basis for a potent new human vaccine adjuvant and further swells the growing family of carbohydrate structures with immunological activity. PMID:21147758

  13. New Food Oligosaccharides via Alternansucrase Acceptor Reactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alternansucrase [EC 2.4.1.140] is a glycosyltransferase from food-grade bacteria that is capable of synthesizing unique polysaccharides and oligosaccharides from sucrose. The enzyme typically produces the high-molecular weight polysaccharide alternan. However, in the presence of low-molecular weig...

  14. Fermentation of xylo-oligosaccharides obtained from wheat bran and Bengal gram husk by lactic acid bacteria and bifidobacteria.

    PubMed

    Madhukumar, M S; Muralikrishna, G

    2012-12-01

    Different strains of Bifidobacteria, Lactobacilli and Pediococci spp. were evaluated for their utilization of xylo-oligosaccharides derived from Bengal gram husk and wheat bran water extractable polysaccharides. The fermentation pattern of xylo-oligosaccharides by bacteria depends on the nature of xylo-oligosaccharides i.e. degree of polymerization and arabinose to xylose ratio as well as the bacterial strain tested, which inturn are very important for designing species-specific prebiotic xylo-oligosaccharides and synbiotic preparations for incorporation in various health foods. All the bacterial strains tested readily utilized xylo-oligosaccharides derived from bengal gram husk and wheat bran as indicated by the increase in (a) turbidity of the culture broth (b) xylanase, xylosidase and arabinosidase activities (c) dry cell mass and (d) the liberation of short chain fatty acids (SCFA). Acetate was found to be the major SCFA produced as the end product of fermentation and its amount varied from 75.4 to 100 mol%. Xylo-oligosaccharides derived from wheat bran were found to have better prebiotic activity compared to the one derived from Bengal gram husk and this can be ascribed to relatively high amount of arabinose. PMID:24293694

  15. Identification of novel isomeric pectic oligosaccharides using hydrophilic interaction chromatography coupled to traveling-wave ion mobility mass spectrometry.

    PubMed

    Leijdekkers, Antonius G M; Huang, Jie-Hong; Bakx, Edwin J; Gruppen, Harry; Schols, Henk A

    2015-03-01

    Separation and characterization of complex mixtures of pectic oligosaccharides still remains challenging and often requires the use of multiple analytical techniques, especially when isomeric structures are present. In this work, it is demonstrated that the coupling of hydrophilic interaction chromatography (HILIC) to traveling-wave ion mobility mass spectrometry (TWIMMS) enabled the simultaneous separation and characterization of complex mixtures of various isomeric pectic oligosaccharides. Labeling of oligosaccharides with 3-aminoquinoline (3-AQ) improved MS-ionization efficiency of the oligosaccharides and reduced the complexity of the product ion mass spectra, without losing resolution of the HILIC separation. In addition, labeling enabled quantification of oligosaccharides on molar basis using in-line fluorescence detection. Isomeric structures were distinguished using TWIMMS. The 3-AQ-HILIC-TWIMMS method was used to characterize a series of isomeric sugar beet rhamnogalacturonan I derived oligosaccharides carrying a glucuronic acid substituent. Thereby, some novel structural features were identified for the first time: glucuronic acid was attached to O-3 or to O-2 of galacturonic acid residues and a single galacturonic acid residue within an oligomer could contain both an acetyl group and a glucuronic acid substituent. PMID:25647688

  16. Enzymatic generation of galactose-rich oligosaccharides/oligomers from potato rhamnogalacturonan I pectic polysaccharides.

    PubMed

    Khodaei, Nastaran; Karboune, Salwa

    2016-04-15

    Potato pulp by-product rich in galactan-rich rhamnogalacturonan I (RG I) was investigated as a new source of oligosaccharides with potential prebiotic properties. The efficiency of selected monocomponent enzymes and multi-enzymatic preparations to generate oligosaccharides/oligomers from potato RG I was evaluated. These overall results of yield were dependent on the activity profile of the multi-enzymatic preparations. Highest oligo-RG I yield of 93.9% was achieved using multi-enzymatic preparation (Depol 670L) with higher hydrolytic activity toward side chains of RG I as compared to its backbone. Main oligo-RG I products were oligosaccharides with DP of 2-12 (79.8-100%), while the oligomers with DP of 13-70 comprised smaller proportion (0.0-20.2%). Galactose (58.9-91.2%, w/w) was the main monosaccharide of oligo-RG I, while arabinose represented 0.0-12.1%. An understanding of the relationship between the activity profile of multi-enzymatic preparations and the yield/DP of oligo-RG I was achieved. This is expected to provide the capability to generate galacto- and galacto(arabino) oligosaccharides and their corresponding oligomers from an abundant by-product. PMID:26616968

  17. Novel process for the coproduction of xylo-oligosaccharides, fermentable sugars, and lignosulfonates from hardwood.

    PubMed

    Huang, Caoxing; Jeuck, Ben; Du, Jing; Yong, Qiang; Chang, Hou-Min; Jameel, Hasan; Phillips, Richard

    2016-11-01

    Many biorefineries have not been commercialized due to poor economic returns from final products. In this work, a novel process has been developed to coproduce valuable sugars, xylo-oligosaccharides, and lignosulfonates from hardwood. The modified process includes a mild autohydrolysis pretreatment, which enables for the recovery of the xylo-oligosaccharides in auto-hydrolysate. Following enzymatic hydrolysis, the residue is sulfomethylated to produce lignosulfonates. Recycling the sulfomethylation residues increased both the glucan recovery and lignosulfonate production. The glucose recovery was increased from 81.7% to 87.9%. Steady state simulation using 100g of hardwood produced 46.7g sugars, 5.9g xylo-oligosaccharides, and 25.7g lignosulfonates, which were significantly higher than that produced from the no-recycling process with 39.1g sugars, 5.9g xylo-oligosaccharides, and 15.0g lignosulfonates. The results indicate that this novel biorefinery process can improve the production of fermentable sugars and lignosulfonate from hardwood as compared to a conventional biorefinery process. PMID:27543951

  18. Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.

    PubMed

    Newburg, D S

    2009-04-01

    This review discusses the role of human milk glycans in protecting infants, but the conclusion that the human milk glycans constitute an innate immune system whereby the mother protects her offspring may have general applicability in all mammals, including species of commercial importance. Infants that are not breastfed have a greater incidence of severe diarrhea and respiratory diseases than those who are breastfed. In the past, this had been attributed primarily to human milk secretory antibodies. However, the oligosaccharides are major components of human milk, and milk is also rich in other glycans, including glycoproteins, mucins, glycosaminoglycans, and glycolipids. These milk glycans, especially the oligosaccharides, are composed of thousands of components. The milk factor that promotes gut colonization by Bifidobacterium bifidum was found to be a glycan, and such prebiotic characteristics may contribute to protection against infectious agents. However, the ability of human milk glycans to protect the neonate seems primarily to be due to their inhibition of pathogen binding to their host cell target ligands. Many such examples include specific fucosylated oligosaccharides and glycans that inhibit specific pathogens. Most human milk oligosaccharides are fucosylated, and their production depends on fucosyltransferase enzymes; mutations in these fucosyltransferase genes are common and underlie the various Lewis blood types in humans. Variable expression of specific fucosylated oligosaccharides in milk, also a function of these genes (and maternal Lewis blood type), is significantly associated with the risk of infectious disease in breastfed infants. Human milk also contains major quantities and large numbers of sialylated oligosaccharides, many of which are also present in bovine colostrum. These could similarly inhibit several common viral pathogens. Moreover, human milk oligosaccharides strongly attenuate inflammatory processes in the intestinal mucosa. These

  19. Oligosaccharides in Urine, Blood, and Feces of Piglets Fed Milk Replacer Containing Galacto-oligosaccharides.

    PubMed

    Difilippo, Elisabetta; Bettonvil, Monique; Willems, Rianne H A M; Braber, Saskia; Fink-Gremmels, Johanna; Jeurink, Prescilla V; Schoterman, Margriet H C; Gruppen, Harry; Schols, Henk A

    2015-12-23

    Human milk oligosaccharides (HMOs) are absorbed into the blood (about 1% of the HMO intake) and subsequently excreted in urine, where they may protect the infant from pathogen infection. As dietary galacto-oligosaccharides (GOS) have partial structural similarities with HMOs, this study investigated the presence of GOS and oligosaccharides originating from milk replacer in blood serum, urine, and cecal and fecal samples of piglets, as a model for human infants. Using liquid chromatography-mass spectrometry and capillary electrophoresis with fluorescence detection, oligosaccharides originating from piglet diet including 3'-sialyllactose and specific GOS ranging from degree of polymerization 3 to 6 were detected in blood serum and in urine of piglets. In blood serum, GOS levels ranged from 16 to 23 μg/mL, representing about 0.1% of the GOS daily intake. In urine, approximately 0.85 g of GOS/g of creatinine was found. Cecum digesta and feces contained low amounts of oligosaccharides, suggesting an extensive GOS intestinal fermentation in piglets. PMID:26621571

  20. Profiling oligosaccharidurias by electrospray tandem mass spectrometry: quantifying reducing oligosaccharides.

    PubMed

    Ramsay, Steven L; Meikle, Peter J; Hopwood, John J; Clements, Peter R

    2005-10-01

    A method to semiquantify urinary oligosaccharides from patients suffering from oligosaccharidurias is presented. 1-Phenyl-3-methyl-5-pyrazolone has been used to derivatize urinary oligosaccharides prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Disease-specific oligosaccharides were identified for several oligosaccharidurias, including GM1 gangliosidosis, GM2 gangliosidosis, sialic acid storage disease, sialidase/neuraminidase deficiency, galactosialidosis, I-cell disease, fucosidosis, Pompe and Gaucher diseases, and alpha-mannosidosis. The oligosaccharides were referenced against the internal standard, methyl lactose, to produce ratios for comparison with control samples. Elevations in specific urinary oligosaccharides were indicative of lysosomal disease and the defective catabolic enzyme. This method has been adapted to enable assay of large sample numbers and could readily be extended to other oligosaccharidurias and to monitor oligosaccharide levels in patients receiving treatment. It also has immediate potential for incorporation into a newborn screening program. PMID:16111643

  1. Detection of N-(1-deoxy-d-fructos-1-yl) Fumonisins B2 and B3 in Corn by High-Resolution LC-Orbitrap MS

    PubMed Central

    Matsuo, Yosuke; Takahara, Kentaro; Sago, Yuki; Kushiro, Masayo; Nagashima, Hitoshi; Nakagawa, Hiroyuki

    2015-01-01

    The existence of glucose conjugates of fumonisin B2 (FB2) and fumonisin B3 (FB3) in corn powder was confirmed for the first time. These “bound-fumonisins” (FB2 and FB3 bound to glucose) were identified as N-(1-deoxy-d-fructos-1-yl) fumonisin B2 (NDfrc-FB2) and N-(1-deoxy-d-fructos-1-yl) fumonisin B3 (NDfrc-FB3) respectively, based on the accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS) analysis. Treatment on NDfrc-FB2 and NDfrc-FB3 with the o-phthalaldehyde (OPA) reagent also supported that d-glucose binding to FB2 and FB3 molecules occurred to their primary amine residues. PMID:26389955

  2. Revision of the oligosaccharide structures of yeast carboxypeptidase Y

    SciTech Connect

    Ballou, L.; Hernandez, L.M.; Alvarado, E.; Ballou, C.E. )

    1990-05-01

    The N-linked oligosaccharides from baker's yeast carboxypeptidase Y were analyzed by {sup 1}H NMR and specific mannosidase digestion and found to be identical to those from the Saccharomyces cerevisiae mnn9 mutant bulk mannoprotein. The results support the view that the mnn mutants make oligosaccharides that are a true reflection of the normal biosynthetic pathway and confirm that a recently revised yeast oligosaccharide structure is applicable to wild-type mannoproteins.

  3. Divergent Synthesis of Heparan Sulfate Oligosaccharides.

    PubMed

    Dulaney, Steven B; Xu, Yongmei; Wang, Peng; Tiruchinapally, Gopinath; Wang, Zhen; Kathawa, Jolian; El-Dakdouki, Mohammad H; Yang, Bo; Liu, Jian; Huang, Xuefei

    2015-12-18

    Heparan sulfates are implicated in a wide range of biological processes. A major challenge in deciphering their structure and activity relationship is the synthetic difficulties to access diverse heparan sulfate oligosaccharides with well-defined sulfation patterns. In order to expedite the synthesis, a divergent synthetic strategy was developed. By integrating chemical synthesis and two types of O-sulfo transferases, seven different hexasaccharides were obtained from a single hexasaccharide precursor. This approach combined the flexibility of chemical synthesis with the selectivity of enzyme-catalyzed sulfations, thus simplifying the overall synthetic operations. In an attempt to establish structure activity relationships of heparan sulfate binding with its receptor, the synthesized oligosaccharides were incorporated onto a glycan microarray, and their bindings with a growth factor FGF-2 were examined. The unique combination of chemical and enzymatic approaches expanded the capability of oligosaccharide synthesis. In addition, the well-defined heparan sulfate structures helped shine light on the fine substrate specificities of biosynthetic enzymes and confirm the potential sequence of enzymatic reactions in biosynthesis. PMID:26574650

  4. Sialylated oligosaccharides O-glycosidically linked to glycoprotein C from herpes simplex virus type 1.

    PubMed

    Dall'Olio, F; Malagolini, N; Speziali, V; Campadelli-Fiume, G; Serafini-Cessi, F

    1985-10-01

    Glycoprotein C (gC) was purified by immunoabsorbent from herpes simplex virus type-1-infected BHK cells labeled with [14C]glucosamine for 11 h and chased for 3 h. Glycopeptides obtained by pronase digestion of gC were fractionated by Bio-Gel filtration and concanavalin A-Sepharose chromatography. Each glycopeptide fraction was analyzed for amino sugar composition by thin-layer chromatography. The majority of radioactivity was recovered as N-acetylglucosamine, but a significant amount of labeled N-acetylgalactosamine was detected and recovered preferentially in some glycopeptide species. Mild alkaline borohydride treatment of the glycopeptides resulted in the release of small degradation products which contained N-acetylgalactosaminitol as the major labeled component and a drastic reduction of N-acetylgalactosamine in the residual glycopeptides. These results demonstrated that gC carries O-glycosidically linked oligosaccharides in addition to the N-linked di- and triantennary glycans previously described (F. Serafini-Cessi, F. Dall'Olio, L. Pereira, and G. Campadelli-Fiume, J. Virol. 51:838-844, 1984). Chromatographic behavior on DEAE-Sephacel chromatography and neuraminidase digestion of O-linked oligosaccharides indicated the presence of two major sialylated species carrying one and two sialic acid residues, respectively. The characterization of a peculiar glycopeptide species supported the notion that some of the O-linked oligosaccharides are bound to a cluster of hydroxyamino acids located near an N-glycosylation site which carries one N-linked diantennary oligosaccharide. PMID:2993643

  5. Oligosaccharides of milk and colostrum in non-human mammals.

    PubMed

    Urashima, T; Saito, T; Nakamura, T; Messer, M

    2001-05-01

    Mammalian milk or colostrum usually contains, in addition to lactose, a variety of neutral and acidic oligosaccharides. Although the oligosaccharides of human milk have been reviewed in several recent publications, those of non-human mammals have received much less attention. This paper reviews the chemical structures and the variety of milk oligosaccharides in species other than humans, including placental mammals (e.g. primates, domestic herbivores, bears and other carnivores, the rat and the elephant) as well as monotremes (platypus and echidna) and marsupials (e.g. wallaby). The gastrointestinal digestion and absorption and the possible biological functions of these oligosaccharides are also discussed. PMID:11925504

  6. The principal fucosylated oligosaccharides of human milk exhibit prebiotic properties on cultured infant microbiota

    PubMed Central

    Yu, Zhuo-Teng; Chen, Ceng; Kling, David E; Liu, Bo; McCoy, John M; Merighi, Massimo; Heidtman, Matthew; Newburg, David S

    2013-01-01

    Breast-fed infant microbiota is typically rich in bifidobacteria. Herein, major human milk oligosaccharides (HMOS) are assessed for their ability to promote the growth of bifidobacteria and to acidify their environment, key features of prebiotics. During in vitro anaerobic fermentation of infant microbiota, supplementation by HMOS significantly decreased the pH even greater than supplementation by fructooligosaccharide (FOS), a prebiotic positive control. HMOS elevated lactate concentrations, increased the proportion of Bifidobacterium spp. in culture, and through their fermentation into organic acids, decreased the proportion of Escherichia and Clostridium perfringens. Three principal components of HMOS, 2′-fucosyllactose, lactodifucotetraose and 3-fucosyllactose, were consumed in these cultures. These three principal oligosaccharides of human milk were then individually tested as supplements for in vitro growth of four individual representative strains of infant gut microbes. Bifidobacterium longum JCM7007 and B. longum ATCC15697 efficiently consumed oligosaccharides and produced abundant lactate and short-chain fatty acids, resulting in significant pH reduction. The specificity of fermentation differed by microbe species and strain and by oligosaccharide structure. Escherichia coli K12 and C. perfringens did not utilize appreciable fucosylated oligosaccharides, and a typical mixture of organic acid fermentation products inhibited their growth. In summary, 2′-fucosyllactose, lactodifucotetraose, and 3-fucosyllactose, when cultured with B. longum JCM7007 and B. longum ATCC15697, exhibit key characteristics of a prebiotic in vitro. If these bifidobacteria are representative of pioneering or keystone species for human microbiota, fucosylated HMOS could strongly promote colonization and maintenance of a mutualist symbiotic microbiome. Thus, these simple glycans could mediate beneficial effects of human milk on infant health. PMID:23028202

  7. Consumption of Human Milk Oligosaccharides by Gut-related Microbes

    PubMed Central

    Marcobal, Angela; Barboza, Mariana; Froehlich, John W.; Block, David E.; German, J. Bruce; Lebrilla, Carlito B.; Mills, David A.

    2010-01-01

    Human milk contains large amounts of complex oligosaccharides that putatively modulate the intestinal microbiota of breast-fed infants by acting as decoy binding sites for pathogens and as prebiotics for enrichment of beneficial bacteria. Several bifidobacterial species have been shown to grow well on human milk oligosaccharides. However, little data exists on other bacterial species. In this work we examined 16 bacterial strains belonging to 10 different genera for growth on human milk oligosaccharides. For this propose, we used a chemically-defined medium, ZMB1, which allows vigorous growth of a number gut–related microorganisms in a fashion similar to complex media. Interestingly, Bifidobacterium longum subsp. infantis, Bacteroides fragilis and Bacteroides vulgatus strains were able to metabolize milk oligosaccharides with high efficiency, while Enterococcus, Streptococcus, Veillonella, Eubacterium, Clostridium, and Escherichia coli strains grew less well or not at all. Mass spectrometry-based glycoprofiling of the oligosaccharide consumption behavior revealed a specific preference for fucosylated oligosaccharides by Bifidobacterium longum subsp. infantis and Bacteroides vulgatus. This work expands the current knowledge of human milk oligosaccharides consumption by gut microbes, revealing bacteroides as avid consumer of this substrate. These results provide insight on how human milk oligosaccharides shape the infant intestinal microbiota. PMID:20394371

  8. Nitric Oxide-Releasing Chitosan Oligosaccharides as Antibacterial Agents

    PubMed Central

    Lu, Yuan; Slomberg, Danielle L.; Schoenfisch, Mark H.

    2014-01-01

    Secondary amine-functionalized chitosan oligosaccharides of different molecular weights (i.e., ~2500, 5000, 10000) were synthesized by grafting 2-methyl aziridine from the primary amines on chitosan oligosaccharides, followed by reaction with nitric oxide (NO) gas under basic conditions to yield N-diazeniumdiolate NO donors. The total NO storage, maximum NO flux, and half-life of the resulting NO-releasing chitosan oligosaccharides were controlled by the molar ratio of 2-methyl aziridine to primary amines (e.g., 1:1, 2:1) and the functional group surrounding the N-diazeniumdiolates (e.g., polyethylene glycol (PEG) chains), respectively. The secondary amine-modified chitosan oligosaccharides greatly increased the NO payload over existing biodegradable macromolecular NO donors. In addition, the water-solubility of the chitosan oligosaccharides enabled their penetration across the extracellular polysaccharides matrix of Pseudomonas aeruginosa biofilms and association with embedded bacteria. The effectiveness of these chitosan oligosaccharides at biofilm eradication was shown to depend on both the molecular weight and ionic characteristics. Low molecular weight and cationic chitosan oligosaccharides exhibited rapid association with bacteria throughout the entire biofilm, leading to enhanced biofilm killing. At concentrations resulting in 5-log killing of bacteria in Pseudomonas aeruginosa biofilms, the NO-releasing and control chitosan oligosaccharides elicited no significant cytotoxicity to mouse fibroblast L929 cells in vitro. PMID:24268196

  9. Binding of oligosaccharides of hyaluronic acid to proteoglycans (Short Communication)

    PubMed Central

    Hardingham, Timothy E.; Muir, Helen

    1973-01-01

    Oligosaccharides derived from hyaluronic acid were shown to inhibit proteoglycan–hyaluronic acid interaction, as measured in a viscometer. The relative inhibition increased with the size of the oligosaccharide and the results suggested that decasaccharides were the smallest fragments able to bind strongly to the proteoglycan. PMID:4273187

  10. Automated assembly of oligosaccharides containing multiple cis-glycosidic linkages.

    PubMed

    Hahm, Heung Sik; Hurevich, Mattan; Seeberger, Peter H

    2016-01-01

    Automated glycan assembly (AGA) has advanced from a concept to a commercial technology that rapidly provides access to diverse oligosaccharide chains as long as 30-mers. To date, AGA was mainly employed to incorporate trans-glycosidic linkages, where C2 participating protecting groups ensure stereoselective couplings. Stereocontrol during the installation of cis-glycosidic linkages cannot rely on C2-participation and anomeric mixtures are typically formed. Here, we demonstrate that oligosaccharides containing multiple cis-glycosidic linkages can be prepared efficiently by AGA using monosaccharide building blocks equipped with remote participating protecting groups. The concept is illustrated by the automated syntheses of biologically relevant oligosaccharides bearing various cis-galactosidic and cis-glucosidic linkages. This work provides further proof that AGA facilitates the synthesis of complex oligosaccharides with multiple cis-linkages and other biologically important oligosaccharides. PMID:27580973

  11. Characterization of a bifidobacterial system that utilizes galacto-oligosaccharides.

    PubMed

    Shigehisa, Akira; Sotoya, Hidetsugu; Sato, Takashi; Hara, Taeko; Matsumoto, Hoshitaka; Matsuki, Takahiro

    2015-07-01

    The galacto-oligosaccharide (GOS) OLIGOMATE 55N (Yakult) is a mixture of oligosaccharides, the main component of which is 4'-galactosyllactose (4'-GL). Numerous reports have shown that GOSs are non-digestible, reach the colon and selectively stimulate the growth of bifidobacteria. The product has been used as a food ingredient and its applications have expanded rapidly. However, the bifidobacterial glycoside hydrolases and transporters responsible for utilizing GOSs have not been characterized sufficiently. In this study, we aimed to identify and characterize genes responsible for metabolizing 4'-GL in Bifidobacterium breve strain Yakult. We attempted to identify B. breve Yakult genes induced by 4'-GL using transcriptional profiling during growth in basal medium containing 4'-GL with a custom microarray. We found that BbrY_0420, which encodes solute-binding protein (SBP), and BbrY_0422, which encodes β-galactosidase, were markedly upregulated relative to that during growth in basal medium containing lactose. Investigation of the substrate specificity of recombinant BbrY_0420 protein using surface plasmon resonance showed that BbrY_0420 protein bound to 4'-GL, but not to 3'-GL and 6'-GL, structural isomers of 4'-GL. Additionally, BbrY_0420 had a strong affinity for 4-galactobiose (4-GB), suggesting that this SBP recognized the non-reducing terminal structure of 4'-GL. Incubation of purified recombinant BbrY_0422 protein with 4'-GL, 3'-GL, 6'-GL and 4-GB revealed that the protein efficiently hydrolysed 4'-GL and 4-GB, but did not digest 3'-GL, 6'-GL or lactose, suggesting that BbrY_0422 digested the bond within Gal1,4-β-Gal. Thus, BbrY_0420 (SBP) and BbrY_0422 (β-galactosidase) had identical, strict substrate specificity, suggesting that they were coupled by co-induction to facilitate the transportation and hydrolysis of 4'-GL. PMID:25903756

  12. Multifunctional fructans and raffinose family oligosaccharides

    PubMed Central

    den Ende, Wim Van

    2013-01-01

    Fructans and raffinose family oligosaccharides (RFOs) are the two most important classes of water-soluble carbohydrates in plants. Recent progress is summarized on their metabolism (and regulation) and on their functions in plants and in food (prebiotics, antioxidants). Interest has shifted from the classic inulin-type fructans to more complex fructans. Similarly, alternative RFOs were discovered next to the classic RFOs. Considerable progress has been made in the understanding of structure–function relationships among different kinds of plant fructan metabolizing enzymes. This helps to understand their evolution from (invertase) ancestors, and the evolution and role of so-called “defective invertases.” Both fructans and RFOs can act as reserve carbohydrates, membrane stabilizers and stress tolerance mediators. Fructan metabolism can also play a role in osmoregulation (e.g., flower opening) and source–sink relationships. Here, two novel emerging roles are highlighted. First, fructans and RFOs may contribute to overall cellular reactive oxygen species (ROS) homeostasis by specific ROS scavenging processes in the vicinity of organellar membranes (e.g., vacuole, chloroplasts). Second, it is hypothesized that small fructans and RFOs act as phloem-mobile signaling compounds under stress. It is speculated that such underlying antioxidant and oligosaccharide signaling mechanisms contribute to disease prevention in plants as well as in animals and in humans. PMID:23882273

  13. C-Glycosyl Analogs of Oligosaccharides

    NASA Astrophysics Data System (ADS)

    Vauzeilles, Boris; Urban, Dominique; Doisneau, Gilles; Beau, Jean-Marie

    This chapter covers the synthesis of a large collection of "C-oligosaccharides ", synthetic analogs of naturally occurring oligosaccharides in which a carbon atom replaces the anomeric, interglycosidic oxygen atom. These non-natural constructs are stable to chemical and enzymatic degradation, and are primarily devised to probe carbohydrate-based biological processes. These mainly target carbohydrate-protein interactions such as the modulation of glycoenzyme (glycosylhydrolases and transferases) activities or the design of ligands for lectin Carbohydrate Recognition Domains. The discussion is based on the key carbon-carbon bond assembling steps on carbohydrate templates: ionic (anionic and cationic chemistries, sigmatropic rearrangements) or radical assemblage, and olefin metathesis. Synthetic schemes in which at least one of the monosaccharide units is constructed by total synthesis or by cyclization of acyclic chiral chains are presented separately in a "partial de novo synthesis" section. The review also provides comments, when they are known, on the conformational and binding properties of these synthetic analogs, as well as their biological behavior when tested.

  14. Mannan Oligosaccharides in Nursery Pig Nutrition and Their Potential Mode of Action

    PubMed Central

    Halas, Veronika; Nochta, Imre

    2012-01-01

    Simple Summary The aim of the paper is to provide a review of mannan oligosaccharide products in relation to their growth promoting effect and mode of action. Mannan oligosaccharide products maintain intestinal integrity and the digestive and absorptive function of the gut in the post-weaning period in pigs and enhance disease resistance by promoting antigen presentation. We find that dietary supplementation has growth promoting effects in pigs kept in a poor hygienic environment, while the positive effect of MOS is not observed in healthy pig herds with high hygienic standards. Abstract Mannan oligosaccharides (MOSs) are often referred to as one of the potential alternatives for antimicrobial growth promoters. The aim of the paper is to provide a review of mannan oligosaccharide products in relation to their growth promoting effect and mode of action based on the latest publications. We discuss the dietary impact of MOSs on (1) microbial changes, (2) morphological changes of gut tissue and digestibility of nutrients, and (3) immune response of pigs after weaning. Dietary MOSs maintain the intestinal integrity and the digestive and absorptive function of the gut in the post-weaning period. Recent results suggest that MOS enhances the disease resistance in swine by promoting antigen presentation facilitating thereby the shift from an innate to an adaptive immune response. Accordingly, dietary MOS supplementation has a potential growth promoting effect in pigs kept in a poor hygienic environment, while the positive effect of MOS is not observed in healthy pig herds with high hygienic standards that are able to maintain a high growth rate after weaning. PMID:26486920

  15. Affinity entrapment of oligosaccharides and glycopeptides using free lectin solution.

    PubMed

    Yodoshi, Masahiro; Oyama, Takehiro; Masaki, Ken; Kakehi, Kazuaki; Hayakawa, Takao; Suzuki, Shigeo

    2011-01-01

    Two procedures were proposed for the specific recovery of fluorescent derivatives of glycoprotein-derived oligosaccharides and tryptic glycopeptides using certain plant lectins. The first was based on the salting out of oligosaccharide-lectin conjugates with ammonium sulfate. Oligosaccharides specifically bound to lectins were recovered free from lectins using ethanol precipitation after dissolution in water. This method enabled group separation of 2-aminopyridine-labeled oligosaccharides derived from ovalbumin to galacto-oligosaccharides and agalacto-oligosaccharides by Ricinus communis agglutinin, and to high mannose- and hybrid-type oligosaccharides by wheat-germ agglutinin. Fractional precipitation based on differences in affinity for concanavalin A was accomplished by adding an appropriate concentration of methyl α-mannoside as an inhibitor. In the second method, tryptic digests of glycoproteins were mixed with a lectin solution, and the glycopeptide-lectin conjugates were specifically trapped on a centrifugal ultrafiltration membrane with cut-off of 10 kD. Trapped glycopeptides, as retentates, were passed through membranes by resuspension in diluted acid. This method is particularly useful for the enrichment of glycopeptides in protease digestion mixtures for glycosylation analyses by liquid chromatography-mass spectrometry. PMID:21478615

  16. Phospho-oligosaccharide dependent phosphorylation of ATP citrate lyase.

    PubMed

    Puerta, J; Mato, J M; Alemany, S

    1990-01-01

    The effect of insulin on ATP citrate lyase phosphorylation has been shown to be mimicked by a phospho-oligosaccharide in intact adipocytes. We demonstrate that the addition of phospho-oligosaccharide to intact adipocytes enhances the phosphorylation of ATP citrate lyase in the same tryptic peptide as insulin does. The addition of phospho-oligosaccharide to an adipocyte extract also results in an increase in ATP citrate lyase phosphorylation but in a different site than that observed in intact cells. The phospho-oligosaccharide-dependent incorporation of phosphate into ATP citrate lyase in intact cells is resistant to isopropanol and acetic acid, but the phosphoenzyme phosphorylated in cell extracts is acid labile. In cell extracts, the addition of phospho-oligosaccharide markedly inhibits ATP hydrolysis, which may explain the effect of this molecule on ATP citrate lyase phosphorylation in broken cells. These results support the hypothesis that this phospho-oligosaccharide mediates some of the effects of insulin on protein phosphorylation. They also indicate that caution should be exercised in interpreting the results obtained by adding phospho-oligosaccharide to broken cell preparations. PMID:2119547

  17. Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes.

    PubMed

    Mravec, Jozef; Kračun, Stjepan K; Rydahl, Maja G; Westereng, Bjørge; Miart, Fabien; Clausen, Mads H; Fangel, Jonatan U; Daugaard, Mathilde; Van Cutsem, Pierre; De Fine Licht, Henrik H; Höfte, Herman; Malinovsky, Frederikke G; Domozych, David S; Willats, William G T

    2014-12-01

    Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems. We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons. PMID:25395456

  18. Extending Synthetic Routes for Oligosaccharides by Enzyme, Substrate and Reaction Engineering

    NASA Astrophysics Data System (ADS)

    Seibel, Jürgen; Jördening, Hans-Joachim; Buchholz, Klaus

    The integration of all relevant tools for bioreaction engineering has been a recent challenge. This approach should notably favor the production of oligo- and polysaccharides, which is highly complex due to the requirements of regio- and stereoselectivity. Oligosaccharides (OS) and polysaccharides (PS) have found many interests in the fields of food, pharmaceuticals, and cosmetics due to different specific properties. Food, sweeteners, and food ingredients represent important sectors where OS are used in major amounts. Increasing attention has been devoted to the sophisticated roles of OS and glycosylated compounds, at cell or membrane surfaces, and their function, e.g., in infection and cancer proliferation. The challenge for synthesis is obvious, and convenient approaches using cheap and readily available substrates and enzymes will be discussed. We report on new routes for the synthesis of oligosaccharides (OS), with emphasis on enzymatic reactions, since they offer unique properties, proceeding highly regio- and stereoselective in water solution, and providing for high yields in general.

  19. Combinational effects of prebiotic oligosaccharides on bifidobacterial growth and host gene expression in a simplified mixed culture model and neonatal mice.

    PubMed

    Ehara, Tatsuya; Izumi, Hirohisa; Tsuda, Muneya; Nakazato, Yuki; Iwamoto, Hiroshi; Namba, Kazuyoshi; Takeda, Yasuhiro

    2016-07-01

    It is important to provide formula-fed infants with a bifidobacteria-enriched gut microbiota similar to those of breastfed infants to ensure intestinal health. Prebiotics, such as certain oligosaccharides, are a useful solution to this problem, but the combinational benefits of these oligosaccharides have not been evaluated. This study investigated the benefits of oligosaccharide combinations and screened for an optimal combination of oligosaccharides to promote healthy gut microbiota of formula-fed infants. In vitro and in vivo experiments were performed to assess the bifidogenic effects of lactulose (LAC) alone and LAC combined with raffinose (RAF) and/or galacto-oligosaccharide (GOS), using a mixed culture model and neonatal mice orally administered with these oligosaccharides and Bifidobacterium breve. In the in vitro culture model, the combination of the three oligosaccharides (LAC-RAF-GOS) significantly increased cell numbers of B. breve and Bifidobacterium longum (P<0·05) compared with either LAC alone or the combination of two oligosaccharides, and resulted in the production of SCFA under anaerobic conditions. In the in vivo experiment, the LAC-RAF-GOS combination significantly increased cell numbers of B. breve and Bacteroidetes in the large intestinal content (P<0·05) and increased acetate concentrations in the caecal content and serum of neonatal mice. Genes related to metabolism and immune responses were differentially expressed in the liver and large intestine of mice administered with LAC-RAF-GOS. These results indicate a synergistic effect of the LAC-RAF-GOS combination on the growth of bifidobacteria and reveal possible benefits of this combination to the gut microbiota and health of infants. PMID:27198516

  20. Structural Identification of O-Linked Oligosaccharides Using Exoglycosidases and MSn Together with UniCarb-DB Fragment Spectra Comparison

    PubMed Central

    Ali, Liaqat; Kenny, Diarmuid T.; Hayes, Catherine A.; Karlsson, Niclas G.

    2012-01-01

    The availability of specific exoglycosidases alongside a spectral library of O-linked oligosaccharide collision induced dissociation (CID) MS fragments, UniCarb-DB, provides a pathway to make the elucidation of O-linked oligosaccharides more efficient. Here, we advise an approach of exoglycosidase-digestion of O-linked oligosaccharide mixtures, for structures that do not provide confirmative spectra. The combination of specific exoglycosidase digestion and MS2 matching of the exoglycosidase products with structures from UniCarb-DB, allowed the assignment of unknown structures. This approach was illustrated by treating sialylated core 2 O-linked oligosaccharides, released from the human synovial glycoprotein (lubricin), with a α2–3 specific sialidase. This methodology demonstrated the exclusive 3 linked nature of the sialylation of core 2 oligosaccharides on lubricin. When specific exoglycosidases were not available, MS3 spectral matching using standards was used. This allowed the unusual 4-linked terminal GlcNAc epitope in a porcine stomach to be identified in the GlcNAc1-4Galβ1–3(GlcNAcβ1-6)GalNAcol structure, indicating the antibacterial epitope GlcNAcα1–4. In total, 13 structures were identified using exoglycosidase and MSn, alongside UniCarb-DB fragment spectra comparison. UniCarb-DB could also be used to identify the specificity of unknown exoglycosidases in human saliva. Endogenous salivary exoglycosidase activity on mucin oligosaccharides could be monitored by comparing the generated tandem MS spectra with those present in UniCarb-DB, showing that oral exoglycosidases were dominated by sialidases with a higher activity towards 3-linked sialic acid rather than 6-linked sialic acid. PMID:24957756

  1. Enzymatic Hydrolysis of Alginate to Produce Oligosaccharides by a New Purified Endo-Type Alginate Lyase

    PubMed Central

    Zhu, Benwei; Chen, Meijuan; Yin, Heng; Du, Yuguang; Ning, Limin

    2016-01-01

    Enzymatic hydrolysis of sodium alginate to produce alginate oligosaccharides has drawn increasing attention due to its advantages of containing a wild reaction condition, excellent gel properties and specific products easy for purification. However, the efficient commercial enzyme tools are rarely available. A new alginate lyase with high activity (24,038 U/mg) has been purified from a newly isolated marine strain, Cellulophaga sp. NJ-1. The enzyme was most active at 50 °C and pH 8.0 and maintained stability at a broad pH range (6.0–10.0) and temperature below 40 °C. It had broad substrate specificity toward sodium alginate, heteropolymeric MG blocks (polyMG), homopolymeric M blocks (polyM) and homopolymeric G blocks (polyG), and possessed higher affinity toward polyG (15.63 mM) as well as polyMG (23.90 mM) than polyM (53.61 mM) and sodium alginate (27.21 mM). The TLC and MS spectroscopy analysis of degradation products suggested that it completely hydrolyzed sodium alginate into oligosaccharides of low degrees of polymerization (DPs). The excellent properties would make it a promising tool for full use of sodium alginate to produce oligosaccharides. PMID:27275826

  2. Enzymatic Hydrolysis of Alginate to Produce Oligosaccharides by a New Purified Endo-Type Alginate Lyase.

    PubMed

    Zhu, Benwei; Chen, Meijuan; Yin, Heng; Du, Yuguang; Ning, Limin

    2016-01-01

    Enzymatic hydrolysis of sodium alginate to produce alginate oligosaccharides has drawn increasing attention due to its advantages of containing a wild reaction condition, excellent gel properties and specific products easy for purification. However, the efficient commercial enzyme tools are rarely available. A new alginate lyase with high activity (24,038 U/mg) has been purified from a newly isolated marine strain, Cellulophaga sp. NJ-1. The enzyme was most active at 50 °C and pH 8.0 and maintained stability at a broad pH range (6.0-10.0) and temperature below 40 °C. It had broad substrate specificity toward sodium alginate, heteropolymeric MG blocks (polyMG), homopolymeric M blocks (polyM) and homopolymeric G blocks (polyG), and possessed higher affinity toward polyG (15.63 mM) as well as polyMG (23.90 mM) than polyM (53.61 mM) and sodium alginate (27.21 mM). The TLC and MS spectroscopy analysis of degradation products suggested that it completely hydrolyzed sodium alginate into oligosaccharides of low degrees of polymerization (DPs). The excellent properties would make it a promising tool for full use of sodium alginate to produce oligosaccharides. PMID:27275826

  3. "Three sources and three component parts" of free oligosaccharides.

    PubMed

    Pismenetskaya, I U; Butters, T D

    2014-01-01

    Metabolism of glycoproteins and glycolipids is accompanied by the appearance of unbound structural analogues of the carbohydrate portion of glycoconjugates or so called free oligosaccharides. There are their several sources inside the cell: 1) multistep pathways of N-glycosylation, 2) the cell quality control and ER-associated degradation of misglycosylated and/or misfolded glycoproteins, 3) lysosomal degradation of mature glycoconjugates. In this review the information about the ways of free oligosaccharides appearance in different cell compartments and details of their structures depending on the source is summarized. In addition, extracellular free oligosaccharides, their structures and changes under normal and pathological conditions are discussed. PMID:25816601

  4. Recent developments in manufacturing oligosaccharides with prebiotic functions.

    PubMed

    Kovács, Zoltán; Benjamins, Eric; Grau, Konrad; Ur Rehman, Amad; Ebrahimi, Mehrdad; Czermak, Peter

    2014-01-01

    The market for prebiotics is steadily growing. To satisfy this increasing worldwide demand, the introduction of effective bioprocessing methods and implementation strategies is required. In this chapter, we review recent developments in the manufacture of galactooligosaccharides (GOS) and fructooligosaccharides (FOS). These well-established oligosaccharides (OS) provide several health benefits and have excellent technological properties that make their use as food ingredients especially attractive. The biosyntheses of lactose-based GOS and sucrose-based FOS show similarities in terms of reaction mechanisms and product formation. Both GOS and FOS can be synthesized using whole cells or (partially) purified enzymes in immobilized or free forms. The biocatalysis results in a final product that consists of OS, unreacted disaccharides, and monosaccharides. This incomplete conversion poses a challenge to manufacturers because an enrichment of OS in this mixture adds value to the product. For removing digestible carbohydrates from OS, a variety of bioengineering techniques have been investigated, including downstream separation technologies, additional bioconversion steps applying enzymes, and selective fermentation strategies. This chapter summarizes the state-of-the-art manufacturing strategies and recent advances in bioprocessing technologies that can lead to new possibilities for manufacturing and purifying sucrose-based FOS and lactose-based GOS. PMID:23942834

  5. High mannose oligosaccharide of phytohemagglutinin is attached to asparagine 12 and the modified oligosaccharide to asparagine 60. [Phaseolus vulgaris

    SciTech Connect

    Sturm, A.; Chrispeels, M.J.

    1986-05-01

    Phytohemagglutinin, the lectin of the common bean Phaseolus vulgaris, has a high mannose and a modified (fucosylated) oligosaccharide on each polypeptide. Fractionation by high performance liquid chromatography of tryptic digests of (/sup 3/H)fucose or (/sup 3/H)glucosamine labeled phytohemagglutinin, followed by amino acid sequencing of the isolated glycopeptides, shows that the high mannose oligosaccharide is attached to Asn/sup 12/ and the modified oligosaccharide to Asn /sup 60/ of the protein. In animal glycoproteins, high mannose chains are rarely found at the N-terminal side of complex chains.

  6. Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway

    PubMed Central

    Hao, Cui; Gao, Lixia; Zhang, Yiran; Wang, Wei; Yu, Guangli; Guan, Huashi; Zhang, Lijuan; Li, Chunxia

    2015-01-01

    Chitosan oligosaccharides (COSs), depolymerized products of chitosan composed of β-(1→4) d-glucosamine units, have broad range of biological activities such as antitumour, antifungal, and antioxidant activities. In this study, peracetylated chitosan oligosaccharides (PACOs) and N-acetylated chitosan oligosaccharides (NACOs) were prepared from the COSs by chemcal modification. The structures of these monomers were identified using NMR and ESI-MS spectra. Their antagonist effects against glutamate-induced PC12 cell death were investigated. The results showed that pretreatment of PC12 cells with the PACOs markedly inhibited glutamate-induced cell death in a concentration-dependent manner. The PACOs were better glutamate antagonists compared to the COSs and the NACOs, suggesting the peracetylation is essential for the neuroprotective effects of chitosan oligosaccharides. In addition, the PACOs pretreatment significantly reduced lactate dehydrogenase release and reactive oxygen species production. It also attenuated the loss of mitochondrial membrane potential. Further studies indicated that the PACOs inhibited glutamate-induced cell death by preventing apoptosis through depressing the elevation of Bax/Bcl-2 ratio and caspase-3 activation. These results suggest that PACOs might be promising antagonists against glutamate-induced neural cell death. PMID:25775423

  7. Multiple applications of ion chromatography oligosaccharide fingerprint profiles to solve a variety of sugar and sugar-biofuel industry problems.

    PubMed

    Eggleston, Gillian; Borges, Eduardo

    2015-03-25

    Sugar crops contain a broad variety of carbohydrates used for human consumption and the production of biofuels and bioproducts. Ion chromatography with integrated pulsed amperometric detection (IC-IPAD) can be used to simultaneously detect mono-, di-, and oligosaccharides, oligosaccharide isomers, mannitol, and ethanol in complex matrices from sugar crops. By utilizing a strong NaOH/NaOAc gradient method over 45 min, oligosaccharides of at least 2-12 dp can be detected. Fingerprint IC oligosaccharide profiles are extremely selective, sensitive, and reliable and can detect deterioration product metabolites from as low as 100 colony-forming units/mL lactic acid bacteria. The IC fingerprints can also be used to (i) monitor freeze deterioration, (ii) optimize harvesting methods and cut-to-crush times, (iii) differentiate between white refined sugar from sugar cane and from sugar beets, (iv) verify the activities of carbohydrate enzymes, (v) select yeasts for ethanol fermentations, and (vi) isolate and diagnose infections and processing problems in sugar factories. PMID:25708094

  8. Synthesis of branched cyclomalto-oligosaccharides using Pseudomonas isoamylase.

    PubMed

    Abe, J; Mizowaki, N; Hizukuri, S; Koizumi, K; Utamura, T

    1986-10-15

    Branched cyclomalto-oligosaccharides (cyclodextrins) were synthesised from cyclomalto-oligosaccharides and maltose or maltotriose through the reverse action of Pseudomonas isoamylase. The reaction rate was greater with maltotriose than with maltose, and with increasing size of the cyclomalto-oligosaccharide (cG6 less than cG7 less than cG8). Maltotriose is effective as both a side-chain donor and acceptor, and three isomers of 6-O-alpha-maltotriosylmaltotriose (branched G6) were formed through mutual condensation, but maltose was effective only as a side-chain donor. Each branched cyclomalto-oligosaccharide and G6 was purified by liquid chromatography, and their structures were determined by chemical, enzymic, and 13C-n.m.r. spectroscopic analyses. PMID:3791296

  9. Stable isotope labeling of oligosaccharide cell surface antigens

    SciTech Connect

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A.

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  10. Preparation and antibacterial activity of oligosaccharides derived from dandelion.

    PubMed

    Qian, Li; Zhou, Yan; Teng, Zhaolin; Du, Chun-Ling; Tian, Changrong

    2014-03-01

    In this study, we prepared oligosaccharides from dandelion (Taraxacum officinale) by hydrolysis with hydrogen peroxide (H2O2) and investigated their antibacterial activity. The optimum hydrolysis conditions, as determined using the response surface methodology, were as follows: reaction time, 5.12h; reaction temperature, 65.53 °C and H2O2 concentration, 3.16%. Under these conditions, the maximum yield of the oligosaccharides reached 25.43%. The sugar content in the sample was 96.8%, and the average degree of polymerisation was approximately 9. The oligosaccharides showed high antibacterial activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus, indicating that dandelion-derived oligosaccharides have the potential to be used as antibacterial agents. PMID:24368113

  11. Synbiotic matrices derived from plant oligosaccharides and polysaccharides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A porous synbiotic matrix was prepared by lyophilization of alginate and pectin or fructan oligosaccharides and polysaccharides cross-linked with calcium. These synbiotic matrices were excellent physical structures to support the growth of Lactobacillus acidophilus (1426) and Lactobacillus reuteri (...

  12. Enhancing the chemoenzymatic synthesis of arabinosylated xylo-oligosaccharides by GH51 α-L-arabinofuranosidase.

    PubMed

    Arab-Jaziri, Faten; Bissaro, Bastien; Tellier, Charles; Dion, Michel; Fauré, Régis; O'Donohue, Michael J

    2015-01-12

    Random mutagenesis was performed on the α-l-arabinofuranosidase of Thermobacillus xylanilyticus in order to enhance its ability to perform transarabinofuranosylation using natural xylo-oligosaccharides as acceptors. To achieve this goal, a two-step, high-throughput digital imaging protocol involving a colorimetric substrate was used to screen a library of 30,000 mutants. In the first step this screen selected for hydrolytically-impaired mutants, and in the second step the screen identified mutants whose global activity was improved in the presence of a xylo-oligosaccharide mixture. Thereby, 199 mutants displaying lowered hydrolytic activity and modified properties were detected. In the presence of these xylo-oligosaccharides, most of the 199 (i.e., 70%) enzymes were less inhibited and some (18) mutants displayed an unambiguous alleviation of inhibition (<25% loss of activity). More precise monitoring of reactions catalyzed by the most promising mutants revealed a significant improvement of the synthesis yields of transglycosylation products (up to 18% compared to 9% for the parental enzyme) when xylobiose was present in the reaction. Genetic analysis of improved mutants revealed that many of the amino acid substitutions that correlate with the modified phenotype are located in the vicinity of the active site, particularly in subsite -1. Consequently, we hypothesize that these mutations modify the active site topology or the molecular interaction network of the l-arabinofuranoside donor substrate, thus impairing the hydrolysis and concomitantly favoring transglycosylation onto natural acceptors. PMID:25464083

  13. Milk Proteins, Peptides, and Oligosaccharides: Effects against the 21st Century Disorders

    PubMed Central

    Hsieh, Chia-Chien; Hernández-Ledesma, Blanca; Fernández-Tomé, Samuel; Weinborn, Valerie; Barile, Daniela; de Moura Bell, Juliana María Leite Nobrega

    2015-01-01

    Milk is the most complete food for mammals, as it supplies all the energy and nutrients needed for the proper growth and development of the neonate. Milk is a source of many bioactive components, which not only help meeting the nutritional requirements of the consumers, but also play a relevant role in preventing various disorders. Milk-derived proteins and peptides have the potential to act as coadjuvants in conventional therapies, addressing cardiovascular diseases, metabolic disorders, intestinal health, and chemopreventive properties. In addition to being a source of proteins and peptides, milk contains complex oligosaccharides that possess important functions related to the newborn's development and health. Some of the health benefits attributed to milk oligosaccharides include prebiotic probifidogenic effects, antiadherence of pathogenic bacteria, and immunomodulation. This review focuses on recent findings demonstrating the biological activities of milk peptides, proteins, and oligosaccharides towards the prevention of diseases of the 21st century. Processing challenges hindering large-scale production and commercialization of those bioactive compounds have been also addressed. PMID:25789308

  14. Major carbohydrate, polyol, and oligosaccharide profiles of agave syrup. Application of this data to authenticity analysis.

    PubMed

    Willems, Jamie L; Low, Nicholas H

    2012-09-01

    Nineteen pure agave syrups representing the three major production regions and four processing facilities in Mexico were analyzed for their major carbohydrate, polyol, and oligosaccharide profiles, as well as their physicochemical properties (pH, °Brix, total acidity, percent total titratable acidity, and color). Additionally, the detection of intentional debasing of agave syrup with four commercial nutritive sweeteners (HFCS 55 and 90, DE 42 and sucrose) was afforded by oligosaccharide profiling employing both high performance anion exchange liquid chromatography with pulsed amperometric detection (HPAE-PAD) and capillary gas chromatography with flame ionization detection (CGC-FID). Results showed that the major carbohydrate and polyol in agave syrups were fructose and inositol with mean concentrations of 84.29% and 0.38%, respectively. Oligosaccharide profiling was extremely successful for adulteration detection with detection limits ranging from 0.5 to 2.0% for the aforementioned debasing agents. Also, all four of these possible adulterants could be detected within a single chromatographic analysis. PMID:22909406

  15. Deproteinated palm kernel cake-derived oligosaccharides: A preliminary study

    NASA Astrophysics Data System (ADS)

    Fan, Suet Pin; Chia, Chin Hua; Fang, Zhen; Zakaria, Sarani; Chee, Kah Leong

    2014-09-01

    Preliminary study on microwave-assisted hydrolysis of deproteinated palm kernel cake (DPKC) to produce oligosaccharides using succinic acid was performed. Three important factors, i.e., temperature, acid concentration and reaction time, were selected to carry out the hydrolysis processes. Results showed that the highest yield of DPKC-derived oligosaccharides can be obtained at a parameter 170 °C, 0.2 N SA and 20 min of reaction time.

  16. Distribution of Heparan Sulfate Oligosaccharides in Murine Mucopolysaccharidosis Type IIIA

    PubMed Central

    Mason, Kerryn; Meikle, Peter; Hopwood, John; Fuller, Maria

    2014-01-01

    Heparan sulfate (HS) catabolism begins with endo-degradation of the polysaccharide to smaller HS oligosaccharides, followed by the sequential action of exo-enzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate. In mucopolysaccharidosis type IIIA (MPS IIIA) the exo-enzyme, N-sulfoglucosamine sulfohydrolase, is deficient resulting in an inability to hydrolyze non-reducing end glucosamine N-sulfate esters. Consequently, partially degraded HS oligosaccharides with non-reducing end glucosamine sulfate esters accumulate. We investigated the distribution of these HS oligosaccharides in tissues of a mouse model of MPS IIIA using high performance liquid chromatography electrospray ionization-tandem mass spectrometry. Oligosaccharide levels were compared to total uronic acid (UA), which was used as a measure of total glycosaminoglycan. Ten oligosaccharides, ranging in size from di- to hexasaccharides, were present in all the tissues examined including brain, spleen, lung, heart, liver, kidney and urine. However, the relative levels varied up to 10-fold, suggesting different levels of HS turnover and storage. The relationship between the di- and tetrasaccharides and total UA was tissue specific with spleen and kidney showing a different disaccharide:total UA ratio than the other tissues. The hexasaccharides showed a stronger correlation with total UA in all tissue types suggesting that hexasaccharides may more accurately reflect the storage burden in these tissues. PMID:25513953

  17. Typing of Blood-Group Antigens on Neutral Oligosaccharides by Negative-Ion Electrospray Ionization Tandem Mass Spectrometry

    PubMed Central

    Zhang, Hongtao; Zhang, Shuang; Tao, Guanjun; Zhang, Yibing; Mulloy, Barbara; Zhan, Xiaobei; Chai, Wengang

    2013-01-01

    Blood-group antigens, such as those containing fucose and bearing the ABO(H)- and Lewis-type determinants expressed on the carbohydrate chains of glycoproteins and glycolipids, and also on unconjugated free oligosaccharides in human milk and other secretions, are associated with various biological functions. We have previously shown the utility of negative-ion electrospay ionization tandem mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) for typing of Lewis (Le) determinants, e.g. Lea, Lex, Leb, and Ley on neutral and sialylated oligosaccharide chains. In the present report we extended the strategy to characterization of blood-group A-, B- and H-determinants on type 1 and type 2, and also on type 4 globoside chains to provide a high sensitivity method for typing of all the major blood-group antigens, including the A, B, H, Lea, Lex, Leb, and Ley determinants, present in oligosaccharides. Using the principles established we identified two minor unknown oligosaccharide components present in the products of enzymatic synthesis by bacterial fermentation. We also demonstrated that the unique fragmentations derived from the D- and 0,2A-type cleavages observed in ESI-CID-MS/MS, which are important for assigning blood-group and chain types, only occur under the negative-ion conditions for reducing sugars but not for reduced alditols or under positive-ion conditions. PMID:23692402

  18. Isolation and Structural Characterization of an Oligosaccharide Produced by Bacillus subtilis in a Maltose-Containing Medium

    PubMed Central

    Shin, Kwang-Soon

    2016-01-01

    Among 116 bacterial strains isolated from Korean fermented foods, one strain (SS-76) was selected for producing new oligosaccharides in a basal medium containing maltose as the sole source of carbon. Upon morphological characterization using scanning electron microscopy, the cells of strain SS-76 appeared rod-shaped; subsequent 16S rRNA gene sequence analysis revealed that strain SS-76 was phylogenetically close to Bacillus subtilis. The main oligosaccharide fraction B extracted from the culture supernatant of B. subtilis SS-76 was purified by high performance liquid chromatography. Subsequent structural analysis revealed that this oligosaccharide consisted only of glucose, and methylation analysis indicated similar proportions of glucopyranosides in the 6-linkage, 4-linkage, and non-reducing terminal positions. Matrix-assisted laser-induced/ionization time-of-flight/mass spectrometry and electrospray ionization-based liquid chromatography-mass spectrometry/mass spectrometry analyses suggested that this oligosaccharide consisted of a trisaccharide unit with 1,6- and 1,4-glycosidic linkages. The anomeric signals in the 1H-nuclear magnetic resonance spectrum corresponded to α-anomeric configurations, and the trisaccharide was finally identified as panose (α-D-glucopyranosyl-1,6-α-D-glucopyranosyl-1,4-D-glucose). These results suggest that B. subtilis SS-76 converts maltose into panose; strain SS-76 may thus find industrial application in the production of panose. PMID:27390729

  19. Isolation and Structural Characterization of an Oligosaccharide Produced by Bacillus subtilis in a Maltose-Containing Medium.

    PubMed

    Shin, Kwang-Soon

    2016-06-01

    Among 116 bacterial strains isolated from Korean fermented foods, one strain (SS-76) was selected for producing new oligosaccharides in a basal medium containing maltose as the sole source of carbon. Upon morphological characterization using scanning electron microscopy, the cells of strain SS-76 appeared rod-shaped; subsequent 16S rRNA gene sequence analysis revealed that strain SS-76 was phylogenetically close to Bacillus subtilis. The main oligosaccharide fraction B extracted from the culture supernatant of B. subtilis SS-76 was purified by high performance liquid chromatography. Subsequent structural analysis revealed that this oligosaccharide consisted only of glucose, and methylation analysis indicated similar proportions of glucopyranosides in the 6-linkage, 4-linkage, and non-reducing terminal positions. Matrix-assisted laser-induced/ionization time-of-flight/mass spectrometry and electrospray ionization-based liquid chromatography-mass spectrometry/mass spectrometry analyses suggested that this oligosaccharide consisted of a trisaccharide unit with 1,6- and 1,4-glycosidic linkages. The anomeric signals in the (1)H-nuclear magnetic resonance spectrum corresponded to α-anomeric configurations, and the trisaccharide was finally identified as panose (α-D-glucopyranosyl-1,6-α-D-glucopyranosyl-1,4-D-glucose). These results suggest that B. subtilis SS-76 converts maltose into panose; strain SS-76 may thus find industrial application in the production of panose. PMID:27390729

  20. Mannose-6-phosphate regulates destruction of lipid-linked oligosaccharides

    PubMed Central

    Gao, Ningguo; Shang, Jie; Huynh, Dang; Manthati, Vijaya L.; Arias, Carolina; Harding, Heather P.; Kaufman, Randal J.; Mohr, Ian; Ron, David; Falck, John R.; Lehrman, Mark A.

    2011-01-01

    Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose3mannose9GlcNAc2-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-α. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses. PMID:21737679

  1. PLANT OLIGOSACCHARIDES ENHANCE WHEAT DEFENCE RESPONSE AGAINST SEPTORIA LEAF BLOTCH.

    PubMed

    Somai-Jemmali, L; Siah, A; Randoux, B; Reignault, Ph; Halama, P; Rodriguez, R; Hamada, W

    2015-01-01

    Our work provides the first evidence for elicitation and protection effects of preventive treatments with oligosaccharides (20%)-based new formulation (Oligos) against Mycosphaerella graminicola, a major pathogen of bread wheat (BW) and durum wheat (DW). In planta Oligos treatment led to strongly reduced hyphal growth, penetration, mesophyll colonization and fructification. During the necrotrophic phase, Oligos also drastically decreased the production of M. graminicola CWDE activities, such as xylanase and glucanase as well as protease activity in both wheat species, suggesting their correlation with disease severity. Concerning plant defence markers, PR2, Chi 4 precursor-, Per- and LOX-1-encoding genes were up-regulated, while glucanase (GLUC), catalase (CAT) and lipoxygenase (LOX) activities and total phenolic compound (PC) accumulation were induced in both (non-inoculated and inoculated contexts. In inoculated context, a localized accumulation of H2O2 and PC at fungal penetration sites and a specific induction of phenylalanine ammonia-Lyase (PAL) enzymatic activity were observed. Moreover, our experiment exhibited some similarities and differences in both wheat species responses. GLUC and CAT activities and H2O2 accumulation were more responsive in DW leaves, while LOX and PAL activities and PC accumulation occurred earlier and to a stronger extent in BW leaves. The tested Oligos formulation showed an interesting resistance induction activity characterized by a high and stable efficiency whatever the wheat species, suggesting it integration in common control strategies against STB on both DW and BW. PMID:27141743

  2. Campylobacter jejuni free oligosaccharides: function and fate.

    PubMed

    Nothaft, Harald; Liu, Xin; Li, Jianjun; Szymanski, Christine M

    2010-01-01

    The Campylobacter jejuni N-linked protein glycosylation pathway produces a heptasaccharide that is added to >65 periplasmic and membrane proteins and is also released into the periplasm as the free oligosaccharide (fOS). The fOS is a novel soluble component of the C. jejuni periplasmic space that exists in 10-fold greater quantities than its asparagine-linked counterpart. Structurally, fOS is the same heptasaccharide that is found attached to asparagine residues on C. jejuni glycoproteins and both glycans are cleaved from the undecaprenylpyrophosphate anchor by the previously identified oligosaccharyltransferase PglB, which we have now shown to be a bifunctional enzyme also displaying hydrolase activity. The fOS levels in C. jejuni, similar to bacterial periplasmic glucans, can be manipulated by altering the salt and osmolyte concentrations in the growth environment. Here, we outline potential functions of fOS and raise new questions about the underlying mechanism involved in PglB-mediated fOS release from its lipid anchor and fOS retention within the C. jejuni periplasm. PMID:21178500

  3. Historical Aspects of Human Milk Oligosaccharides1234

    PubMed Central

    Kunz, Clemens

    2012-01-01

    This review focuses on important observations regarding infant health around 1900 when breastfeeding was not considered a matter of importance. The discovery of lactobacilli and bifidobacteria and their relevance for health and disease was an important milestone leading to a decrease in infant mortality in the first year of life. At the same time, pediatricians realized that the fecal composition of breast-fed and bottle-fed infants differed. Observations indicated that this difference is linked to milk composition, particularly due to the milk carbohydrate fraction. Circa 1930, a human milk carbohydrate fraction called gynolactose was identified. This was the starting point of research on human milk oligosaccharides (HMO). In the following years, the first HMO were identified and their functions investigated. Studies after 1950 focused on the identification of various HMO as the bifidus factor in human milk. In the following 30 years, a tremendous amount of research was done with regard to the characterization of individual HMO and HMO patterns in milk. In this short introduction to the history of HMO research, which ends circa 1980, some outstanding scientists in pediatrics and chemistry and their pioneering contributions to research in the field of HMO are presented. PMID:22585922

  4. The Ability of Bifidobacteria To Degrade Arabinoxylan Oligosaccharide Constituents and Derived Oligosaccharides Is Strain Dependent

    PubMed Central

    Rivière, Audrey; Moens, Frédéric; Selak, Marija; Maes, Dominique; Weckx, Stefan

    2014-01-01

    Arabinoxylan oligosaccharides (AXOS) are prebiotic carbohydrates with promising health-promoting properties that stimulate the activity of specific colon bacteria, in particular bifidobacteria. However, the mechanisms by which bifidobacterial strains break down these compounds in the colon is still unknown. This study investigates AXOS consumption of a large number of bifidobacterial strains (36), belonging to 11 different species, systematically. To determine their degradation mechanisms, all strains were grown on a mixture of arabinose and xylose, xylo-oligosaccharides, and complex AXOS molecules as the sole added energy sources. Based on principal component and cluster analyses of their different arabinose substituent and/or xylose backbone consumption patterns, five clusters that were species independent could be distinguished among the bifidobacterial strains tested. In parallel, the strains were screened for the presence of genes encoding several putative AXOS-degrading enzymes, but no clear-cut correlation could be made with the different degradation mechanisms. The intra- and interspecies differences in the consumption patterns of AXOS indicate that bifidobacterial strains could avoid competition among each other or even could cooperate jointly to degrade these complex prebiotics. The knowledge gained on the AXOS degradation mechanisms in bifidobacteria can be of importance in the rational design of prebiotics with tailor-made composition and thus increased specificity in the colon. PMID:24141124

  5. In-depth characterization of N-linked oligosaccharides using fluoride-mediated negative ion microfluidic chip LC-MS.

    PubMed

    Ni, Wenqin; Bones, Jonathan; Karger, Barry L

    2013-03-19

    Characterization of N-glycans by liquid chromatography-positive electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS) using a microfluidic chip packed with porous graphitized carbon (PGC) represents a rapidly developing area in oligosaccharide analysis. Positive ion ESI-MS generates B/Y-type glycosidic fragment ions under collisional-induced dissociation (CID). Although these ions facilitate glycan sequencing, they provide little information on linkage and positional isomers. Isomer identification in these cases is by retention on the PGC stationary phase where the specific structural isomers can, in principle, be separated. In this paper, we broaden the applicability of the PGC microfluidic chip/MS platform by implementing fluoride-mediated negative ESI-MS. Ammonium fluoride, added to the mobile phase, aids in the formation of pseudomolecular oligosaccharide anions due to the ability of fluoride to abstract a proton from the glycan structure. The negative charge results in the generation of C-type glycosidic fragments, highly informative A-type cross-ring fragment ions, and additional gas-phase ion reaction products (e.g., D- and E-type ions), which, when combined, lead to in-depth oligosaccharide characterization, including linkage and positional isomers. Due to the separation of anomers by the PGC phase, comparison of oligosaccharides with an intact reducing terminus to their experimentally prepared corresponding alditols was performed, revealing a more sensitive MS and, especially, MS/MS analysis from the glycans with a free reducing end. Fluoride also ensured recovery of charged oligosaccharides from the PGC stationary phase. Application to the characterization of N-glycans released from polyclonal human and murine serum IgG is presented to demonstrate the effectiveness of the chip/negative ESI approach. PMID:23398125

  6. Enzymatic degradation of oligosaccharides in pinto bean flour.

    PubMed

    Song, Danfeng; Chang, Sam K C

    2006-02-22

    The use of dry edible beans is limited due to the presence of flatulence factors, the raffinose oligosaccharides. Our objective was to investigate the process for the removal of oligosaccharides from pinto bean using enzymatic treatment and to compare it to removal by soaking and cooking methods. Crude enzyme preparation was produced by six fungal species on wheat bran- and okara-based substrates with soy tofu whey. The loss of raffinose oligosaccharides after soaking pinto beans for 16 h at the room temperature was 10%, after cooking for 90 min was 52%, and after autoclaving for 30 min was 58%. On the other hand, the treatment using crude alpha-galactosidase (60 U mL(-1)) produced by Aspergillus awamori NRRL 4869 from wheat bran-based substrate with soy tofu whey on pinto bean flour for 2 h completely hydrolyzed raffinose oligosaccharides. These results supported that the enzymatic treatment was the most effective among various processing methods tested for removing the raffinose oligosaccharides, and hence, crude alpha-galactosidases from fungi have potential use in the food industry. PMID:16478251

  7. Synthesis of oligosaccharides using per-O-trimethylsilyl-glycosyl iodides as glycosyl donor.

    PubMed

    Wang, Hong; Cui, Yanli; Zou, Rong; Cheng, Zhaodong; Yao, Weirong; Mao, Yangyi; Zhang, Yongmin

    2016-06-01

    Trimethylsilyl (TMS) protecting group has been found to be very useful for the simultaneous protection of both the glycosyl donor- and the acceptor-substrates in oligosaccharide synthesis. Thus, while the per-O-trimethylsilylated glycosyl iodides served as the glycosyl donor, those bearing selectively exposed primary hydroxyl groups were found suitable as the glycosyl acceptor for the reaction. The cheap and commercially available trialkylamine, triethylamine was found to be an effective promoter for the glycosylation. Importantly, the reaction was α-stereospecific and gave the products in 58%-78% yields. PMID:27077820

  8. A C4-oxidizing Lytic Polysaccharide Monooxygenase Cleaving Both Cellulose and Cello-oligosaccharides*

    PubMed Central

    Isaksen, Trine; Westereng, Bjørge; Aachmann, Finn L.; Agger, Jane W.; Kracher, Daniel; Kittl, Roman; Ludwig, Roland; Haltrich, Dietmar; Eijsink, Vincent G. H.; Horn, Svein J.

    2014-01-01

    Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61–3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end. PMID:24324265

  9. A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides.

    PubMed

    Isaksen, Trine; Westereng, Bjørge; Aachmann, Finn L; Agger, Jane W; Kracher, Daniel; Kittl, Roman; Ludwig, Roland; Haltrich, Dietmar; Eijsink, Vincent G H; Horn, Svein J

    2014-01-31

    Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61-3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end. PMID:24324265

  10. Natural variability in bovine milk oligosaccharides from Danish Jersey and Holstein-Friesian breeds

    PubMed Central

    Sundekilde, Ulrik K; Barile, Daniela; Meyrand, Mickael; Poulsen, Nina A; Larsen, Lotte B; Lebrilla, Carlito B.; Bruce, German J.; Bertram, Hanne C

    2012-01-01

    Free oligosaccharides are key components of human milk and play multiple roles in the health of the neonate, by stimulating growth of selected beneficial bacteria in the gut, participating in development of the brain and exerting anti-pathogenic activity. However, the concentration of oligosaccharides is low in mature bovine milk, normally used for infant formula, compared with both human colostrum and mature human milk. Characterization of bovine milk oligosaccharides in different breeds is crucial for the identification of viable sources for oligosaccharide purification. An improved source of oligosaccharides can lead to infant formula with improved oligosaccharide functionality. In the present study we have analyzed milk oligosaccharides by high-performance liquid chromatography chip quadrupole time-of-flight mass spectrometry and performed a detailed data analysis using both univariate and multivariate methods. Both statistical tools revealed several differences in oligosaccharide profiles between milk samples from the two Danish breeds; Jersey and Holstein-Friesians. Jersey milk contained higher relative amounts of both sialylated and the more complex neutral fucosylated oligosaccharides, while the Holstein-Friesian milk had higher abundance of smaller and simpler neutral oligosaccharides. The statistical analyses revealed that Jersey milk contain significantly higher levels of fucosylated oligosaccharides than Holstein-Friesian milk. Jersey milk also possesses oligosaccharides with a higher degree of complexity and functional residues (fucose and sialic acid) suggesting it may therefore offer advantages in term of a wider array of bioactivities. PMID:22632419

  11. Natural variability in bovine milk oligosaccharides from Danish Jersey and Holstein-Friesian breeds.

    PubMed

    Sundekilde, Ulrik K; Barile, Daniela; Meyrand, Mickael; Poulsen, Nina A; Larsen, Lotte B; Lebrilla, Carlito B; German, J Bruce; Bertram, Hanne C

    2012-06-20

    Free oligosaccharides are key components of human milk and play multiple roles in the health of the neonate, by stimulating growth of selected beneficial bacteria in the gut, participating in development of the brain, and exerting antipathogenic activity. However, the concentration of oligosaccharides is low in mature bovine milk, normally used for infant formula, compared with both human colostrum and mature human milk. Characterization of bovine milk oligosaccharides in different breeds is crucial for the identification of viable sources for oligosaccharide purification. An improved source of oligosaccharides can lead to infant formula with improved oligosaccharide functionality. In the present study we have analyzed milk oligosaccharides by high-performance liquid chromatography chip quadrupole time-of-flight mass spectrometry and performed a detailed data analysis using both univariate and multivariate methods. Both statistical tools revealed several differences in oligosaccharide profiles between milk samples from the two Danish breeds, Jersey and Holstein-Friesians. Jersey milk contained higher relative amounts of both sialylated and the more complex neutral fucosylated oligosaccharides, while the Holstein-Friesian milk had higher abundance of smaller and simpler neutral oligosaccharides. The statistical analyses revealed that Jersey milk contains levels of fucosylated oligosaccharides significantly higher than that of Holstein-Friesian milk. Jersey milk also possesses oligosaccharides with a higher degree of complexity and functional residues (fucose and sialic acid), suggesting it may therefore offer advantages in term of a wider array of bioactivities. PMID:22632419

  12. Novel arabinan and galactan oligosaccharides from dicotyledonous plants.

    PubMed

    Wefers, Daniel; Tyl, Catrin E; Bunzel, Mirko

    2014-01-01

    Arabinans and galactans are neutral pectic side chains and an important part of the cell walls of dicotyledonous plants. To get a detailed insight into their fine structure, various oligosaccharides were isolated from quinoa, potato galactan, and sugar beet pulp after enzymatic treatment. LC-MS(2) and one- and two-dimensional NMR spectroscopy were used for unambiguous structural characterization. It was demonstrated that arabinans contain β-(1→3)-linked arabinobiose as a side chain in quinoa seeds, while potato galactan was comprised of β-(1→4)-linked galactopyranoses which are interspersed with α-(1→4)-linked arabinopyranoses. Additionally, an oligosaccharide with two adjacent arabinofuranose units O2-substituted with two ferulic acid monomers was characterized. The isolated oligosaccharides gave further insight into the structures of pectic side chains and may have an impact on plant physiology and dietary fiber fermentation. PMID:25426490

  13. Novel arabinan and galactan oligosaccharides from dicotyledonous plants

    NASA Astrophysics Data System (ADS)

    Wefers, Daniel; Tyl, Catrin; Bunzel, Mirko

    2014-11-01

    Arabinans and galactans are neutral pectic side chains and an important part of the cell walls of dicotyledonous plants. To get a detailed insight into their fine structure, various oligosaccharides were isolated from quinoa, potato galactan, and sugar beet pulp after enzymatic treatment. LC-MS2 and one- and two-dimensional NMR spectroscopy were used for unambiguous structural characterization. It was demonstrated that arabinans contain β-(1→3)-linked arabinobiose as a side chain in quinoa seeds, while potato galactan was comprised of β-(1→4)-linked galactopyranoses which are interspersed with α-(1→4)-linked arabinopyranoses. Additionally, an oligosaccharide with two adjacent arabinofuranose units O2-substituted with two ferulic acid monomers was characterized. The isolated oligosaccharides gave further insight into the structures of pectic side chains and may have an impact on plant physiology and dietary fiber fermentation.

  14. Novel arabinan and galactan oligosaccharides from dicotyledonous plants

    PubMed Central

    Wefers, Daniel; Tyl, Catrin E.; Bunzel, Mirko

    2014-01-01

    Arabinans and galactans are neutral pectic side chains and an important part of the cell walls of dicotyledonous plants. To get a detailed insight into their fine structure, various oligosaccharides were isolated from quinoa, potato galactan, and sugar beet pulp after enzymatic treatment. LC-MS2 and one- and two-dimensional NMR spectroscopy were used for unambiguous structural characterization. It was demonstrated that arabinans contain β-(1→3)-linked arabinobiose as a side chain in quinoa seeds, while potato galactan was comprised of β-(1→4)-linked galactopyranoses which are interspersed with α-(1→4)-linked arabinopyranoses. Additionally, an oligosaccharide with two adjacent arabinofuranose units O2-substituted with two ferulic acid monomers was characterized. The isolated oligosaccharides gave further insight into the structures of pectic side chains and may have an impact on plant physiology and dietary fiber fermentation. PMID:25426490

  15. Purification and characterization of a xyloglucan oligosaccharide-specific xylosidase from pea seedlings

    SciTech Connect

    O'Neill, R.A.; Albersheim, P.; Darvill, A.G. )

    1989-12-05

    An {alpha}-xylosidase that acts on oligosaccharide fragments of xyloglucan, a plant cell wall polysaccharide, was purified from pea (Pisum sativum) epicotyls that had been treated with an auxin analog. The enzyme had an apparent molecular mass of 85,000 Da according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 79,000 Da according to gel-permeation chromatography under nondenaturing conditions. The purified xylosidase consisted of a series of closely related, enzymatically active proteins with isoelectric points ranging from about pH 7.35 to 7.7; the xylosidases were separated by chromatofocusing. The pH optimum of the mixed xylosidase was 4.9-5.1. The substrate specificity of the xylosidase mixture was determined by purification and structural characterization of the products of treating xyloglucan-oligosaccharide substrates with the enzyme. Characterization of the substrates and products included elution volume from a gel-permeation column, glycosyl residue and glycosyl linkage composition analyses, fast atom bombardment-mass spectrometry, and {sup 1}H NMR spectroscopy. The enzyme specifically cleaved only one of the {alpha}-xylosidic linkages of xyloglucan-oligosaccharide substrates, the one attached to a 6-linked glucosyl residue, not those attached to the 4,6-linked glucosyl residues. The enzyme was unable to cleave the xylosidic linkage of p-nitrophenyl-{alpha}-D-xylopyranoside or the {alpha}-xylosidic linkage to C-6 of glucose in the disaccharide isoprimeverose. The enzyme was also unable to release measurable amounts of xylose from large xyloglucan polymers.

  16. Synthetic oligosaccharides as heparin-mimetics displaying anticoagulant properties.

    PubMed

    Avci, Fikri Y; Karst, Nathalie A; Linhardt, Robert J

    2003-01-01

    Heparin and low molecular weight heparins are major clinical anticoagulants and the drugs of choice for the treatment of deep venous thrombosis. The discovery of an antithrombin binding domain in heparin focused interest on understanding the mechanism of heparin's antithrombotic/ anticoagulant activity. Various heparin-mimetic oligosaccharides have been prepared in an effort to replace polydisperse heparin and low molecular weight heparins with a structurally-defined anticoagulant. The goal of attaining a heparin-mimetic with no unwanted side-effects has also provided motivation for these efforts. This article reviews structure-activity relationship (SAR) of structurally-defined heparin-mimetic oligosaccharides. PMID:14529394

  17. Family 18 chitinase-oligosaccharide substrate interaction: subsite preference and anomer selectivity of Serratia marcescens chitinase A.

    PubMed Central

    Aronson, Nathan N; Halloran, Brian A; Alexyev, Mikhail F; Amable, Lauren; Madura, Jeffry D; Pasupulati, Lakshminarasimhulu; Worth, Catherine; Van Roey, Patrick

    2003-01-01

    The sizes and anomers of the products formed during the hydrolysis of chitin oligosaccharides by the Family 18 chitinase A (ChiA) from Serratia marcescens were analysed by hydrophilic interaction chromatography using a novel approach in which reactions were performed at 0 degrees C to stabilize the anomer conformations of the initial products. Crystallographic studies of the enzyme, having the structure of the complex of the ChiA E315L (Glu315-->Leu) mutant with a hexasaccharide, show that the oligosaccharide occupies subsites -4 to +2 in the substrate-binding cleft, consistent with the processing of beta-chitin by the release of disaccharide at the reducing end. Products of the hydrolysis of hexa- and penta-saccharides by wild-type ChiA, as well as by two mutants of the residues Trp275 and Phe396 important in binding the substrate at the +1 and +2 sites, show that the substrates only occupy sites -2 to +2 and that additional N -acetyl-D-glucosamines extend beyond the substrate-binding cleft at the reducing end. The subsites -3 and -4 are not used in this four-site binding mode. The explanation for these results is found in the high importance of individual binding sites for the processing of short oligosaccharides compared with the cumulative recognition and processive hydrolysis mechanism used to digest natural beta-chitin. PMID:12932195

  18. Gluco-oligomers initially formed by the reuteransucrase enzyme of Lactobacillus reuteri 121 incubated with sucrose and malto-oligosaccharides.

    PubMed

    Dobruchowska, Justyna M; Meng, Xiangfeng; Leemhuis, Hans; Gerwig, Gerrit J; Dijkhuizen, Lubbert; Kamerling, Johannis P

    2013-09-01

    The probiotic bacterium Lactobacillus reuteri 121 produces a complex, branched (1 → 4, 1 → 6)-α-D-glucan as extracellular polysaccharide (reuteran) from sucrose (Suc), using a single glucansucrase/glucosyltransferase (GTFA) enzyme (reuteransucrase). To gain insight into the reaction/product specificity of the GTFA enzyme and the mechanism of reuteran formation, incubations with Suc and/or a series of malto-oligosaccharides (MOSs) (degree of polymerization (DP2-DP6)) were followed in time. The structures of the initially formed products, isolated via high-performance anion-exchange chromatography, were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and 1D/2D (1)H/(13)C NMR spectroscopy. Incubations with Suc only, acting as both donor and acceptor, resulted in elongation of Suc with glucose (Glc) units via alternating (α1 → 4) and (α1 → 6) linkages, yielding linear gluco-oligosaccharides up to at least DP ~ 12. Simultaneously with the ensemble of oligosaccharides, polymeric material was formed early on, suggesting that alternan fragments longer than DP ~ 12 have higher affinity with the GTFA enzyme and are quickly extended, yielding high-molecular-mass branched reuteran (4 × 10(7) Da). MOSs (DP2-DP6) in the absence of Suc turned out to be poor substrates. Incubations of GTFA with Suc plus MOSs as substrates resulted in preferential elongation of MOSs (acceptors) with Glc units from Suc (donor). This apparently reflects the higher affinity of GTFA for MOSs compared with Suc. In accordance with the GTFA specificity, most prominent products were oligosaccharides with an (α1 → 4)/(α1 → 6) alternating structure. PMID:23804502

  19. Role of human milk oligosaccharides in Group B Streptococcus colonisation.

    PubMed

    Andreas, Nicholas J; Al-Khalidi, Asmaa; Jaiteh, Mustapha; Clarke, Edward; Hyde, Matthew J; Modi, Neena; Holmes, Elaine; Kampmann, Beate; Mehring Le Doare, Kirsty

    2016-08-01

    Group B Streptococcus (GBS) infection is a major cause of morbidity and mortality in infants. The major risk factor for GBS disease is maternal and subsequent infant colonisation. It is unknown whether human milk oligosaccharides (HMOs) protect against GBS colonisation. HMO production is genetically determined and linked to the Lewis antigen system. We aimed to investigate the association between HMOs and infant GBS colonisation between birth and postnatal day 90. Rectovaginal swabs were collected at delivery, as well as colostrum/breast milk, infant nasopharyngeal and rectal swabs at birth, 6 days and days 60-89 postpartum from 183 Gambian mother/infant pairs. GBS colonisation and serotypes were determined using culture and PCR. (1)H nuclear magnetic resonance spectroscopy was used to characterise the mother's Lewis status and HMO profile in breast milk. Mothers who were Lewis-positive were significantly less likely to be colonised by GBS (X (2)=12.50, P<0.001). Infants of Lewis-positive mothers were less likely GBS colonised at birth (X (2)=4.88 P=0.03) and more likely to clear colonisation between birth and days 60-89 than infants born to Lewis-negative women (P=0.05). There was no association between Secretor status and GBS colonisation. In vitro work revealed that lacto-N-difucohexaose I (LNDFHI) correlated with a reduction in the growth of GBS. Our results suggest that HMO such as LNDFHI may be a useful adjunct in reducing maternal and infant colonisation and hence invasive GBS disease. Secretor status offers utility as a stratification variable in GBS clinical trials. PMID:27588204

  20. Role of human milk oligosaccharides in Group B Streptococcus colonisation

    PubMed Central

    Andreas, Nicholas J; Al-Khalidi, Asmaa; Jaiteh, Mustapha; Clarke, Edward; Hyde, Matthew J; Modi, Neena; Holmes, Elaine; Kampmann, Beate; Mehring Le Doare, Kirsty

    2016-01-01

    Group B Streptococcus (GBS) infection is a major cause of morbidity and mortality in infants. The major risk factor for GBS disease is maternal and subsequent infant colonisation. It is unknown whether human milk oligosaccharides (HMOs) protect against GBS colonisation. HMO production is genetically determined and linked to the Lewis antigen system. We aimed to investigate the association between HMOs and infant GBS colonisation between birth and postnatal day 90. Rectovaginal swabs were collected at delivery, as well as colostrum/breast milk, infant nasopharyngeal and rectal swabs at birth, 6 days and days 60–89 postpartum from 183 Gambian mother/infant pairs. GBS colonisation and serotypes were determined using culture and PCR. 1H nuclear magnetic resonance spectroscopy was used to characterise the mother's Lewis status and HMO profile in breast milk. Mothers who were Lewis-positive were significantly less likely to be colonised by GBS (X2=12.50, P<0.001). Infants of Lewis-positive mothers were less likely GBS colonised at birth (X2=4.88 P=0.03) and more likely to clear colonisation between birth and days 60–89 than infants born to Lewis-negative women (P=0.05). There was no association between Secretor status and GBS colonisation. In vitro work revealed that lacto-N-difucohexaose I (LNDFHI) correlated with a reduction in the growth of GBS. Our results suggest that HMO such as LNDFHI may be a useful adjunct in reducing maternal and infant colonisation and hence invasive GBS disease. Secretor status offers utility as a stratification variable in GBS clinical trials. PMID:27588204

  1. The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy.

    PubMed

    Green, E D; Adelt, G; Baenziger, J U; Wilson, S; Van Halbeek, H

    1988-12-01

    The structures of the entire population of sialylated asparagine-linked oligosaccharides present on bovine fetuin were elucidated. Asparagine-linked oligosaccharides were released from fetuin with N-glycanase, radiolabeled by reduction with NaB[3H]4, and fractionated by anion-exchange high performance liquid chromatography (HPLC), ion-suppression amine adsorption HPLC, and concanavalin A affinity chromatography. The 3H-labeled oligosaccharide fractions obtained were analyzed by 500-MHz 1H nuclear magnetic resonance spectroscopy, revealing the presence of 23 distinct oligosaccharide structures. These oligosaccharides differed in extent of sialylation (3% mono-, 35% di-, 54% tri-, and 8% tetrasialylated), number of peripheral branches (17% di- and 83% tribranched), linkage (alpha 2,3 versus alpha 2,6) and location of sialic acid moieties, and linkage (beta 1,4 versus beta 1,3) of galactose residues. This represents the first time that the asparagine-linked oligosaccharides of fetuin have been successfully fractionated and characterized as sialylated species. The sialylated oligosaccharides derived from fetuin were also used to further define the specificities of the lectins leukoagglutinating phytohemagglutinin and Ricinus communis agglutinin I. The behavior of these oligosaccharides during lectin affinity HPLC further establishes the structural features which predominate in the interaction of oligosaccharides with leukoagglutinating phytohemagglutinin and R. communis agglutinin I. PMID:2461366

  2. In Vitro Fermentation of caprine milk oligosaccharides by bifidobacteria isolated from breast-fed infants

    PubMed Central

    Thum, Caroline; Roy, Nicole C; McNabb, Warren C; Otter, Don E; Cookson, Adrian L

    2015-01-01

    This study was conducted to investigate the catabolism and fermentation of caprine milk oligosaccharides (CMO) by selected bifidobacteria isolated from 4 breast-fed infants. Seventeen bifidobacterial isolates consisting of 3 different species (Bifidobacterium breve, Bifidobacterium longum subsp. longum and Bifidobacterium bifidum) were investigated. A CMO-enriched fraction (CMOF) (50% oligosaccharides, 10% galacto-oligosaccharides (GOS), 20% lactose, 10% glucose and 10% galactose) from caprine cheese whey was added to a growth medium as a sole source of fermentable carbohydrate. The inclusion of the CMOF was associated with increased bifidobacterial growth for all strains compared to glucose, lactose, GOS, inulin, oligofructose, 3'-sialyl-lactose and 6'-sialyl-lactose. Only one B. bifidum strain (AGR2166) was able to utilize the sialyl-CMO, 3'-sialyl-lactose and 6'-sialyl-lactose, as carbohydrate sources. The inclusion of CMOF increased the production of acetic and lactic acid (P < 0.001) after 36 h of anaerobic fermentation at 37°C, when compared to other fermentable substrates. Two B. bifidum strains (AGR2166 and AGR2168) utilised CMO, contained in the CMOF, to a greater extent than B. breve or B. longum subsp longum isolates, and this increased CMO utilization was associated with enhanced sialidase activity. CMOF stimulated bifidobacterial growth when compared to other tested fermentable carbohydrates and also increased the consumption of mono- and disaccharides, such as galactose and lactose present in the CMOF. These findings indicate that the dietary consumption of CMO may stimulate the growth and metabolism of intestinal Bifidobacteria spp. including B. bifidum typically found in the large intestine of breast-fed infants. PMID:26587678

  3. In Vitro Fermentation of caprine milk oligosaccharides by bifidobacteria isolated from breast-fed infants.

    PubMed

    Thum, Caroline; Roy, Nicole C; McNabb, Warren C; Otter, Don E; Cookson, Adrian L

    2015-01-01

    This study was conducted to investigate the catabolism and fermentation of caprine milk oligosaccharides (CMO) by selected bifidobacteria isolated from 4 breast-fed infants. Seventeen bifidobacterial isolates consisting of 3 different species (Bifidobacterium breve, Bifidobacterium longum subsp. longum and Bifidobacterium bifidum) were investigated. A CMO-enriched fraction (CMOF) (50% oligosaccharides, 10% galacto-oligosaccharides (GOS), 20% lactose, 10% glucose and 10% galactose) from caprine cheese whey was added to a growth medium as a sole source of fermentable carbohydrate. The inclusion of the CMOF was associated with increased bifidobacterial growth for all strains compared to glucose, lactose, GOS, inulin, oligofructose, 3'-sialyl-lactose and 6'-sialyl-lactose. Only one B. bifidum strain (AGR2166) was able to utilize the sialyl-CMO, 3'-sialyl-lactose and 6'-sialyl-lactose, as carbohydrate sources. The inclusion of CMOF increased the production of acetic and lactic acid (P < 0.001) after 36 h of anaerobic fermentation at 37 °C, when compared to other fermentable substrates. Two B. bifidum strains (AGR2166 and AGR2168) utilised CMO, contained in the CMOF, to a greater extent than B. breve or B. longum subsp longum isolates, and this increased CMO utilization was associated with enhanced sialidase activity. CMOF stimulated bifidobacterial growth when compared to other tested fermentable carbohydrates and also increased the consumption of mono- and disaccharides, such as galactose and lactose present in the CMOF. These findings indicate that the dietary consumption of CMO may stimulate the growth and metabolism of intestinal Bifidobacteria spp. including B. bifidum typically found in the large intestine of breast-fed infants. PMID:26587678

  4. Simultaneous analysis of heparosan oligosaccharides by isocratic liquid chromatography with charged aerosol detection/mass spectrometry.

    PubMed

    Ji, Xiaohu; Hu, Guixin; Zhang, Qiongyan; Wang, Fengshan; Liu, Chunhui

    2016-11-01

    Uncovering the biological roles of heparosan oligosaccharides requires a simple and robust method for their separation and identification. We reported on systematic investigations of the retention behaviors of synthetic heparosan oligosaccharides on porous graphitic carbon (PGC) column by HPLC with charged aerosol detection. Oligosaccharides were strongly retained by PGC material in water-acetonitrile mobile phase, and eluted by trifluoroacetic acid occurring as narrow peaks. Addition of small fraction of methanol led to better selectivity of PGC to oligosaccharides than acetonitrile modifier alone, presumably, resulting from displacement of methanol to give different chemical environment at the PGC surface. Van't-Hoff plots demonstrated that retention behaviors highly depended on the column temperature and oligosaccharide moieties. By implementing the optimal MeOH content and temperature, a novel isocratic elution method was successfully developed for baseline resolution and identification of seven heparosan oligosaccharides using PGC-HPLC-CAD/MS. This approach allows for rapid analysis of heparosan oligosaccharides from various sources. PMID:27516280

  5. Screening Substrate Properties of Microorganisms for Biosensor Detection of Oligosaccharides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oligosaccharides feature high biological activity ensuring their wide application in the biotechnology, food, and cosmetic industries. On the other hand they are considered environmental pollutants. The study outlines a biosensor approach to detect these substances which is important from above st...

  6. Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens.

    PubMed

    Gerwig, G J; Kamerling, J P; Vliegenthart, J F; Morag, E; Lamed, R; Bayer, E A

    1992-04-15

    The multiple cellulase-containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O-linked mainly to a 230-kDa subunit. The oligosaccharide chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by one- and two-dimensional (correlation, homonuclear Hartmann-Hahn, rotating-frame nuclear Overhauser enhancement) 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast-atom-bombardment mass spectrometry. The following carbohydrate structures could be established: [formula: see text] The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B. cellulosolvens and of Clostridium thermocellum [Gerwig, G. J., Kamerling, J. P., Vliegenthart, J. F. G., Morag (Morgenstern), E., Lamed, R. & Bayer, E. A. (1991) Eur. J. Biochem. 196, 115-122], having 3, 5 and 6 as common elements. The furanose form of a terminal alpha-D-galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome-like entity of B. cellulosolvens. PMID:1572372

  7. Capillary electrophoresis of sialylated oligosaccharides in milk from different species.

    PubMed

    Monti, Lucia; Cattaneo, Tiziana Maria Piera; Orlandi, Mario; Curadi, Maria Claudia

    2015-08-28

    Oligosaccharides are relevant components of human milk, which have been quite well studied for their pre-biotic effect and their capacity in stimulating the immune system. Since oligosaccharides from milk of non-human mammals received so far less attention, the aim of this work was the application of capillary electrophoresis (CE) for the analysis of sialylated oligosaccharides in cow, goat and equine (mare and donkey) milk to possibly identify potential sources of oligosaccharides to use as health promoting ingredients in functional foods. Human milk was used as reference milk. A recent CE technique was applied to resolve and quantify 3-sialyllactose (3-SL), 6-sialyllactose (6-SL) and disialyl-lacto-N-tetraose (DSLNT). Analysis of non-human milk samples confirmed differences among species and individuals: DSLNT, which was the most abundant compound in human milk (455-805μg/mL) was missing in most of the samples. In most cases, 3-SL showed to be the most concentrated of the quantified analytes, with values ranging from 12 to 77μg/mL. PMID:26228851

  8. Conversion of yeast mannan polysaccharide in mannose oligosaccharides with a thiopropargyl linker at the pseudo-reducing end.

    PubMed

    Marzaioli, Alberto M; Bedini, Emiliano; Lanzetta, Rosa; Parrilli, Michelangelo; De Castro, Cristina

    2014-01-13

    Polysaccharides are useful sources of preformed glycosyl linkages and therefore convenient building blocks can be prepared from them upon application of a proper degradation and derivatization scheme. Following this idea, the α-(1→6) linkages of this polysaccharide were broken applying acetolysis reaction and the pseudo-reducing position of the resulting acetylated mannose oligosaccharides was functionalized with a thiopropargyl appendage through thiourea activation of the related glycosyl iodides. Deacetylation of the final products and optimization of the purification procedures are discussed. The best yields were observed combining size exclusion chromatography and HPLC. Nine different oligosaccharides, from mono- to tetrasaccharides, mainly α-configured at the pseudo-reducing terminal, were obtained in pure form, presenting the propargyl appendage, a feature that makes them suitable substrates for successive functionalizations. PMID:24275083

  9. Thermopressurized diluted phosphoric acid pretreatment of ligno(hemi)cellulose to make free sugars and nutraceutical oligosaccharides.

    PubMed

    Tiboni, Marcela; Grzybowski, Adelia; Baldo, Gizele Rejane; Dias, Edson Flausino; Tanner, Robert D; Kornfield, Julia Ann; Fontana, José Domingos

    2014-06-01

    Ligno(hemi)cellulosics (L(h)Cs) as sugarcane bagasse and loblolly pine sawdust are currently being used to produce biofuels such as bioethanol and biobutanol through fermentation of free sugars that are often obtained enzymatically. However, this bioconversion requires a pretreatment to solubilize the hemicellulose fractions, thus facilitating the action of the cellulolytic enzymes. Instead of the main free monosaccharides used in these current models, the modulation of thermopressurized orthophosphoric acid as a pretreatment, in the ranges of 3-12 atm and pH 1.5-2.5, can produce nondigestible oligosaccharides (NDOS) such as xylo-oligosaccharides (XOS) because heteroxylan is present in both types of hardwood and softwood hemicelluloses. A comparative thin-layer chromatographic analysis of the hydrolytic products showed the best conditions for NDOS production to be 7 atm/water, pH 2.25 and 2.50, and 8.5 atm/water for both sources. Particular hydrolysates from 7 atm (171 °C) at pHs 2.25 and 2.50 both for cane bagasse and pine sawdust, with respective oligosaccharide contents of 57 and 59 %, once mixed in a proportion of 1:1 for each plant source, were used in vitro as carbon sources for Bifidobacterium or Lactobacillus. Once both bacteria attained the stationary phase of growth, an unforeseen feature emerged: the preference of B. animalis for bagasse hydrolysates and, conversely, the preference of L. casei for pine hydrolysates. Considering the fact that nutraceutical oligosaccharides from both hemicelluloses correspond to higher value-added byproducts, the technology using a much diluted thermopressurized orthophosphoric acid pretreatment becomes an attractive choice for L(h)Cs. PMID:24747989

  10. Milk Oligosaccharide Variation in Sow Milk and Milk Oligosaccharide Fermentation in Piglet Intestine.

    PubMed

    Difilippo, Elisabetta; Pan, Feipeng; Logtenberg, Madelon; Willems, Rianne H A M; Braber, Saskia; Fink-Gremmels, Johanna; Schols, Henk Arie; Gruppen, Harry

    2016-03-16

    Porcine milk oligosaccharides (PMOs) were analyzed in six colostrum and two mature milk samples from Dutch Landrace sows. In total, 35 PMOs were recognized of which 13 were new for the PMO literature: neutral HexNAc-Hex, β4'-galactosyllactose, putative GalNAc(α/β1-3)Gal(β1-4)Glc, lacto-N-fucopentaose-II, lacto-N-tetraose, galactose substituted lacto-N-neohexaose, lacto-N-hexaose and difucosyl-lacto-N-hexaose, and acidic Neu5Ac(α2-6)GlcNAc(β1-3)Gal(β1-4)Glc, sialyllacto-N-tetraose-a and -b, Neu5Ac2-Hex3, and sialyllacto-N-fucopentaose-II. PMOs were analyzed using capillary electrophoresis with laser-induced florescence detection or mass spectrometry and using liquid chromatography with mass spectrometry. Interindividual variation regarding PMO presence and concentration was observed between porcine milks. Within a limited sample set, a 43% decrease of the major PMOs was found during a 1 w lactation period. Interestingly, while some PMOs decreased, some other PMOs increased in concentration. PMOs were also monitored in fecal samples of suckling piglets. In feces of 1-2 d old piglets, few intact PMOs were found, indicating considerable PMO fermentation at early stage of life. PMID:26882005

  11. Chitosan and its oligosaccharide derivatives (chito-oligosaccharides) as feed supplements in poultry and swine nutrition.

    PubMed

    Swiatkiewicz, S; Swiatkiewicz, M; Arczewska-Wlosek, A; Jozefiak, D

    2015-02-01

    Chitosan is a non-toxic polyglucosamine, widespread in nature, which is deacetylated to varying degrees form of chitin, a component of exoskeleton of shrimps, crabs and insects. Because chitosan contains reactive functional groups, that is, amino acids and hydroxyl groups, it is characterised by antimicrobial, anti-inflammatory, anti-oxidative, antitumor, immunostimulatory and hypocholesterolemic properties when fed as dietary additive for farm animals. This article reviews and discusses the results of studies on the effects of dietary chitosan and its oligosaccharide derivatives on performance and metabolic response in poultry and pigs, that is, haematological, biochemical and immunological blood characteristics, microbiological profile of intestines, intestinal morphology and digestibility of nutrients, as well as on the quality of meat and eggs. The results of most of the experiments presented in this review indicate that chitosan used as a feed additive for poultry and pigs has some beneficial, biological effects, including immunomodulatory, anti-oxidative, antimicrobial and hypocholesterolemic properties. These properties of chitosan, unlike many other kinds of feed additives, were often reflected in improved growth performance (body weight gain and/or feed conversion ratio) of young animals, that is, broiler chickens and weaned pigs. PMID:25041091

  12. Cranberry (Vaccinium macrocarpon) oligosaccharides decrease biofilm formation by uropathogenic Escherichia coli

    PubMed Central

    Sun, Jiadong; Marais, Jannie P. J.; Khoo, Christina; LaPlante, Kerry; Vejborg, Rebecca M.; Givskov, Michael; Tolker-Nielsen, Tim; Seeram, Navindra P.; Rowley, David C.

    2015-01-01

    The preventive effects of the American cranberry (Vaccinium macrocarpon) against urinary tract infections are supported by extensive studies which have primarily focused on its phenolic constituents. Herein, a phenolic-free carbohydrate fraction (designated cranf1b-F2) was purified from cranberry fruit using ion exchange and size exclusion chromatography. MALDI-TOF-MS analysis revealed that the cranf1b-F2 constituents are predominantly oligosaccharides possessing various degrees of polymerisation and further structural analysis (by GC-MS and NMR) revealed mainly xyloglucan and arabinan residues. In antimicrobial assays, cranf1b-F2 (at 1.25 mg/mL concentration) reduced biofilm production by the uropathogenic Escherichia coli CFT073 strain by over 50% but did not inhibit bacterial growth. Cranf1b-F2 (ranging from 0.625 - 10 mg/mL) also inhibited biofilm formation of the non-pathogenic E. coli MG1655 strain up to 60% in a concentration-dependent manner. These results suggest that cranberry oligosaccharides, in addition to its phenolic constituents, may play a role in its preventive effects against urinary tract infections. PMID:26613004

  13. Water soluble biocompatible vesicles based on polysaccharides and oligosaccharides inclusion complexes for carotenoid delivery.

    PubMed

    Polyakov, Nikolay E; Kispert, Lowell D

    2015-09-01

    Since carotenoids are highly hydrophobic, air- and light-sensitive hydrocarbon compounds, developing methods for increasing their bioavailability and stability towards irradiation and reactive oxygen species is an important goal. Application of inclusion complexes of "host-guest" type with polysaccharides and oligosaccharides such as arabinogalactan, cyclodextrins and glycyrrhizin minimizes the disadvantages of carotenoids when these compounds are used in food processing (colors and antioxidant capacity) as well as for production of therapeutic formulations. Cyclodextrin complexes which have been used demonstrated enhanced storage stability but suffered from poor solubility. Polysaccharide and oligosaccharide based inclusion complexes play an important role in pharmacology by providing increased solubility and stability of lipophilic drugs. In addition they are used as drug delivery systems to increase absorption rate and bioavailability of the drugs. In this review we summarize the existing data on preparation methods, analysis, and chemical reactivity of carotenoids in inclusion complexes with cyclodextrin, arabinogalactan and glycyrrhizin. It was demonstrated that incorporation of carotenoids into the "host" macromolecule results in significant changes in their physical and chemical properties. In particular, polysaccharide complexes show enhanced photostability of carotenoids in water solutions. A significant decrease in the reactivity towards metal ions and reactive oxygen species in solution was also detected. PMID:26005157

  14. Investigation on the Synthesis of Shigella flexneri Specific Oligosaccharides Using Disaccharides as Potential Transglucosylase Acceptor Substrates.

    PubMed

    Salamone, Stéphane; Guerreiro, Catherine; Cambon, Emmanuelle; Hargreaves, Jason M; Tarrat, Nathalie; Remaud-Siméon, Magali; André, Isabelle; Mulard, Laurence A

    2015-11-20

    Chemo-enzymatic strategies hold great potential for the development of stereo- and regioselective syntheses of structurally defined bioactive oligosaccharides. Herein, we illustrate the potential of the appropriate combination of a planned chemo-enzymatic pathway and an engineered biocatalyst for the multistep synthesis of an important decasaccharide for vaccine development. We report the stepwise investigation, which led to an efficient chemical conversion of allyl α-d-glucopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3)-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside, the product of site-specific enzymatic α-d-glucosylation of a lightly protected non-natural disaccharide acceptor, into a pentasaccharide building block suitable for chain elongation at both ends. Successful differentiation between hydroxyl groups features the selective acylation of primary alcohols and acetalation of a cis-vicinal diol, followed by a controlled per-O-benzylation step. Moreover, we describe the successful use of the pentasaccharide intermediate in the [5 + 5] synthesis of an aminoethyl aglycon-equipped decasaccharide, corresponding to a dimer of the basic repeating unit from the O-specific polysaccharide of Shigella flexneri 2a, a major cause of bacillary dysentery. Four analogues of the disaccharide acceptor were synthesized and evaluated to reach a larger repertoire of O-glucosylation patterns encountered among S. flexneri type-specific polysaccharides. New insights on the potential and limitations of planned chemo-enzymatic pathways in oligosaccharide synthesis are provided. PMID:26340432

  15. Efficient synthesis and activity of beneficial intestinal flora of two lactulose-derived oligosaccharides.

    PubMed

    Zhu, Zhen-Yuan; Cui, Di; Gao, Hui; Dong, Feng-Ying; Liu, Xiao-Cui; Liu, Fei; Chen, Lu; Zhang, Yong-Min

    2016-05-23

    Lactulose is considered as a prebiotic because it promotes the intestinal proliferation of Lactobacillus acidophilus which is added to various milk products. Moreover, lactulose is used in pharmaceuticals as a gentle laxative and to treat hyperammonemia. This study was aimed at the total synthesis of two Lactulose-derived oligosaccharides: one is 3-O-β-d-galactopyranosyl-d-fructose, d-fructose and β-d-galactose bounded together with β-1,3-glycosidic bound, the other is 1-O-β-d-galactopyranosyl-d-fructose, d-fructose and β-d-galactose bounded together with β-1,1-glycosidic bound, which were accomplished in seven steps from d-fructose and β-d-galactose and every step of yield above 75%. This synthetic route provided a practical and effective synthetic strategy for galactooligosaccharides, starting from commercially available monosaccharides. Then we evaluated on their prebiotic properties in the search for potential agents of regulating and improving the intestinal flora of human. The result showed that the prebiotic properties of Lactulose-derived oligosaccharides was much better than Lactulose. Among them, 3-O-β-d-galactopyranosyl-d-fructose displayed the most potent activity of proliferation of L. acidophilus. PMID:26974370

  16. Advances in Analysis of Human Milk Oligosaccharides123

    PubMed Central

    Ruhaak, L. Renee; Lebrilla, Carlito B.

    2012-01-01

    Oligosaccharides in human milk strongly influence the composition of the gut microflora of neonates. Because it is now clear that the microflora play important roles in the development of the infant immune system, human milk oligosaccharides (HMO) are studied frequently. Milk samples contain complex mixtures of HMO, usually comprising several isomeric structures that can be either linear or branched. Traditionally, HMO profiling was performed using HPLC with fluorescence or UV detection. By using porous graphitic carbon liquid chromatography MS, it is now possible to separate and identify most of the isomers, facilitating linkage-specific analysis. Matrix-assisted laser desorption ionization time-of-flight analysis allows fast profiling, but does not allow isomer separation. Novel MS fragmentation techniques have facilitated structural characterization of HMO that are present at lower concentrations. These techniques now facilitate more accurate studies of HMO consumption as well as Lewis blood group determinations. PMID:22585919

  17. Preparation and antioxidant activities of oligosaccharides from Crassostrea gigas.

    PubMed

    Wu, Shengjun; Huang, Xiaolian

    2017-02-01

    Oligosaccharides were prepared from Crassostrea gigas by hydrolysis of polysaccharide in C. gigas with peroxide oxygen (H2O2). The hydrolysates were cleared of protein, filtered, ultrafiltered and precipitated with absolute ethanol to give C. gigas oligosaccharides (CGOs). Factors affecting CGO yields, i.e., reaction time, temperature, and H2O2 concentration, were optimised as follows: 2.96h reaction time, 84.71°C reaction temperature, and 2.46% H2O2 concentration. Under these conditions, the maximum yield of CGOs reached 10.61%. The CGOs were then partially characterised by Fourier transform infrared spectroscopy, UV spectroscopy, monosaccharide composition, and antioxidant activities. Results indicate that CGOs possessed strong hydroxyl radical activity, 2,2-diphenyl-β-picrylhydrazyl-radical-scavenging activity and reducing capacity at a concentration of 100μg/mL. PMID:27596415

  18. Release of feruloylated oligosaccharides from wheat bran through submerged fermentation by edible mushrooms.

    PubMed

    Xie, Chunyan; Wu, Zhiyan; Guo, Hongzhen; Gu, Zhenxin

    2014-07-01

    Wheat bran, a by-product of the flour industry, is believed to be a raw material for the production of feruloylated oligosaccharides (FOs) because of its high content of conjiont ferulic acid (FA). Studies were carried out to identify edible mushrooms that are able to release FOs from wheat bran. All the six tested mushrooms (Pleurotus ostreatus, Hericium erinaceum, Auricularia auricula, Cordyceps militaris, Agrocybe chaxingu, and Ganoderma lucium) were found to release FOs, and Agrocybe chaxingu had the highest yield, reaching 35.4 µM in wheat bran broth. Enzymes detection showed that these species secreted extracellular enzymes during fermentation, including cellulase and xylanase. Agrocybe chaxingu secreted the significant amount of xylanase (180 mU ml(-1) ), which was responsible for the release of FOs from wheat bran, while Hericium erinaceum secreted FA esterase which could disassemble FOs. PMID:24123163

  19. The chromatographic analysis of oligosaccharides and preparation of 1-kestose and nystose in yacon.

    PubMed

    Zhu, Zhen-Yuan; Lian, Hong-Yu; Si, Chuan-Ling; Liu, Yang; Liu, Nian; Chen, Jing; Ding, Li-Na; Yao, Qiang; Zhang, Yongmin

    2012-05-01

    The thin-layer chromatographic analysis of the crude oligosaccharides extracted from yacon revealed the presence of glucose, fructose, sucrose, 1-kestose and nystose. The qualitative and quantitative analysis was carried out on oligosaccharides by high pressure liquid chromatography and the results showed that the contents of d-glucose, fructose, sucrose, 1-kestose, nystose and 1-fructofuranosyl nystose in oligosaccharides were 38.30%, 16.44%, 14.58%, 12.29%, 12.17%, 6.20%, respectively. The content of the fructooligosaccharides in oligosaccharides was 30.66%. The crude oligosaccharides were separated and purified by silica gel column chromatography. The two fractions obtained from crude oligosaccharides were 1-kestose and nystose, which were identified by mass spectra. The yield of 1-kestose and nystose were 10.36% and 9.73%, respectively. The purity of 1-kestose was 82.9% and of nystose was 73.6%. PMID:22013906

  20. Gliding Motility of Mycoplasma mobile on Uniform Oligosaccharides

    PubMed Central

    Kasai, Taishi; Hamaguchi, Tasuku

    2015-01-01

    ABSTRACT The binding and gliding of Mycoplasma mobile on a plastic plate covered by 53 uniform oligosaccharides were analyzed. Mycoplasmas bound to and glided on only 21 of the fixed sialylated oligosaccharides (SOs), showing that sialic acid is essential as the binding target. The affinities were mostly consistent with our previous results on the inhibitory effects of free SOs and suggested that M. mobile recognizes SOs from the nonreducing end with four continuous sites as follows. (i and ii) A sialic acid at the nonreducing end is tightly recognized by tandemly connected two sites. (iii) The third site is recognized by a loose groove that may be affected by branches. (iv) The fourth site is recognized by a large groove that may be enhanced by branches, especially those with a negative charge. The cells glided on uniform SOs in manners apparently similar to those of the gliding on mixed SOs. The gliding speed was related inversely to the mycoplasma's affinity for SO, suggesting that the detaching step may be one of the speed determinants. The cells glided faster and with smaller fluctuations on the uniform SOs than on the mixtures, suggesting that the drag caused by the variation in SOs influences gliding behaviors. IMPORTANCE Mycoplasma is a group of bacteria generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide in the direction of the protrusion. These procedures are essential for parasitism. Usually, mycoplasmas glide on mixed sialylated oligosaccharides (SOs) derived from glycoprotein and glycolipid. Since gliding motility on uniform oligosaccharides has never been observed, this study gives critical information about recognition and interaction between receptors and SOs. PMID:26148712

  1. Eukaryotic Oligosaccharyltransferase Generates Free Oligosaccharides during N-Glycosylation*

    PubMed Central

    Harada, Yoichiro; Buser, Reto; Ngwa, Elsy M.; Hirayama, Hiroto; Aebi, Markus; Suzuki, Tadashi

    2013-01-01

    Asparagine (N)-linked glycosylation regulates numerous cellular activities, such as glycoprotein quality control, intracellular trafficking, and cell-cell communications. In eukaryotes, the glycosylation reaction is catalyzed by oligosaccharyltransferase (OST), a multimembrane protein complex that is localized in the endoplasmic reticulum (ER). During N-glycosylation in the ER, the protein-unbound form of oligosaccharides (free oligosaccharides; fOSs), which is structurally related to N-glycan, is released into the ER lumen. However, the enzyme responsible for this process remains unidentified. Here, we demonstrate that eukaryotic OST generates fOSs. Biochemical and genetic analyses using mutant strains of Saccharomyces cerevisiae revealed that the generation of fOSs is tightly correlated with the N-glycosylation activity of OST. Furthermore, we present evidence that the purified OST complex can generate fOSs by hydrolyzing dolichol-linked oligosaccharide, the glycan donor substrate for N-glycosylation. The heterologous expression of a single subunit of OST from the protozoan Leishmania major in S. cerevisiae demonstrated that this enzyme functions both in N-glycosylation and generation of fOSs. This study provides insight into the mechanism of PNGase-independent formation of fOSs. PMID:24062310

  2. Cellulase-assisted extraction of oligosaccharides from defatted rice bran.

    PubMed

    Patindol, J; Wang, L; Wang, Y-J

    2007-11-01

    Defatted rice bran was subjected to cellulase treatment in order to increase its extractable oligosaccharides. Various combinations of enzyme concentration (0%, 0.5%, 1.0%, and 2.0%), temperature (room, 30, 40, and 50 degrees C), and time (1, 3, 5, and 16 h) were tested to identify the optimum extraction conditions. The saccharide content and composition of the extracts were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Digestibility was assayed in vitro with human salivary and porcine pancreatic alpha-amylases. Extraction yield ranged from 13.4% (without cellulase) to 39.9% (with 2% cellulase). Total carbohydrates, reducing sugars, and crude protein of the dried extracts ranged from 69.2% to 87.2%, 18.7% to 62.3%, and 7.1% to 22.3%, respectively. Mono- and disaccharides constituted more than 50% of the total carbohydrates in the extracts. Inherent oligosaccharides and those produced by cellulolysis made up less than 25%. The in vitro digestibility of the extracts by alpha-amylases was lower compared with that of the original rice bran sample and potato dextrin, which could be attributed to the increased concentrations of oligosaccharides and reducing sugars. PMID:18034713

  3. Laminin oligosaccharides play a pivotal role in cell spreading.

    PubMed

    Tanzer, M L; Giniger, M S; Chandrasekaran, S

    1993-01-01

    The basement membrane glycoprotein laminin promotes cell adhesion, spreading and neurite outgrowth. We can uncouple cell adhesion and spreading (or neurite outgrowth) when unglycosylated laminin is used as a substratum. Mouse melanoma cells, B16F1 line, readily attach to unglycosylated laminin but fail to spread once adherent. Spreading can be restored by titration with glycosylated laminin or with laminin glycopeptides. When the laminin substratum is absent in the test chambers, the cells do not adhere when either intact laminin or its glycopeptides are then added. Analyses show that these added substances are recoverable from the culture medium and do not bind to the chamber surfaces. Use of selective inhibitors which interfere with carbohydrate processing yields several glycoforms of laminin which we have isolated and examined for their ability to support cell adhesion and spreading. Laminin which is enriched in high mannose oligosaccharides is much more effective in promoting cell spreading than laminin which is enriched in hybrid oligosaccharides. These results are consistent with earlier studies which showed that ConA, which primarily recognizes mannose residues, could also uncouple cell adhesion and spreading. Although mono- and disaccharides failed to restore cell spreading, we have found that addition of various mannose oligosaccharides to adherent cells effectively reestablishes their spreading behavior. The extent of cell spreading which is achieved by the added saccharides is related to their amount, their duration of addition, and their molecular structures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8165563

  4. Separation of radiolabelled glycosaminoglycan oligosaccharides by polyacrylamide-gel electrophoresis.

    PubMed Central

    Hampson, I N; Gallagher, J T

    1984-01-01

    Glycosaminoglycan oligosaccharides generated by treatment of biosynthetically radiolabelled dermatan sulphate and hyaluronic acid with chondroitin AC lyase or testicular hyaluronidase may be resolved into a series of discrete bands by polyacrylamide-gel electrophoresis. Bands were identified by fixation in glacial acetic acid containing 20% (w/v) 2,5-diphenyloxazole followed by fluorography. The bands represented glycans which differed in size by one disaccharide unit. For the larger oligosaccharides (decasaccharides and above) of similar charge: mass ratio, there was a linear relationship between electrophoretic mobility and log Mr. However, the smaller species showed anomalous migration patterns. Consideration of the structures of the fragments produced by the different enzyme treatments suggests that copolymeric and homopolymeric oligosaccharides may be separated by polyacrylamide-gel electrophoresis. There are many potential applications of this technique, foremost amongst them being studies on the molecular size heterogeneity and patterns of enzyme-mediated depolymerization of native glycosaminoglycan chains and investigations into rates of polymer chain elongation and post-polymerization modification reactions so essential to glycosaminoglycan function. Images Fig. 1. Fig. 2. Fig. 5. Fig. 6. PMID:6477495

  5. Synthesis of heparin-like oligosaccharides on polymer supports.

    PubMed

    Ojeda, Rafael; Terentí, Olimpia; de Paz, José-Luis; Martín-Lomas, Manuel

    2004-01-01

    The biological functions of a variety of proteins are regulated by heparan sulfate glycosaminoglycans. In order to facilitate the elucidation of the molecular basis of glycosaminoglycan-protein interactions we have developed syntheses of heparin-like oligosaccharides on polymer supports. A completely stereoselective strategy previously developed by us for the synthesis of these oligosaccharides in solution has been extended to the solid phase using an acceptor-bound approach. Both a soluble polymer support and a polyethylene glycol-grafted polystyrene resin have been used and different strategies for the attachment of the acceptor to the support have been explored. The attachment of fully protected disaccharide building blocks to a soluble support through the carboxylic group of the uronic acid unit by a succinic ester linkage, the use of trichloroacetimidates as glycosylating agents and of a functionalized Merryfield type resin for the capping process allowed for the construction of hexasaccharide and octasaccharide fragments containing the structural motif of the regular region of heparin. This strategy may facilitate the synthesis of glycosaminoglycan oligosaccharides by using the required building blocks in the glycosylation sequence. PMID:15486451

  6. Milk Oligosaccharides over Time of Lactation from Different Dog Breeds

    PubMed Central

    Macias Rostami, Shirin; Bénet, Thierry; Spears, Julie; Reynolds, Arleigh; Satyaraj, Ebenezer; Sprenger, Norbert; Austin, Sean

    2014-01-01

    The partnership of humans and dogs goes back to over 10'000 years, yet relatively little is known about a dog's first extra-uterine nutrition particularly when it comes to milk oligosaccharides. We set out to identify and quantify milk oligosaccharides over the course of lactation from different dog breeds (Labrador retriever, Schnauzer and 3 Alaskan husky crossbreeds). To this end, 2 different chromatographic methods with fluorescence and mass spectrometry detection were developed and one was validated for quantification. Besides lactose and lactose-sulphate, we identified 2 different trisaccharides composed of 3 hexose units, 3′sialyllactose (3′SL), 6′sialyllactose (6′SL), 2′fucosyllactose (2′FL), and a tetrasaccharide composed of 2 hexoses, an N-acetylhexosamine and a deoxyhexose. 3′SL was present at the highest levels in milk of all dog breeds starting at around 7.5 g/L and dropping to about 1.5 g/L in the first 10 days of lactation. 6′SL was about 10 times less abundant and 2′FL and the tetrasaccharide had rather varying levels in the milk of the different breeds with the tetrasaccharide only detectable in the Alaskan husky crossbreeds. The longitudinal and quantitative data of milk oligosaccharides from different dog breeds are an important basis to further our understanding on their specific biological roles and also on the specific nutritional requirements of lactating puppies. PMID:24924915

  7. Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium, Flammeovirga sp. Strain MY04: Effects of Module Truncation on Biochemical Characteristics, Alginate Degradation Patterns, and Oligosaccharide-Yielding Properties

    PubMed Central

    Han, Wenjun; Gu, Jingyan; Cheng, Yuanyuan; Liu, Huihui; Li, Yuezhong

    2015-01-01

    Alginate lyases are important tools for oligosaccharide preparation, medical treatment, and energy bioconversion. Numerous alginate lyases have been elucidated. However, relatively little is known about their substrate degradation patterns and product-yielding properties, which is a limit to wider enzymatic applications and further enzyme improvements. Herein, we report the characterization and module truncation of Aly5, the first alginate lyase obtained from the polysaccharide-degrading bacterium Flammeovirga. Aly5 is a 566-amino-acid protein and belongs to a novel branch of the polysaccharide lyase 7 (PL7) superfamily. The protein rAly5 is an endolytic enzyme of alginate and associated oligosaccharides. It prefers guluronate (G) to mannuronate (M). Its smallest substrate is an unsaturated pentasaccharide, and its minimum product is an unsaturated disaccharide. The final alginate digests contain unsaturated oligosaccharides that generally range from disaccharides to heptasaccharides, with the tetrasaccharide fraction constituting the highest mass concentration. The disaccharide products are identified as ΔG units. While interestingly, the tri- and tetrasaccharide fractions each contain higher proportions of ΔG to ΔM ends, the larger final products contain only ΔM ends, which constitute a novel oligosaccharide-yielding property of guluronate lyases. The deletion of the noncatalytic region of Aly5 does not alter its M/G preference but significantly decreases the enzymatic activity and enzyme stability. Notably, the truncated protein accumulates large final oligosaccharide products but yields fewer small final products than Aly5, which are codetermined by its M/G preference to and size enlargement of degradable oligosaccharides. This study provides novel enzymatic properties and catalytic mechanisms of a guluronate lyase for potential uses and improvements. PMID:26519393

  8. High-performance liquid chromatography of monosaccharides and oligosaccharides in a complex biological matrix.

    PubMed

    Peelen, G O; de Jong, J G; Wevers, R A

    1991-11-01

    Analysis of oligosaccharides in complex biological matrices is hampered by the fact that oligosaccharides, closely related in structure, are difficult to separate from each other and that conventional detection procedures (refraction index and uv detection) are not specific enough for carbohydrates. Prepurification of samples by procedures like desalting or gel filtration is often used but can lead to the loss of specific oligosaccharides. We have used pellicular anion chromatography in combination with a postcolumn reaction for reducing carbohydrates based on 4-aminobenzoylhydrazide. This procedure not only detected normal mono- and oligosaccharides but N-acetylhexosamines and reducing N-acetylhexosamine containing oligosaccharides as well. A sensitivity of about 20-25 pmol for non-GlcNAc containing mono- or oligosaccharides and between 30-50 pmol for GlcNAc or oligosaccharides with GlcNAc at the reducing side was reached. The postcolumn detection was compared with pulsed amperometric detection and appeared to be more specific for mono- and oligosaccharides. Except for deproteination to protect the column, no further sample preparation was needed with this system for our application (urines). In this way pellicular anion chromatography in combination with this postcolumn reaction reaction to be a sensitive and specific HPLC procedure for analysis of monosaccharides and oligosaccharides in complex biological matrices. PMID:1799219

  9. An Update on Oligosaccharides and Their Esters from Traditional Chinese Medicines: Chemical Structures and Biological Activities

    PubMed Central

    Chen, Xiang-Yang; Wang, Ru-Feng; Liu, Bin

    2015-01-01

    A great number of naturally occurring oligosaccharides and oligosaccharide esters have been isolated from traditional Chinese medicinal plants, which are used widely in Asia and show prominent curative effects in the prevention and treatment of kinds of diseases. Numerous in vitro and in vivo experiments have revealed that oligosaccharides and their esters exhibited various activities, including antioxidant, antidepressant, cytotoxic, antineoplastic, anti-inflammatory, neuroprotective, cerebral protective, antidiabetic, plant growth-regulatory, and immunopotentiating activities. This review summarizes the investigations on the distribution, chemical structures, and bioactivities of natural oligosaccharides and their esters from traditional Chinese medicines between 2003 and 2013. PMID:25861364

  10. Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin.

    PubMed

    Takahashi, N; Hitotsuya, H; Hanzawa, H; Arata, Y; Kurihara, Y

    1990-05-15

    The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five oligosaccharides obtained are a series of compounds with xylose-containing common structural core, Xyl beta 1----2 (Man alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures containing monoantennary complex-type were characterized. (formula; see text) PMID:2335505

  11. Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin: distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

    SciTech Connect

    Green, E.D.; Baenziger, J.U.

    1988-01-05

    The asparagine-linked oligosaccharides on the pituitary glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) consist of a heterogeneous array of neutral, sulfated, sialylated, and sulfated/sialylated structures. In this study, the authors determined the relative quantities of the various asparagine-linked oligosaccharides on LH, FSH, and TSH from these three animal species. The proportions of sulfated versus sialylated oligosaccharides varied markedly among the different hormones. Both hormone- and animal species-specific differences in the types and distributions of sulfated, sialylated, and sulfated/sialylated structures were evident. In particular, LH and FSH, which are synthesized in the same pituitary cell and bear ..cap alpha..-subunits with the identical amino acid sequence, contained significantly different distributions of sulfated and sialylated oligosaccharides. For all three animal species, the ratio of sialylated to sulfated oligosaccharides differed by >10-fold for LH and FSH, with sulfated structures dominating on LH and sialylated structures on FSH. Sialylated oligosaccharides were also heterogeneous with respect to sialic acid linkage (..cap alpha..2,3 versus ..cap alpha..2,6). The differences in oligosaccharide structures among the various pituitary glycoprotein hormones as well as among the various glycosylation sites within a single hormone support the hypothesis that glycosylation may serve important functional roles in the expression and/or regulation of hormone bioactivity.

  12. Cellular effects of deoxynojirimycin analogues: inhibition of N-linked oligosaccharide processing and generation of free glucosylated oligosaccharides

    PubMed Central

    2004-01-01

    In the accompanying paper [Mellor, Neville, Harvey, Platt, Dwek and Butters (2004) Biochem. J. 381, 861–866] we treated HL60 cells with N-alk(en)yl-deoxynojirimycin (DNJ) compounds to inhibit glucosphingolipid (GSL) biosynthesis and identified a number of non-GSL-derived, small, free oligosaccharides (FOS) most likely produced due to inhibition of the oligosaccharide-processing enzymes α-glucosidases I and II. When HL60 cells were treated with concentrations of N-alk(en)ylated DNJ analogues that inhibited GSL biosynthesis completely, N-butyl- and N-nonyl-DNJ inhibited endoplasmic reticulum (ER) glucosidases I and II, but octadecyl-DNJ did not, probably due to the lack of ER lumen access for this novel, long-chain derivative. Glucosidase inhibition resulted in the appearance of free Glc1–3Man structures, which is evidence of Golgi glycoprotein endomannosidase processing of oligosaccharides with retained glucose residues. Additional large FOS was also detected in cells following a 16 h treatment with N-butyl- and N-nonyl-DNJ. When these FOS structures (>30, including >20 species not present in control cells) were characterized by enzyme digests and MALDI-TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS, all were found to be polymannose-type oligosaccharides, of which the majority were glucosylated and had only one reducing terminal GlcNAc (N-acetylglucosamine) residue (FOS-GlcNAc1), demonstrating a cytosolic location. These results support the proposal that the increase in glucosylated FOS results from enzyme-mediated cytosolic cleavage of oligosaccharides from glycoproteins exported from the ER because of misfolding or excessive retention. Importantly, the present study characterizes the cellular properties of DNJs further and demonstrates that side-chain modifications allow selective inhibition of protein and lipid glycosylation pathways. This represents the most detailed characterization of the FOS structures arising from ER

  13. Development and Application of Multidimensional HPLC Mapping Method for O-linked Oligosaccharides

    PubMed Central

    Yagi, Hirokazu; Ohno, Erina; Kondo, Sachiko; Yoshida, Atsuhiro; Kato, Koichi

    2011-01-01

    Glycosylation improves the solubility and stability of proteins, contributes to the structural integrity of protein functional sites, and mediates biomolecular recognition events involved in cell-cell communications and viral infections. The first step toward understanding the molecular mechanisms underlying these carbohydrate functionalities is a detailed characterization of glycan structures. Recently developed glycomic approaches have enabled comprehensive analyses of N-glycosylation profiles in a quantitative manner. However, there are only a few reports describing detailed O-glycosylation profiles primarily because of the lack of a widespread standard method to identify O-glycan structures. Here, we developed an HPLC mapping method for detailed identification of O-glycans including neutral, sialylated, and sulfated oligosaccharides. Furthermore, using this method, we were able to quantitatively identify isomeric products from an in vitro reaction catalyzed by N-acetylglucosamine-6O-sulfotransferases and obtain O-glycosylation profiles of serum IgA as a model glycoprotein. PMID:24970123

  14. Characterization of sugar beet pectic-derived oligosaccharides obtained by enzymatic hydrolysis.

    PubMed

    Combo, Agnan Marie Michel; Aguedo, Mario; Quiévy, Nicolas; Danthine, Sabine; Goffin, Dorothée; Jacquet, Nicolas; Blecker, Christophe; Devaux, Jacques; Paquot, Michel

    2013-01-01

    Three pectic oligosaccharides (POS) obtained by enzymatic hydrolysis of sugar beet pectin by combining endopolygalacturonase and pectinmethylesterase, were characterized using high performance liquid chromatography, thermogravimetric analysis, Fourier transform infrared spectroscopy, differential scanning calorimetry and X-ray diffraction. According to chromatographic analyses, POS are composed of mixture of polymers with different molecular weights and different galacturonic acid contents. The thermal analysis showed no major variation in thermal behavior regarding POS composition but showed that POS were more sensitive to thermal degradation than the parent pectin as well as the deesterified pectin. No change in composition of the gaseous products was obtained through TGA-FTIR analysis. The X-ray pattern of POS clearly indicated a considerable decrease in crystallinity when compared to the native pectin. Thus, thermal characterization of POS may have practical repercussions if the formulation in which POS is incorporated is submitted to a high temperature treatment. PMID:22986181

  15. A versatile and scalable strategy for glycoprofiling bifidobacterial consumption of human milk oligosaccharides

    PubMed Central

    LoCascio, Riccardo G.; Niñonuevo, Milady R.; Kronewitter, Scott R.; Freeman, Samara L.; German, J. Bruce; Lebrilla, Carlito B.; Mills, David A.

    2009-01-01

    Summary Human milk contains approximately 200 complex oligosaccharides believed to stimulate the growth and establishment of a protective microbiota in the infant gut. The lack of scalable analytical techniques has hindered the measurement of bacterial metabolism of these and other complex prebiotic oligosaccharides. An in vitro, multi‐strain, assay capable of measuring kinetics of bacterial growth and detailed oligosaccharide consumption analysis by FTICR‐MS was developed and tested simultaneously on 12 bifidobacterial strains. For quantitative consumption, deuterated and reduced human milk oligosaccharide (HMO) standards were used. A custom software suite developed in house called Glycolyzer was used to process the large amounts of oligosaccharide mass spectra automatically with 13C corrections based on de‐isotoping protocols. High growth on HMOs was characteristic of Bifidobacterium longum biovar infantis strains, which consumed nearly all available substrates, while other bifidobacterial strains tested, B. longum bv. longum, B. adolescentis, B. breve and B. bifidum, showed low or only moderate growth ability. Total oligosaccharide consumption ranged from a high of 87% for B. infantis JCM 7009 to only 12% for B. adolescentis ATCC 15703. A detailed analysis of consumption glycoprofiles indicated strain‐specific capabilities towards differential metabolism of milk oligosaccharides. This method overcomes previous limitations in the quantitative, multi‐strain analysis of bacterial metabolism of HMOs and represents a novel approach towards understanding bacterial consumption of complex prebiotic oligosaccharides. PMID:21261928

  16. Degradation of Misfolded Endoplasmic Reticulum Glycoproteins in Saccharomyces cerevisiae Is Determined by a Specific Oligosaccharide Structure

    PubMed Central

    Jakob, Claude A.; Burda, Patricie; Roth, Jürgen; Aebi, Markus

    1998-01-01

    In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal α1,2 glucose and the first α1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man8GlcNAc2 oligosaccharide. Reduced rate of degradation was mainly observed for mis- folded CPY bearing Man6GlcNAc2, Man7GlcNAc2 and Man9GlcNAc2 oligosaccharides, whereas Man8GlcNAc2 and, to a lesser extent, Man5GlcNAc2 oligosaccharides supported degradation. These results suggest a role for the Man8GlcNAc2 oligosaccharide in the degradation process. They may indicate the presence of a Man8GlcNAc2-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae. PMID:9732283

  17. Understanding the fundamental mechanism behind accumulation of oligosaccharides during high solids loading enzymatic hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During enzymatic hydrolysis of biomass, polysaccharides are cleaved by glycosyl hydrolases to soluble oligosaccharides and further hydrolyzed by ß-glucosidase, ß-xylosidase and other enzymes to monomeric sugars. However, commercial enzyme mixtures do not hydrolyze all of these oligosaccharides and v...

  18. Detection of milk oligosaccharides in plasma of infants

    PubMed Central

    Ruhaak, L. Renee; Stroble, Carol; Underwood, Mark A.; Lebrilla, Carlito B.

    2014-01-01

    Human Milk Oligosaccharides (HMO) are one of the major components of human milk. HMO are non-digestible by the human gut, where they are known to play important functions as prebiotics and decoys for binding pathogens. Moreover, it has been proposed that HMO may provide sialic acids to the infant that are important in brain development, however this would require absorption of HMO into the bloodstream. HMO have consistently been found in the urine of humans and other mammals, suggesting systemic absorption. Here we present a procedure for the profiling of milk oligosaccharides (MO) in plasma samples obtained from 13 term infants hospitalized for surgery for congenital heart disease. The method comprises protein denaturation, oligosaccharide reduction and porous graphitized carbon solid phase extraction for purification followed by analysis using nHPLC-PGC-chip-TOF-MS. Approximately 15 free MO were typically observed in the plasma of human infants, including LNT, LDFP, LNFT, 3’SL, 6’SL, 3’SLN and 6’SLN, of which the presence was confirmed using fragmentation studies. A novel third isomer of SLN, not found in human or bovine milk was also consistently detected. Differences in the free MO profiles were observed between infants that were totally formula-fed and infants that received at least some part breast milk. Our results indicate that free MO similar in structure to those found in human milk and urine are present in the blood of infants. The method and results presented here will facilitate further research toward the possible roles of free MO in the development of the infant. PMID:25059723

  19. Hyaluronidase and Hyaluronan Oligosaccharides Promote Neurological Recovery after Intraventricular Hemorrhage

    PubMed Central

    Vinukonda, Govindaiah; Dohare, Preeti; Arshad, Arslan; Zia, Muhammad T.; Panda, Sanjeet; Korumilli, Ritesh; Kayton, Robert; Hascall, Vincent C.; Lauer, Mark E.

    2016-01-01

    Intraventricular hemorrhage (IVH) in premature infants results in inflammation, arrested oligodendrocyte progenitor cell (OPC) maturation, and reduced myelination of the white matter. Hyaluronan (HA) inhibits OPC maturation and complexes with the heavy chain (HC) of glycoprotein inter-α-inhibitor to form pathological HA (HC–HA complex), which exacerbates inflammation. Therefore, we hypothesized that IVH would result in accumulation of HA, and that either degradation of HA by hyaluronidase treatment or elimination of HCs from pathological HA by HA oligosaccharide administration would restore OPC maturation, myelination, and neurological function in survivors with IVH. To test these hypotheses, we used the preterm rabbit model of glycerol-induced IVH and analyzed autopsy samples from premature infants. We found that total HA levels were comparable in both preterm rabbit pups and human infants with and without IVH, but HA receptors—CD44, TLR2, TLR4—were elevated in the forebrain of both humans and rabbits with IVH. Hyaluronidase treatment of rabbits with IVH reduced CD44 and TLR4 expression, proinflammatory cytokine levels, and microglia infiltration. It also promoted OPC maturation, myelination, and neurological recovery. HC–HA and tumor necrosis factor-stimulated gene-6 were elevated in newborns with IVH; and depletion of HC–HA levels by HA oligosaccharide treatment reduced inflammation and enhanced myelination and neurological recovery in rabbits with IVH. Hence, hyaluronidase or HA oligosaccharide treatment represses inflammation, promotes OPC maturation, and restores myelination and neurological function in rabbits with IVH. These therapeutic strategies might improve the neurological outcome of premature infants with IVH. SIGNIFICANCE STATEMENT Approximately 12,000 premature infants develop IVH every year in the United States, and a large number of survivors with IVH develop cerebral palsy and cognitive deficits. The onset of IVH induces inflammation

  20. Potent fluoro-oligosaccharide probes of adhesion in Toxoplasmosis.

    PubMed

    Allman, Sarah A; Jensen, Henrik H; Vijayakrishnan, Balakumar; Garnett, James A; Leon, Ester; Liu, Yan; Anthony, Daniel C; Sibson, Nicola R; Feizi, Ten; Matthews, Stephen; Davis, Benjamin G

    2009-10-12

    Unnatural, NMR- and MRI-active fluorinated sugar probes, designed and synthesised to bind to the pathogenic protein TgMIC1 from Toxoplasma gondii, were found to display binding potency equal to and above that of the natural ligand. Dissection of the binding mechanism and modes, including the first X-ray crystal structures of a fluoro-oligosaccharide bound to a lectin, demonstrate that it is possible to create effective fluorinated probe ligands for the study of, and perhaps intervention in, sugar-protein binding events. PMID:19750531

  1. Fluorous-assisted chemoenzymatic synthesis of heparan sulfate oligosaccharides.

    PubMed

    Cai, Chao; Dickinson, Demetria M; Li, Lingyun; Masuko, Sayaka; Suflita, Matt; Schultz, Victor; Nelson, Shawn D; Bhaskar, Ujjwal; Liu, Jian; Linhardt, Robert J

    2014-04-18

    The chemoenzymatic synthesis of heparan sulfate tetrasaccharide (1) and hexasaccharide (2) with a fluorous tag attached at the reducing end is reported. The fluorous tert-butyl dicarbonate ((F)Boc) tag did not interfere with enzymatic recognition for both elongation and specific sulfation, and flash purification was performed by standard fluorous solid-phase extraction (FSPE). Based on an (F)Boc attached disaccharide as acceptor, a series of partial N-sulfated, 6-O-sulfated heparan sulfate oligosaccharides were successfully synthesized employing fluorous techniques. PMID:24697306

  2. Hyaluronidase and Hyaluronan Oligosaccharides Promote Neurological Recovery after Intraventricular Hemorrhage.

    PubMed

    Vinukonda, Govindaiah; Dohare, Preeti; Arshad, Arslan; Zia, Muhammad T; Panda, Sanjeet; Korumilli, Ritesh; Kayton, Robert; Hascall, Vincent C; Lauer, Mark E; Ballabh, Praveen

    2016-01-20

    Intraventricular hemorrhage (IVH) in premature infants results in inflammation, arrested oligodendrocyte progenitor cell (OPC) maturation, and reduced myelination of the white matter. Hyaluronan (HA) inhibits OPC maturation and complexes with the heavy chain (HC) of glycoprotein inter-α-inhibitor to form pathological HA (HC-HA complex), which exacerbates inflammation. Therefore, we hypothesized that IVH would result in accumulation of HA, and that either degradation of HA by hyaluronidase treatment or elimination of HCs from pathological HA by HA oligosaccharide administration would restore OPC maturation, myelination, and neurological function in survivors with IVH. To test these hypotheses, we used the preterm rabbit model of glycerol-induced IVH and analyzed autopsy samples from premature infants. We found that total HA levels were comparable in both preterm rabbit pups and human infants with and without IVH, but HA receptors--CD44, TLR2, TLR4--were elevated in the forebrain of both humans and rabbits with IVH. Hyaluronidase treatment of rabbits with IVH reduced CD44 and TLR4 expression, proinflammatory cytokine levels, and microglia infiltration. It also promoted OPC maturation, myelination, and neurological recovery. HC-HA and tumor necrosis factor-stimulated gene-6 were elevated in newborns with IVH; and depletion of HC-HA levels by HA oligosaccharide treatment reduced inflammation and enhanced myelination and neurological recovery in rabbits with IVH. Hence, hyaluronidase or HA oligosaccharide treatment represses inflammation, promotes OPC maturation, and restores myelination and neurological function in rabbits with IVH. These therapeutic strategies might improve the neurological outcome of premature infants with IVH. Significance statement: Approximately 12,000 premature infants develop IVH every year in the United States, and a large number of survivors with IVH develop cerebral palsy and cognitive deficits. The onset of IVH induces inflammation of the

  3. Core Oligosaccharide of Plesiomonas shigelloides PCM 2231 (Serotype O17) Lipopolysaccharide—Structural and Serological Analysis

    PubMed Central

    Maciejewska, Anna; Lukasiewicz, Jolanta; Kaszowska, Marta; Man-Kupisinska, Aleksandra; Jachymek, Wojciech; Lugowski, Czeslaw

    2013-01-01

    The herein presented complete structure of the core oligosaccharide of lipopolysaccharide (LPS) P. shigelloides Polish Collection of Microorganisms (PCM) 2231 (serotype O17) was investigated by 1H, 13C NMR spectroscopy, mass spectrometry, chemical analyses and serological methods. The core oligosaccharide is composed of an undecasaccharide, which represents the second core type identified for P. shigelloides serotype O17 LPS. This structure is similar to that of the core oligosaccharide of P. shigelloides strains 302-73 (serotype O1) and 7-63 (serotype O17) and differs from these only by one sugar residue. Serological screening of 55 strains of P. shigelloides with the use of serum against identified core oligosaccharide conjugated with bovine serum albumin (BSA) indicated the presence of similar structures in the LPS core region of 28 O-serotypes. This observation suggests that the core oligosaccharide structure present in strain PCM 2231 could be the most common type among P. shigelloides lipopolysaccharides. PMID:23389090

  4. Disruption and eradication of P. aeruginosa biofilms using nitric oxide-releasing chitosan oligosaccharides

    PubMed Central

    Reighard, Katelyn P.; Hill, David B.; Dixon, Graham A.; Worley, Brittany; Schoenfisch, Mark H.

    2015-01-01

    Biofilm disruption and eradication were investigated as a function of nitric oxide- (NO) releasing chitosan oligosaccharide dose with results compared to control (ie non-NO-releasing) chitosan oligosaccharides and tobramycin. Quantification of biofilm expansion/contraction and multiple-particle tracking microrheology were used to assess the structural integrity of the biofilm before and after antibacterial treatment. While tobramycin had no effect on the physical properties of the biofilm, NO-releasing chitosan oligosaccharides exhibited dose-dependent behavior with biofilm degradation. Control chitosan oligosaccharides increased biofilm elasticity, indicating that the scaffold may mitigate the biofilm disrupting power of nitric oxide somewhat. The results from this study indicate that nitric oxide-releasing chitosan oligosaccharides act as dual-action therapeutics capable of eradicating and physically disrupting P. aeruginosa biofilms. PMID:26610146

  5. Protection of probiotic bacteria in synbiotic matrices

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Probiotics, like Lactobacillus acidophilus, Lactobacillus reuteri, Bifidobacterium breve, Bifidobacterium longum, when encapsulated with prebiotic fibers such as fructo-oligosaccharides (FOS), inulin (I) and pectic-oligosaccharides (POS), formed a synbiotic matrix system that protected the bacteria ...

  6. Cell surface oligosaccharide modulation during differentiation: VI. The effect of biomodulation on the senescent and neoplastic cell phenotype.

    PubMed

    Mann, P L; Busse, S C; Griffey, R H; Laubscher, K

    1992-01-01

    These investigations test the hypothesis developed previously, that there are biomolecules which control and integrate cellular differentiation. Our specific interest in cellular differentiation lies in the area of what we refer to as basal or primitive cellular differentiation mechanisms. These mechanisms are common to all cells, and are required for simple recognition and growth regulation. We have investigated two models, the IMR-90 human fetal lung fibroblast model as a representative of normal growth control, and the CG model, canine glioma cells, a transplantable growth transformed cell line. These two models represent normal, and aberrant cellular differentiation control. In previous studies we have shown that the arrangement of the cell surface oligosaccharide structure on these cell types are predictive of phenotypic transition. We have developed, and partially characterized a series of BIOMODULATORS (BM) which delay the onset of display of neoplastic cells. Three classes of BIOMODULATOR have been explored; (1) a large molecular weight natural product (25-35 kDa), PokeWeed Mitogen (PWM); (2) a small molecular weight natural product (500 Da) Cellular Activator and Differentiator (CAD) and a number of natural and synthetic analogs; and (3) an indolizidine alkaloid natural product, Swainsonine (Sw) which has a known cellular target (oligosaccharide biosynthesis). Preliminary data is presented which structurally links some of these BIOMODULATORS in terms of their effective stereochemistry. These BIOMODULATORS, when used before PDL 38, prevent the cell surface oligosaccharide display changes typical of morphological senescence and delay their onset to PDL 100 or more. These BIOMODULATORS also appear to have regulatory effects on the neoplastic cell models. This re-regulation results in increases in generation time and an increase in the ability of these cells to be recognized by cytotoxic lymphocytes. Proton NMR linewidth measurements of the fraction of 'bound

  7. Carbohydrate-deficient glycoprotein syndrome: not an N-linked oligosaccharide processing defect, but an abnormality in lipid-linked oligosaccharide biosynthesis?

    PubMed Central

    Powell, L D; Paneerselvam, K; Vij, R; Diaz, S; Manzi, A; Buist, N; Freeze, H; Varki, A

    1994-01-01

    The carbohydrate-deficient glycoprotein syndrome (CDGS) is a developmental disease associated with an abnormally high isoelectric point of serum transferrin. Carbohydrate analyses of this glycoprotein initially suggested a defect in N-linked oligosaccharide processing, although more recent studies indicate a defect in the attachment of these sugar chains to the protein. We studied both serum glycoproteins and fibroblast-derived [2-3H]mannose-labeled oligosaccharides from CDGS patients and normal controls. While there was a decrease in the glycosylation of serum glycoproteins of affected individuals, differences were not seen in either monosaccharide composition or oligosaccharide structures. The lectin-binding profiles of glycopeptides from [2-3H]-mannose-labeled fibroblasts were likewise indistinguishable. However, the incorporation of [2-3H]mannose into both glycoproteins and the dolichol-linked oligosaccharide precursor was significantly reduced. Thus, at least in some patients, CDGS is not due to a defect in processing of N-linked oligosaccharides, but rather to defective synthesis and transfer of nascent dolichol-linked oligosaccharide precursors. This abnormality could result in both a failure to glycosylate some sites on some proteins, as well as secondary abnormalities in overall glycoprotein processing and/or function. PMID:7962535

  8. Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin: structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

    SciTech Connect

    Green, E.D.; Baenziger, J.U.

    1988-01-05

    The authors have elucidated the structures of the anionic asparagine-linked oligosaccharides present on the glycoprotein hormones lutropin (luteinizing hormone), follitropin (follicle-stimulating hormone), and thyrotropin (thyroid-stimulating hormone). Purified hormones, isolated from bovine, ovine, and human pituitaries, were digested with N-glycanase, and the released oligosaccharides were reduced with NaB(/sup 3/H)/sub 4/. The /sup 3/H-labeled oligosaccharides from each hormone were then fractionated by anion-exchange high performance liquid chromatography (HPLC) into populations differing in the number of sulfate and/or sialic acid moieties. The sulfated, sialylated, and sulfated/sialylated structures, which together comprised 67-90% of the asparagine-linked oligosaccharides on the pituitary glycoprotein hormones, were highly heterogeneous and displayed hormone- as well as animal species-specific features. A previously uncharacterized dibranched oligosaccharide, bearing one residue each of sulfate and sialic acid, was found on all of the hormones except bovine lutropin. In this study, they describe the purification and detailed structural characterizations of the sulfated, sialylated, and sulfated/sialylated oligosaccharides found on lutropin, follitropin, and thyrotropin from several animal species.

  9. The CebE/MsiK Transporter is a Doorway to the Cello-oligosaccharide-mediated Induction of Streptomyces scabies Pathogenicity.

    PubMed

    Jourdan, Samuel; Francis, Isolde Maria; Kim, Min Jung; Salazar, Joren Jeico C; Planckaert, Sören; Frère, Jean-Marie; Matagne, André; Kerff, Frédéric; Devreese, Bart; Loria, Rosemary; Rigali, Sébastien

    2016-01-01

    Streptomyces scabies is an economically important plant pathogen well-known for damaging root and tuber crops by causing scab lesions. Thaxtomin A is the main causative agent responsible for the pathogenicity of S. scabies and cello-oligosaccharides are environmental triggers that induce the production of this phytotoxin. How cello-oligosaccharides are sensed or transported in order to induce the virulent behavior of S. scabies? Here we report that the cellobiose and cellotriose binding protein CebE, and MsiK, the ATPase providing energy for carbohydrates transport, are the protagonists of the cello-oligosaccharide mediated induction of thaxtomin production in S. scabies. Our work provides the first example where the transport and not the sensing of major constituents of the plant host is the central mechanism associated with virulence of the pathogen. Our results allow to draw a complete pathway from signal transport to phytotoxin production where each step of the cascade is controlled by CebR, the cellulose utilization regulator. We propose the high affinity of CebE to cellotriose as possible adaptation of S. scabies to colonize expanding plant tissue. Our work further highlights how genes associated with primary metabolism in nonpathogenic Streptomyces species have been recruited as basic elements of virulence in plant pathogenic species. PMID:27250236

  10. The CebE/MsiK Transporter is a Doorway to the Cello-oligosaccharide-mediated Induction of Streptomyces scabies Pathogenicity

    PubMed Central

    Jourdan, Samuel; Francis, Isolde Maria; Kim, Min Jung; Salazar, Joren Jeico C.; Planckaert, Sören; Frère, Jean-Marie; Matagne, André; Kerff, Frédéric; Devreese, Bart; Loria, Rosemary; Rigali, Sébastien

    2016-01-01

    Streptomyces scabies is an economically important plant pathogen well-known for damaging root and tuber crops by causing scab lesions. Thaxtomin A is the main causative agent responsible for the pathogenicity of S. scabies and cello-oligosaccharides are environmental triggers that induce the production of this phytotoxin. How cello-oligosaccharides are sensed or transported in order to induce the virulent behavior of S. scabies? Here we report that the cellobiose and cellotriose binding protein CebE, and MsiK, the ATPase providing energy for carbohydrates transport, are the protagonists of the cello-oligosaccharide mediated induction of thaxtomin production in S. scabies. Our work provides the first example where the transport and not the sensing of major constituents of the plant host is the central mechanism associated with virulence of the pathogen. Our results allow to draw a complete pathway from signal transport to phytotoxin production where each step of the cascade is controlled by CebR, the cellulose utilization regulator. We propose the high affinity of CebE to cellotriose as possible adaptation of S. scabies to colonize expanding plant tissue. Our work further highlights how genes associated with primary metabolism in nonpathogenic Streptomyces species have been recruited as basic elements of virulence in plant pathogenic species. PMID:27250236

  11. Significance of galactinol and raffinose family oligosaccharide synthesis in plants

    PubMed Central

    Sengupta, Sonali; Mukherjee, Sritama; Basak, Papri; Majumder, Arun L.

    2015-01-01

    Abiotic stress induces differential expression of genes responsible for the synthesis of raffinose family of oligosaccharides (RFOs) in plants. RFOs are described as the most widespread D-galactose containing oligosaccharides in higher plants. Biosynthesis of RFOs begin with the activity of galactinol synthase (GolS; EC 2.4.1.123), a GT8 family glycosyltransferase that galactosylates myo-inositol to produce galactinol. Raffinose and the subsequent higher molecular weight RFOs (Stachyose, Verbascose, and Ajugose) are synthesized from sucrose by the subsequent addition of activated galactose moieties donated by Galactinol. Interestingly, GolS, the key enzyme of this pathway is functional only in the flowering plants. It is thus assumed that RFO synthesis is a specialized metabolic event in higher plants; although it is not known whether lower plant groups synthesize any galactinol or RFOs. In higher plants, several functional importance of RFOs have been reported, e.g., RFOs protect the embryo from maturation associated desiccation, are predominant transport carbohydrates in some plant families, act as signaling molecule following pathogen attack and wounding and accumulate in vegetative tissues in response to a range of abiotic stresses. However, the loss-of-function mutants reported so far fail to show any perturbation in those biological functions. The role of RFOs in biotic and abiotic stress is therefore still in debate and their specificity and related components remains to be demonstrated. The present review discusses the biology and stress-linked regulation of this less studied extension of inositol metabolic pathway. PMID:26379684

  12. De Novo Sequencing of Complex Mixtures of Heparan Sulfate Oligosaccharides.

    PubMed

    Huang, Rongrong; Zong, Chengli; Venot, Andre; Chiu, Yulun; Zhou, Dandan; Boons, Geert-Jan; Sharp, Joshua S

    2016-05-17

    Here, we describe the first sequencing method of a complex mixture of heparan sulfate tetrasaccharides by LC-MS/MS. Heparin and heparan sulfate (HS) are linear polysaccharides that are modified in a complex manner by N- and O-sulfation, N-acetylation, and epimerization of the uronic acid. Heparin and HS are involved in various essential cellular communication processes. The structural analysis of these glycosaminoglycans is challenging due to the lability of their sulfate groups, the high heterogeneity of modifications, and the epimerization of the uronic acids. While advances in liquid chromatography (LC) and mass spectrometry (MS) have enabled compositional profiling of HS oligosaccharide mixtures, online separation and detailed structural analysis of isomeric and epimeric HS mixtures has not been achieved. Here, we report the development and evaluation of a chemical derivatization and tandem mass spectrometry method that can separate and identify isomeric and epimeric structures from complex mixtures. A series of well-defined synthetic HS tetrasaccharides varying in sulfation patterns and uronic acid epimerization were analyzed by chemical derivatization and LC-MS/MS. These synthetic compounds made it possible to establish relationships between HS structure, chromatographic behavior and MS/MS fragmentation characteristics. Using the analytical characteristics determined through the analysis of the synthetic HS tetrasaccharide standards, an HS tetrasacharide mixture derived from natural sources was successfully sequenced. This method represents the first sequencing of complex mixtures of HS oligosaccharides, an essential milestone in the analysis of structure-function relationships of these carbohydrates. PMID:27087275

  13. Recent advances on separation and characterization of human milk oligosaccharides.

    PubMed

    Mantovani, Veronica; Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola

    2016-06-01

    Free human milk oligosaccharides (HMOs) are unique due to their highly complex nature and important emerging biological and protective functions during early life such as prebiotic activity, pathogen deflection, and epithelial and immune cell modulation. Moreover, four genetically determined heterogeneous HMO secretory groups are known to be based on their structure and composition. Over the years, several analytical techniques have been applied to characterize and quantitate HMOs, including nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), high pH anion-exchange chromatography, off-line and on-line mass spectrometry (MS), and capillary electrophoresis (CE). Even if these techniques have proven to be efficient and simple, most glycans have no significant UV absorption and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved chromatographic/electrophoretic profile. Consequently, the analysis by HPLC/CE of derivatized milk oligosaccharides with different chromophoric active tags has been developed. However, UV or fluorescence detection does not provide specific structural information and this is a key point in particular related to the highly complex nature of the milk glycan mixtures. As a consequence, for a specific determination of complex mixtures of oligomers, analytical separation is usually required with evaluation by means of MS, which has been successfully applied to HMOs, resulting in efficient compositional analysis and profiling in various milk samples. This review aims to give an overview of the current state-of-the-art techniques used in HMO analysis. PMID:26801168

  14. Chemical characterization of oligosaccharides in the milk of six species of New and Old World monkeys.

    PubMed

    Goto, Kohta; Fukuda, Kenji; Senda, Akitsugu; Saito, Tadao; Kimura, Kazumasa; Glander, Kenneth E; Hinde, Katie; Dittus, Wolfgang; Milligan, Lauren A; Power, Michael L; Oftedal, Olav T; Urashima, Tadasu

    2010-10-01

    Human and great ape milks contain a diverse array of milk oligosaccharides, but little is known about the milk oligosaccharides of other primates, and how they differ among taxa. Neutral and acidic oligosaccharides were isolated from the milk of three species of Old World or catarrhine monkeys (Cercopithecidae: rhesus macaque (Macaca mulatta), toque macaque (Macaca sinica) and Hamadryas baboon (Papio hamadryas)) and three of New World or platyrrhine monkeys (Cebidae: tufted capuchin (Cebus apella) and Bolivian squirrel monkey (Saimiri boliviensis); Atelidae: mantled howler (Alouatta palliata)). The milks of these species contained 6-8% total sugar, most of which was lactose: the estimated ratio of oligosaccharides to lactose in Old World monkeys (1:4 to 1:6) was greater than in New World monkeys (1:12 to 1:23). The chemical structures of the oligosaccharides were determined mainly by (1)H-NMR spectroscopy. Oligosaccharides containing the type II unit (Gal(β1-4)GlcNAc) were found in the milk of the rhesus macaque, toque macaque, Hamadryas baboon and tufted capuchin, but oligosaccharides containing the type I unit (Gal(β1-3)GlcNAc), which have been found in human and many great ape milks, were absent from the milk of all species studied. Oligosaccharides containing Lewis x (Gal(β1-4)[Fuc(α1-3)]GlcNAc) and 3-fucosyl lactose (3-FL, Gal(β1-4)[Fuc(α1-3)]Glc) were found in the milk of the three cercopithecid monkey species, while 2-fucosyl lactose (5'-FL, Fuc(α1-2)Gal(β1-4)Glc) was absent from all species studied. All of these milks contained acidic oligosaccharides that had N-acetylneuraminic acid as part of their structures, but did not contain oligosaccharides that had N-glycolylneuraminic acid, in contrast to the milk or colostrum of great apes which contain both types of acidic oligosaccharides. Two GalNAc-containing oligosaccharides, lactose 3'-O-sulfate and lacto-N-novopentaose I (Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc) were found only in the milk

  15. Site-specific analysis of N-linked oligosaccharides of recombinant lysosomal arylsulfatase A produced in different cell lines.

    PubMed

    Schröder, Stephan; Matthes, Frank; Hyden, Pia; Andersson, Claes; Fogh, Jens; Müller-Loennies, Sven; Braulke, Thomas; Gieselmann, Volkmar; Matzner, Ulrich

    2010-02-01

    Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency of the lysosomal enzyme arylsulfatase A (ASA). Enzyme replacement therapy (ERT) is a therapeutic option for MLD and other lysosomal disorders. This therapy depends on N-linked oligosaccharide-mediated delivery of intravenously injected recombinant enzyme to the lysosomes of patient cells. Because of the importance of N-linked oligosaccharide side chains in ERT, we examined the composition of the three N-linked glycans of four different recombinant ASAs in a site-specific manner. Depending on the culture conditions and the cell line expressing the enzyme, we detected a high variability of the high-mannose-type N-glycans which prevail at all glycosylation sites. Our data show that the composition of the glycans is largely determined by substantial trimming in the medium. The susceptibility for trimming is different for the glycans at the three N-glycosylation sites. Interestingly, which of the glycans is most susceptible to trimming also depends on production conditions. CHO cells cultured under bioreactor conditions yielded recombinant ASA with the most preserved N-glycan structures, the highest mannose-6-phosphate content and the highest similarity to non-recombinant enzyme. Notably, roughly one-third of the N-glycans released from the three glycosylation sites were fucosylated. In the last years, numerous recombinant lysosomal enzymes were used for preclinical ERT trials. Our data show that the oligosaccharide structures were very different in these trials making it difficult to draw common conclusions from the various investigations. PMID:19864504

  16. Assignment of the Stereochemistry and Anomeric Configuration of Sugars within Oligosaccharides Via Overlapping Disaccharide Ladders Using MSn

    NASA Astrophysics Data System (ADS)

    Konda, Chiharu; Londry, Frank A.; Bendiak, Brad; Xia, Yu

    2014-08-01

    A systematic approach is described that can pinpoint the stereo-structures (sugar identity, anomeric configuration, and location) of individual sugar units within linear oligosaccharides. Using a highly modified mass spectrometer, dissociation of linear oligosaccharides in the gas phase was optimized along multiple-stage tandem dissociation pathways (MSn, n = 4 or 5). The instrument was a hybrid triple quadrupole/linear ion trap mass spectrometer capable of high-efficiency bidirectional ion transfer between quadrupole arrays. Different types of collision-induced dissociation (CID), either on-resonance ion trap or beam-type CID could be utilized at any given stage of dissociation, enabling either glycosidic bond cleavages or cross-ring cleavages to be maximized when wanted. The approach first involves optimizing the isolation of disaccharide units as an ordered set of overlapping substructures via glycosidic bond cleavages during early stages of MSn, with explicit intent to minimize cross-ring cleavages. Subsequently, cross-ring cleavages were optimized for individual disaccharides to yield key diagnostic product ions ( m/ z 221). Finally, fingerprint patterns that establish stereochemistry and anomeric configuration were obtained from the diagnostic ions via CID. Model linear oligosaccharides were derivatized at the reducing end, allowing overlapping ladders of disaccharides to be isolated from MSn. High confidence stereo-structural determination was achieved by matching MSn CID of the diagnostic ions to synthetic standards via a spectral matching algorithm. Using this MSn ( n = 4 or 5) approach, the stereo-structures, anomeric configurations, and locations of three individual sugar units within two pentasaccharides were successfully determined.

  17. MALDI Q-TOF CID MS for Diagnostic Ion Screening of Human Milk Oligosaccharide Samples

    PubMed Central

    Jovanović, Marko; Tyldesley-Worster, Richard; Pohlentz, Gottfried; Peter-Katalinić, Jasna

    2014-01-01

    Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1–3 or 1–4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted. PMID:24743894

  18. Preparation and characterisation of the oligosaccharides derived from Chinese water chestnut polysaccharides.

    PubMed

    Wu, Sheng-Jun; Yu, Lin

    2015-08-15

    Hydrogen peroxide (H2O2) is a strong oxidant that cleaves glycosidic bonds in polysaccharides. In this study, the oligosaccharides were prepared by removing the starch from Chinese water chestnuts through hydrolysis using α-amylase and then hydrolysing the remaining polysaccharides with H2O2, during which the oligosaccharide yield was monitored. The yield of oligosaccharide was affected by reaction time, temperature, and H2O2 concentration. Extended reaction times, high temperatures, and high H2O2 concentrations decreased oligosaccharide yield. Under optimum conditions (i.e., reaction time of 4h, reaction temperature of 80°C, and 2.5% H2O2 concentration), the maximum oligosaccharide yield was 3.91%. The oligosaccharides derived from Chinese water chestnuts polysaccharides exhibited strong hydroxyl and 2,2-diphenyl-β-picrylhydrazyl radical scavenging activity when applied at a concentration of 100 μg/mL. The results indicate that the oligosaccharides derived from Chinese water chestnuts polysaccharides possessed good antioxidant properties and can be developed as a new dietary supplement and functional food. PMID:25794714

  19. Detection and quantitation of low abundance oligosaccharides in recombinant monoclonal antibodies.

    PubMed

    Ponniah, Gomathinayagam; Nowak, Christine; Gonzalez, Nidia; Miano, Dino; Liu, Hongcheng

    2015-03-01

    Oligosaccharides are critical for structural integrity, stability, and biological functions of recombinant monoclonal antibodies. It is relatively easy to characterize, quantify, and determine the impact of major glycoforms. While challenging to detect and quantify, certain low abundance oligosaccharides are highly relevant to the stability and functions of recombinant monoclonal antibodies. Methods were established in this study based on enzymatic digestion to consolidate peaks of the same type of oligosaccharides by removing heterogeneity and thus increase detectability of low abundance peaks. Endo H was used to collapse high mannose oligosaccharides to a single peak of GlcNAc for ease of detection and quantitation. β-Galactosidase and β-N-acetylhexosaminidase were used to convert complex oligosaccharides into two peaks containing either GlcNAc2Man3Fuc or GlcNAc2Man3, which simplified the chromatograms and data analysis. More importantly, low abundance hybrid oligosaccharides can only be detected and qualified after β-galactosidase and β-N-acetylhexosaminidase digestion. Detection and quantitation of low abundance oligosaccharides can also be achieved using a combination of all three enzymes. These methods can be applied to the development of recombinant monoclonal antibody therapeutics. PMID:25647617

  20. Insulin-induced phospho-oligosaccharide stimulates amino acid transport in isolated rat hepatocytes.

    PubMed Central

    Varela, I; Avila, M; Mato, J M; Hue, L

    1990-01-01

    The ability of the insulin-induced phospho-oligosaccharide to stimulate amino acid transport was studied in isolated rat hepatocytes. At low alpha-aminoisobutyric acid concentrations (0.1 mM), both 100 nM-insulin and 10 microM-phospho-oligosaccharide doubled amino acid uptake after 2 h of incubation. This stimulation was prevented by 0.1 mM-cycloheximide or 5 micrograms of actinomycin D/ml, indicating that the phospho-oligosaccharide, like insulin, was acting via the synthesis of a high-affinity transport component. The effects of the phospho-oligosaccharide and of insulin were blocked by Ins2P (2.5 mM), but not by myo-inositol, inositol hexaphosphoric acid or several monosaccharides such as mannose, glucosamine and galactose. Both the temporal effect on amino acid entry and the extent of stimulation of this process by the phospho-oligosaccharide indicate that this molecule mimics, and may mediate, some of the long-term actions of insulin. However, the effects of phospho-oligosaccharide and insulin were not exactly the same, since the effect of insulin, but not of the phospho-oligosaccharide, was additive with that of glucagon. PMID:2185744

  1. Structural requirements for sulfation of asparagine-linked oligosaccharides of lutropin.

    PubMed Central

    Green, E D; Morishima, C; Boime, I; Baenziger, J U

    1985-01-01

    Human and bovine pituitary glycoprotein hormones (lutropin, follitropin, and thyrotropin) contain varying amounts of N-acetylgalactosamine and sulfate. The sulfate on asparagine-linked oligosaccharides of bovine lutropin (bLH) is present exclusively on GalNAc in the sequence GalNAc(beta 1-4)GlcNAc(beta 1-2)Man alpha. We have examined the structural requirements for sulfation of bLH oligosaccharides by using a reconstituted cell-free system. After cleavage from the protein, oligosaccharides containing the sequence GalNAc(beta 1-4)Glc-NAc(beta 1-2)Man alpha were sulfated by enzymes in pituitary membranes. Addition of one or two sulfates was observed, depending upon the number of GalNAc acceptor sites on the oligosaccharide. Neither GalNAc alone nor oligosaccharides devoid of GalNAc were sulfated. Membranes from placenta or liver did not sulfate oligosaccharides released from bLH, indicating that the sulfating activity is pituitary-specific. The lack of peptide dependence for sulfation, in conjunction with the oligosaccharide specificity, suggests that the sequence GalNAc(beta 1-4)GlcNAc(beta 1-2)Man alpha contains the recognition signal for the sulfotransferase(s). PMID:3865202

  2. Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins.

    PubMed

    Tsuda, E; Goto, M; Murakami, A; Akai, K; Ueda, M; Kawanishi, G; Takahashi, N; Sasaki, R; Chiba, H; Ishihara, H

    1988-07-26

    The structures of the N-linked oligosaccharides of the urinary erythropoietin (u-EPO) purified from urine of aplastic anemic patients were analyzed and compared with those for recombinant erythropoietin (r-EPO) prepared with baby hamster kidney (BHK) cells. Asparagine-linked neutral oligosaccharides were released from each EPO protein by N-oligosaccharide glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high-performance liquid chromatography (HPLC) on an ODS silica column. More than 8 and 13 kinds of oligosaccharide fractions for u-EPO and r-EPO (BHK), respectively, were completely separated by the one-step HPLC procedure. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amide-silica column. Furthermore, high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy and methylation analyses were carried out in the case of r-EPO (BHK).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3179269

  3. Simultaneous synthesis of mixtures of lactulose and galacto-oligosaccharides and their selective fermentation.

    PubMed

    Guerrero, Cecilia; Vera, Carlos; Acevedo, Fernando; Illanes, Andrés

    2015-09-10

    Lactulose and galacto-oligosaccharides (GOS) are well recognized prebiotics derived from lactose. In the synthesis of lactulose with β-galactosidases GOS are also produced, but the ratio of lactulose and GOS in the product can be tuned at will, depending on the operation conditions, so to obtain an optimal product distribution in terms of prebiotic potential. The selectivity of fermentation of each carbohydrate alone as well as mixtures of both was determined using pH-controlled anaerobic batch cultures with faecal inoculum. Within the experimental range considered, lactulose/GOS molar ratio of 4 resulted in the highest selectivity for Bifidobacterium and Lactobacillus/Enterococcus, so this ratio was selected as the target for the synthesis of lactulose from fructose and lactose with Aspergillus oryzae β-galactosidase. Synthesis was optimized using response surface methodology, considering temperature, initial concentrations of acceptor sugars and fructose/lactose molar ratio as key variables, with the aim of maximizing lactulose yield at the optimal product distribution in terms of prebiotic potential (lactulose/GOS molar ratio of 4). Under optimal conditions (50°C, 50%w/w total initial concentrations of sugars and fructose/lactose molar ratio of 6.44), lactulose yield of 0.26g of lactulose produced per g of initial lactose was obtained at the optimal product distribution. PMID:26080080

  4. A comparative study of the size-heterogeneous high mannose oligosaccharides of some insect vitellins.

    PubMed

    Nordin, J H; Gochoco, C H; Wojchowski, D M; Kunkel, J G

    1984-01-01

    Comparative studies of the carbohydrate component from vitellins of the cockroaches Blattella germanica, Blaberus discoidalis, Periplaneta americana and Simploce capitata and the locust Locusta migratoria have been conducted. Chemical, enzymatic and chromatographic analyses show that each vitellin contains variably processed high mannose type oligosaccharides. While all have a common size range they occur as two distinct classes based on the proportion of individual saccharides present. Oligosaccharide size distribution is not a characteristic of an individual animal but of the species. Because oligosaccharide heterogeneity also occurs in B. germanica vitellogenin (the hemolymph precursor of vitellin), it does not result from structural changes during or after its uptake by the egg. PMID:6509925

  5. Human Milk Oligosaccharides: Evolution, Structures and Bioselectivity as Substrates for Intestinal Bacteria

    PubMed Central

    German, J. Bruce; Freeman, Samara L.; Lebrilla, Carlito B.; Mills, David A.

    2010-01-01

    Human milk contains a high concentration of diverse soluble oligosaccharides that are carbohydrate polymers formed from a relatively small number of different monosaccharides. Novel methods combining liquid chromatography with high resolution mass spectrometry have identified approximately 200 unique oligosaccharides structures varying from 3 to 22 sugars. The increasing structural complexity of oligosaccharides follows the general pattern of mammalian and primate evolution though the concentration and diversity of these structures in homo sapiens are strikingly more abundant. There is also considerable diversity among different human mothers in the structures of oligosaccharides. Milks from randomly selected mothers contain as few as 23 and as many as 130 different oligosaccharides. The functional implications of this diversity are not yet known. Despite the role of milk to serve as a sole nutrient source for mammalian infants, the majority of the oligosaccharides in milk are not digestible by human infants. This apparent paradox raises the obvious questions about the functions of these oligosaccharides and how their diverse molecular structures affect their functions. The nutritional function that is most frequently attributed to milk oligosaccharides is to serve as prebiotics –a form of indigestible carbohydrate that is selectively fermented by desirable gut microflora. This function was tested by purifying human milk oligosaccharides and providing these as the sole carbon source to various intestinal bacteria. Indeed, the selectively of providing the complex mixture of oligosaccharides pooled from dozens of human milk samples is remarkable. Among a variety of Bifidobacteria tested only Bifidobacteria longum biovar infantis was able to grow extensively on human milk oligosaccharides as sole carbon source. The genomic sequence of this strain revealed approximately 700 genes that are unique to infantis, including a variety of co-regulated glycosidases, relative

  6. High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis1

    PubMed Central

    Laremore, Tatiana N.; Ly, Mellisa; Solakyildirim, Kemal; Zagorevski, Dmitri V.; Linhardt, Robert J.

    2010-01-01

    Separation of milligram amounts of heparin oligosaccharides ranging in degree of polymerization from 4 to 32 is achieved within 6 hours using continuous-elution polyacrylamide gel electrophoresis (CE-PAGE) on commercially available equipment. The purity and structural integrity of CE-PAGE-separated oligosaccharides are confirmed by strong-anion exchange high-pressure liquid chromatography, electrospray ionization Fourier transform mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure-function relationship studies. PMID:20211145

  7. Can an ancestral condition for milk oligosaccharides be determined? Evidence from the Tasmanian echidna (Tachyglossus aculeatus setosus).

    PubMed

    Oftedal, Olav T; Nicol, Stewart C; Davies, Noel W; Sekii, Nobuhiro; Taufik, Epi; Fukuda, Kenji; Saito, Tadao; Urashima, Tadasu

    2014-09-01

    The monotreme pattern of egg-incubation followed by extended lactation represents the ancestral mammalian reproductive condition, suggesting that monotreme milk may include saccharides of an ancestral type. Saccharides were characterized from milk of the Tasmanian echidna Tachyglossus aculeatus setosus. Oligosaccharides in pooled milk from late lactation were purified by gel filtration and high-performance liquid chromatography using a porous graphitized carbon column and characterized by (1)H NMR spectroscopy; oligosaccharides in smaller samples from early and mid-lactation were separated by ultra-performance liquid chromatography and characterized by negative electrospray ionization mass spectrometry (ESI-MS) and tandem collision mass spectroscopy (MS/MS) product ion patterns. Eight saccharides were identified by (1)H NMR: lactose, 2'-fucosyllactose, difucosyllactose (DFL), B-tetrasaccharide, B-pentasaccharide, lacto-N-fucopentaose III (LNFP3), 4-O-acetyl-3'-sialyllactose [Neu4,5Ac(α2-3)Gal(β1-4)Glc] and 4-O-acetyl-3'-sialyl-3-fucosyllactose [Neu4,5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]Glc]. Six of these (all except DFL and LNFP3) were present in early and mid-lactation per ESI-MS, although some at trace levels. Four additional oligosaccharides examined by ESI-MS and MS/MS are proposed to be 3'-sialyllactose [Neu5Ac(α2-3)Gal(β1-4)Glc], di-O-acetyl-3'-sialyllactose [Neu4,5,UAc3(α2-3)Gal(β1-4)Glc where U = 7, 8 or 9], 4-O-acetyl-3'-sialyllactose sulfate [Neu4,5Ac(α2-3)Gal(β1-4)GlcS, where position of the sulfate (S) is unknown] and an unidentified 800 Da oligosaccharide containing a 4-O-acetyl-3'-sialyllactose core. 4-O-acetyl-3'-sialyllactose was the predominant saccharide at all lactation stages. 4-O-Acetylation is known to protect sialyllactose from bacterial sialidases and may be critical to prevent microbial degradation on the mammary areolae and/or in the hatchling digestive tract so that sialyllactose can be available for enterocyte uptake. The ability to

  8. Oligosaccharides derived from bovine articular cartilage keratan sulfates after keratanase II digestion: implications for keratan sulfate structural fingerprinting.

    PubMed

    Brown, G M; Huckerby, T N; Morris, H G; Abram, B L; Nieduszynski, I A

    1994-04-26

    Keratan sulfate chains were isolated from bovine articular cartilage (6-8-year-old animals) and digested with keratanase II, an endo-beta-N-acetylglucosaminidase [Nakazawa, K., Ito, M., Yamagata, T., & Suzuki, S. (1989) in Keratan Sulphate: Chemistry, Biology and Chemical Pathology (Greiling, H., & Scott, J. E., Eds.) pp 99-110, The Biochemical Society, London]. Twenty-five borohydride-reduced oligosaccharides were purified chromatographically and characterized by one- and two-dimensional NMR spectroscopy. From the structures of these oligosaccharides the following conclusions can be drawn about the mode of action of keratanase II: (1) The enzyme cleaves the beta (1-->3)-glycosidic bond between 6-O-sulfated N-acetyl-glucosamine and galactose, the major products being mono- and disulfated disaccharides. (2) Larger oligosaccharides containing keratanase II susceptible bonds are produced which are resistant to further degradation, e.g., tetrasaccharides from the sulfated poly(N-acetyllactosamine) repeat sequence, fucose-containing penta- and hexasaccharides, and hexa- and heptasaccharides from the linkage region. (3) The enzyme cleaves the beta (1-->3)-glycosidic bond of a fucosylated 6-O-sulfated N-acetylglucosamine. (4) Sialic acid-containing capping fragments are always recovered as pentasaccharides, despite the presence of an apparently susceptible bond. Two new elements of skeletal keratan sulfate structure, namely, the highly sulfated cap NeuAc alpha 2-3Gal(6S) beta 1-4GlcNAc (6S) beta 1-3Gal(6S) beta 1-4GlcNAc (6S)-ol and the difucosylated sequence Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)-ol, have been identified. A structural model for articular cartilage keratan sulfate is proposed. The potential of the enzyme keratanase II for the structural fingerprinting of subnanogram quantities both of keratan sulfates and of sulfated oligosaccharide selectin ligands is discussed. PMID:8161543

  9. Breast Milk Oligosaccharides: Structure-Function Relationships in the Neonate

    PubMed Central

    Smilowitz, Jennifer T.; Lebrilla, Carlito B.; Mills, David A.; German, J. Bruce; Freeman, Samara L.

    2015-01-01

    In addition to providing complete postnatal nutrition, breast milk is a complex biofluid that delivers bioactive components for the growth and development of the intestinal and immune systems. Lactation is a unique opportunity to understand the role of diet in shaping the intestinal environment including the infant microbiome. Of considerable interest is the diversity and abundance of milk glycans that are energetically costly for the mammary gland to produce yet indigestible by infants. Milk glycans comprise free oligosaccharides, glycoproteins, glycopeptides, and glycolipids. Emerging technological advances are enabling more comprehensive, sensitive, and rapid analyses of these different classes of milk glycans. Understanding the impact of inter- and intraindividual glycan diversity on function is an important step toward interventions aimed at improving health and preventing disease. This review discusses the state of technology for glycan analysis and how specific structure-function knowledge is enhancing our understanding of early nutrition in the neonate. PMID:24850388

  10. PITOMBA: Parameter Interface for Oligosaccharide Molecules Based on Atoms.

    PubMed

    Rusu, Victor H; Baron, Riccardo; Lins, Roberto D

    2014-11-11

    A novel four-bead coarse-grained (CG) model for carbohydrates denoted PITOMBA was devised using a bottom-up approach based on the atomistic GROMOS 53A6GLYC force field and on experimental thermodynamical data. The model was developed to be used in conjunction with the SPC CG water model (J. Chem. Phys. 2011, 134, 084110) and the GROMOS force field functional form. Explicit electrostatic interactions are considered by assigning point charges to each CG bead. Validation of the model is presented to a variety of structural and thermodynamic properties for mono- and oligosaccharides in solution. In addition, the model development philosophy allows for prompt extensions to include hexopyranose chains with diverse glycosidic linkages and branches. PMID:26584387

  11. Plant growth-promoting oligosaccharides produced from tomato waste.

    PubMed

    Suzuki, Toshisada; Tomita-Yokotani, Kaori; Tsubura, Hirokazu; Yoshida, Shigeki; Kusakabe, Isao; Yamada, Kosumi; Miki, Yoichi; Hasegawa, Koji

    2002-01-01

    Tomato juice waste was hydrolyzed with acid. Tomato juice waste (500 g; wet weight) was heated with 0.5 N HCl (2.5 l) at 70 degrees C for 4 h. After neutralization, the growth-promoting extracts (300 g; dry weight) in the plants were produced from the tomato waste. The acid extract significantly promoted the growth of cockscomb (Celosia argentea L.) and tomato (Lycopersicon esculentum L.) seedlings. We have recognized potent plant growth-promoting substances in the acid extract from tomato waste. The most effective components in the active fraction were almost all oligogalacturonic acids (DP 6-12). This paper is the first report that plant growth-promoting oligosaccharides can be directly produced from tomato juice waste. It is possible that the substances from the tomato waste can become useful plant growth regulators in the agriculture field in the future. PMID:11762911

  12. How a plant lectin recognizes high mannose oligosaccharides.

    PubMed

    Garcia-Pino, Abel; Buts, Lieven; Wyns, Lode; Imberty, Anne; Loris, Remy

    2007-08-01

    The crystal structure of Pterocarpus angolensis seed lectin is presented in complex with a series of high mannose (Man) oligosaccharides ranging from Man-5 to Man-9. Despite that several of the nine Man residues of Man-9 have the potential to bind in the monosaccharide-binding site, all oligomannoses are bound in the same unique way, employing the tetrasaccharide sequence Manalpha(1-2)Manalpha(1-6)[Manalpha(1-3)]Manalpha(1-. Isothermal titration calorimetry titration experiments using Man-5, Man-9, and the Man-9-containing glycoprotein soybean (Glycine max) agglutinin as ligands confirm the monovalence of Man-9 and show a 4-times higher affinity for Man-9 when it is presented to P. angolensis seed lectin in a glycoprotein context. PMID:17556509

  13. Breast milk oligosaccharides: structure-function relationships in the neonate.

    PubMed

    Smilowitz, Jennifer T; Lebrilla, Carlito B; Mills, David A; German, J Bruce; Freeman, Samara L

    2014-01-01

    In addition to providing complete postnatal nutrition, breast milk is a complex biofluid that delivers bioactive components for the growth and development of the intestinal and immune systems. Lactation is a unique opportunity to understand the role of diet in shaping the intestinal environment including the infant microbiome. Of considerable interest is the diversity and abundance of milk glycans that are energetically costly for the mammary gland to produce yet indigestible by infants. Milk glycans comprise free oligosaccharides, glycoproteins, glycopeptides, and glycolipids. Emerging technological advances are enabling more comprehensive, sensitive, and rapid analyses of these different classes of milk glycans. Understanding the impact of inter- and intraindividual glycan diversity on function is an important step toward interventions aimed at improving health and preventing disease. This review discusses the state of technology for glycan analysis and how specific structure-function knowledge is enhancing our understanding of early nutrition in the neonate. PMID:24850388

  14. Synthetic heparin and heparan sulfate oligosaccharides and their protein interactions.

    PubMed

    Zulueta, Medel Manuel L; Lin, Shu-Yi; Hu, Yu-Peng; Hung, Shang-Cheng

    2013-12-01

    Heparin and heparan sulfate bind a host of basic proteins that take advantage of the sugar's dense structural information. The significance of these interactions in various aspects of development, physiology, and disease stimulated keen interest in evaluating structure-activity relationships. The well-defined heparin and heparan sulfate oligosaccharides needed for these studies can be mainly accessed by chemical synthesis and, more recently by chemoenzymatic means. The various synthetic strategies available to chemical synthesis have recently enabled the acquisition of several regular and irregular sequences, including a number of dodecasaccharides, through improved coupling methods and judicial protecting group manipulations. Controlled chain elongation and critical application of modification enzymes allowed the generation of well-defined constructs via chemoenzymatic synthesis. Investigations of various protein interactions with the synthetic constructs delivered valuable information that could aid future drug development endeavors. PMID:24182748

  15. Alginate oligosaccharides: enzymatic preparation and antioxidant property evaluation.

    PubMed

    Falkeborg, Mia; Cheong, Ling-Zhi; Gianfico, Carlo; Sztukiel, Katarzyna Magdalena; Kristensen, Kasper; Glasius, Marianne; Xu, Xuebing; Guo, Zheng

    2014-12-01

    Alginate oligosaccharides (AOs) prepared from alginate, by alginate lyase-mediated depolymerization, were structurally characterized by mass spectrometry, infrared spectrometry and thin layer chromatography. Studies of their antioxidant activities revealed that AOs were able to completely (100%) inhibit lipid oxidation in emulsions, superiorly to ascorbic acid (89% inhibition). AOs showed radical scavenging activity towards ABTṠ, hydroxyl, and superoxide radicals, which might explain their excellent antioxidant activity. The radical scavenging activity is suggested to originate mainly from the presence of the conjugated alkene acid structure formed during enzymatic depolymerization. According to the resonance hybrid theory, the parent radicals of AOs are delocalized through allylic rearrangement, and as a consequence, the reactive intermediates are stabilized. AOs were weak ferrous ion chelators. This work demonstrated that AOs obtained from a facile enzymatic treatment of abundant alginate is an excellent natural antioxidant, which may find applications in the food industry. PMID:24996323

  16. Structure analysis and laxative effects of oligosaccharides isolated from bananas.

    PubMed

    Wang, Juan; Huang, Hui Hua; Cheng, Yan Feng; Yang, Gong Ming

    2012-10-01

    Banana oligosaccharides (BOS) were extracted with water, and then separated and purified using column chromatography. Gel penetration chromatography was used to determine the molecular weights. Thin layer chromatogram and capillary electrophoresis were employed to analyze the monosaccharide composition. The indican bond and structure of the BOS molecule were determined using Fourier transform infrared spectroscopy and nuclear magnetic resonance. Results showed that BOS were probably composed of eight β-D-pyran glucose units linked with 1→6 indican bonds. The laxative effects of BOS were investigated in mice using the method described in "Handbook of Technical Standards for Testing and Assessment of Health Food in China." The length of the small intestine over which a carbon suspension solution advanced in mice treated with low-, middle-, and high-dose BOS was significantly greater than that in the model group, suggesting that BOS are effective in accelerating the movement of the small intestine. PMID:23039112

  17. Human milk oligosaccharides: Every baby needs a sugar mama

    PubMed Central

    Bode, Lars

    2012-01-01

    Human milk oligosaccharides (HMOs) are a family of structurally diverse unconjugated glycans that are highly abundant in and unique to human milk. Originally, HMOs were discovered as a prebiotic “bifidus factor” that serves as a metabolic substrate for desired bacteria and shapes an intestinal microbiota composition with health benefits for the breast-fed neonate. Today, HMOs are known to be more than just “food for bugs”. An accumulating body of evidence suggests that HMOs are antiadhesive antimicrobials that serve as soluble decoy receptors, prevent pathogen attachment to infant mucosal surfaces and lower the risk for viral, bacterial and protozoan parasite infections. In addition, HMOs may modulate epithelial and immune cell responses, reduce excessive mucosal leukocyte infiltration and activation, lower the risk for necrotizing enterocolitis and provide the infant with sialic acid as a potentially essential nutrient for brain development and cognition. Most data, however, stem from in vitro, ex vivo or animal studies and occasionally from association studies in mother–infant cohorts. Powered, randomized and controlled intervention studies will be needed to confirm relevance for human neonates. The first part of this review introduces the pioneers in HMO research, outlines HMO structural diversity and describes what is known about HMO biosynthesis in the mother's mammary gland and their metabolism in the breast-fed infant. The second part highlights the postulated beneficial effects of HMO for the breast-fed neonate, compares HMOs with oligosaccharides in the milk of other mammals and in infant formula and summarizes the current roadblocks and future opportunities for HMO research. PMID:22513036

  18. Isolation and characterization of feruloylated arabinoxylan oligosaccharides from the perennial cereal grain intermediate wheat grass (Thinopyrum intermedium).

    PubMed

    Schendel, Rachel R; Becker, Andreas; Tyl, Catrin E; Bunzel, Mirko

    2015-04-30

    In comparison to the annual grain crops dominating current agricultural production, perennial grain species require fewer chemical and energy inputs and improve soil health and erosion control. The possibility for producing sustainable grain harvests from marginal land areas is motivating research initiatives to integrate perennial grains into commercial cropping and food processing systems. In this study, the feruloylated arabinoxylans from intermediate wheat grass (Thinopyrum intermedium, IWG), a promising perennial grain candidate in agronomic screening studies, were investigated. Insoluble fiber isolated from IWG whole grain flour was subjected to either mildly acidic (50 mM TFA, 100 °C, 2 h) or enzymatic (Driselase) hydrolysis. The liberated feruloylated arabinoxylan oligosaccharides were concentrated with Amberlite XAD-2, separated with gel chromatography (Sephadex LH-20, water), and purified with reversed-phase HPLC (C18, water-MeOH gradient). Thirteen feruloylated oligosaccharides were isolated (including eight structures described for the first time) and identified by LC-ESI-MS and NMR. Linkage-type analysis via methylation analysis, as well as the monosaccharide and phenolic acid profiles of the IWG insoluble fiber were also determined. IWG feruloylated arabinoxylans have a relatively simple structure with only short feruloylated side chains, a lower backbone substitution rate than annual rye and wheat varieties, and a moderate phenolic acid content. PMID:25699975

  19. Linkage Determination of Linear Oligosaccharides by MSn (n > 2) Collision-Induced Dissociation of Z1 Ions in the Negative Ion Mode

    NASA Astrophysics Data System (ADS)

    Konda, Chiharu; Bendiak, Brad; Xia, Yu

    2013-12-01

    Obtaining unambiguous linkage information between sugars in oligosaccharides is an important step in their detailed structural analysis. An approach is described that provides greater confidence in linkage determination for linear oligosaccharides based on multiple-stage tandem mass spectrometry (MSn, n >2) and collision-induced dissociation (CID) of Z1 ions in the negative ion mode. Under low energy CID conditions, disaccharides 18O-labeled on the reducing carbonyl group gave rise to Z1 product ions (m/z 163) derived from the reducing sugar, which could be mass-discriminated from other possible structural isomers having m/z 161. MS3 CID of these m/z 163 ions showed distinct fragmentation fingerprints corresponding to the linkage types and largely unaffected by sugar unit identities or their anomeric configurations. This unique property allowed standard CID spectra of Z1 ions to be generated from a small set of disaccharide samples that were representative of many other possible isomeric structures. With the use of MSn CID (n = 3 - 5), model linear oligosaccharides were dissociated into overlapping disaccharide structures, which were subsequently fragmented to form their corresponding Z1 ions. CID data of these Z1 ions were collected and compared with the standard database of Z1 ion CID using spectra similarity scores for linkage determination. As the proof-of-principle tests demonstrated, we achieved correct determination of individual linkage types along with their locations within two trisaccharides and a pentasaccharide.

  20. Synthetic glycosylation of proteins using N-(beta-saccharide) iodoacetamides: applications in site-specific glycosylation and solid-phase enzymic oligosaccharide synthesis.

    PubMed Central

    Wong, S Y; Guile, G R; Dwek, R A; Arsequell, G

    1994-01-01

    A simple and efficient synthetic glycosylation method suitable for use in solid-phase enzymic oligosaccharide synthesis and site-specific glycosylation of recombinant proteins to produce defined glycoforms is described. This strategy utilizes N-(beta-saccharide) haloacetamides for attaching oligosaccharides specifically to cysteine residues of proteins in solution to form neoglycoproteins. The alkylation reaction was tested using N-(beta-chitotriose) bromoacetamide and an unprotected synthetic hexapeptide containing a single cysteine residue. The glycosylated product was confirmed by amino acid and hexosamine analyses as well as laser desorption mass spectrometry. Similarly N-(beta-chitotriose) iodoacetamide was covalently linked to non-reduced BSA to produce a defined glycoform of this protein. The specific attachment of chitotriose at the single cysteine residue in non-reduced serum albumin was suggested by Ellman's assay for free thiols. This was verified by amino acid sequencing of tryptic glycopeptide derived from this neoglycoprotein. Multiple sugar attachment was accomplished using fully reduced serum albumin as demonstrated by the formation of two neoglycoproteins using iodoacetamide derivatives of galactose beta 1-3-N-acetylgalactosamine (Gal beta 1-3GalNAc) and the major xylose/fucose-class plant-type oligosaccharide of horseradish peroxidase. These two neoglycoproteins with an average of 18-21 sugar residues attached were assayed positively for binding to peanut agglutinin and a sugar-specific anti-(horseradish peroxidase) monoclonal antibody YZ1/2.23 respectively. Sialylation of the neoglycoprotein containing Gal beta 1-3GalNAc was accomplished using alpha-2,3-sialyltransferase and radiolabelled CMP-N-acetylneuraminic acid. Significantly, glycan attachment using this conjugation method is reversible as demonstrated by the release of oligosaccharides from these two neoglycoproteins using hydrazinolysis. Therefore this method could provide invaluable

  1. A new sequencing approach for N-unsubstituted heparin/heparan sulfate oligosaccharides.

    PubMed

    Liang, Qun Tao; Xiao, Xiao Mao; Lin, Jian Hui; Wei, Zheng

    2015-07-01

    The rare N-unsubstituted glucosamine (GlcNH(3)(+)) residues in heparan sulfate (HS) have important biological and pathophysiological roles. Because of their low natural abundance, the use of chemically generated, structurally defined, N-unsubstituted heparin/HS oligosaccharides can greatly contribute to the investigation of their natural role in HS. However, the sequencing of mixtures of chemically generated oligosaccharides presents major challenges due to the difficulties in separating isomers and the available detection methods. In this study, we developed and validated a simple and sensitive method for the sequence analysis of N-unsubstituted heparin/HS oligosaccharides. This protocol involves pH 4 nitrous acid (HNO(2)) degradation, size-exclusion HPLC and ion-pair reversed-phase liquid chromatography-ion trap/time-of-flight mass spectrometry (IPRP-LC-ITTOF MS). We unexpectedly found that absorbance at 232 nm (normally used for specific detection of C4-C5 unsaturated oligosaccharides) was, in most cases, still sufficiently sensitive to also simultaneously detect saturated oligosaccharides during HPLC, thus simplifying the positional analysis of GlcNH(3)(+)) residues. The IPRP-LC-ITTOF MS system can supply further structural information leading to full sequence determination of the original oligosaccharide. This new methodology has been used to separate and sequence a variety of chemically generated, N-unsubstituted dp6 species containing between 1 and 3 GlcNH(3)(+)) residues per oligosaccharide in different positional combinations. This strategy offers possibilities for the sequencing of natural N-unsubstituted oligosaccharides from HS and should also be applicable, with minor modification, for sequencing at N-sulfated residues using alternative pH 1.5 HNO(2) scission. PMID:25677303

  2. Templated Oligosaccharide Synthesis: The Linker Effect on the Stereoselectivity of Glycosylation

    PubMed Central

    Pornsuriyasak, Papapida; Jia, Xiao G.; Kaeothip, Sophon; Demchenko, Alexei V.

    2016-01-01

    A new method for intramolecular oligosaccharide synthesis that is conceptually related to the general molecular clamp approach is introduced. Exceptional α-selectivity has been achieved in a majority of applications. Unlike other related concepts, this approach is based on the bisphenol A template, which allows one to connect multiple building blocks to perform templated oligosaccharide synthesis with complete stereoselectivity. This principle was demonstrated by the synthesis of an α,α-linked trisaccharide. PMID:27115718

  3. Immobilization of fluorous oligosaccharide recognized by influenza virus on polytetrafluoroethylene filter.

    PubMed

    Tojino, Mami; Mori, Masako; Kasuya, Maria Carmelita Z; Hatanaka, Kenichi; Kawaguchi, Atsushi; Nagata, Kyosuke; Shirai, Takashi; Mizuno, Mamoru

    2012-01-15

    The lactoside with PEG-fluorous tag was introduced to BHK-21(C-13) cells to generate a GM3-type oligosaccharide (Siaα2-3Galβ1-4Glc). The GM3-type oligosaccharide obtained was easily immobilized by spotting onto commercially available polytetrafluoroethylene (PTFE) filter through non-covalent fluorous affinity and simply assessed by dot blot method using the interaction of carbohydrate- with proteins which recognize sialic acid such as virus membrane proteins. PMID:22177082

  4. Core oligosaccharide of Escherichia coli B-the structure required for bacteriophage T4 recognition.

    PubMed

    Kaszowska, Marta; Niedziela, Tomasz; Maciejewska, Anna; Lukasiewicz, Jolanta; Jachymek, Wojciech; Lugowski, Czeslaw

    2015-09-01

    The structure of Escherichia coli B strain PCM 1935 core oligosaccharide has been investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF MS and ESI MS(n). It was concluded that the core oligosaccharide is a pentasaccharide with the following structure: ESI MS/MS analysis revealed that the glycine (a minor component) is linked to the →3,7)-l-α-d-Hepp-(1→ residue. PMID:26091777

  5. Effects of Xylo-Oligosaccharides on Broiler Chicken Performance and Microbiota

    PubMed Central

    De Maesschalck, C.; Eeckhaut, V.; Maertens, L.; De Lange, L.; Marchal, L.; Nezer, C.; De Baere, S.; Croubels, S.; Daube, G.; Dewulf, J.; Haesebrouck, F.; Ducatelle, R.; Taminau, B.

    2015-01-01

    In broiler chickens, feed additives, including prebiotics, are widely used to improve gut health and to stimulate performance. Xylo-oligosaccharides (XOS) are hydrolytic degradation products of arabinoxylans that can be fermented by the gut microbiota. In the current study, we aimed to analyze the prebiotic properties of XOS when added to the broiler diet. Administration of XOS to chickens, in addition to a wheat-rye-based diet, significantly improved the feed conversion ratio. XOS significantly increased villus length in the ileum. It also significantly increased numbers of lactobacilli in the colon and Clostridium cluster XIVa in the ceca. Moreover, the number of gene copies encoding the key bacterial enzyme for butyrate production, butyryl-coenzyme A (butyryl-CoA):acetate CoA transferase, was significantly increased in the ceca of chickens administered XOS. In this group of chickens, at the species level, Lactobacillus crispatus and Anaerostipes butyraticus were significantly increased in abundance in the colon and cecum, respectively. In vitro fermentation of XOS revealed cross-feeding between L. crispatus and A. butyraticus. Lactate, produced by L. crispatus during XOS fermentation, was utilized by the butyrate-producing Anaerostipes species. These data show the beneficial effects of XOS on broiler performance when added to the feed, which potentially can be explained by stimulation of butyrate-producing bacteria through cross-feeding of lactate and subsequent effects of butyrate on gastrointestinal function. PMID:26092452

  6. Fractionation of a galacto-oligosaccharides solution at low and high temperature using nanofiltration

    PubMed Central

    Pruksasri, Suwattana; Nguyen, Thu-Ha; Haltrich, Dietmar; Novalin, Senad

    2015-01-01

    Like in many applications, solutions of high sugar content can cause serious problems due to microorganism contaminations. Hence, the main aim of this work was to study a nanofiltration process for GOS purification at 5 °C and 60 °C that may circumvent or reduce potential microbial growth. Process performances and rejection behaviors of monosaccharide as well as individual GOS components were compared. Operating at 5 °C is more advantageous especially with respect to the oligosaccharide (OS) recovery yield. Using a NF membrane (NP030) at 45 bar, a product purity of 85% (based on monosaccharide content) and an OS recovery yield of 82% could be achieved. However, a low average permeate flux of 3 L/m2 h had to be accepted. A diafiltration step improved product purity to 90% with 30% losses of OS. A qualitative theoretical discussion shows that a possible change of the pore radius distribution depending on temperature could play a role in solute rejection as well as selectivity. PMID:26681914

  7. Effects of Xylo-Oligosaccharides on Broiler Chicken Performance and Microbiota.

    PubMed

    De Maesschalck, C; Eeckhaut, V; Maertens, L; De Lange, L; Marchal, L; Nezer, C; De Baere, S; Croubels, S; Daube, G; Dewulf, J; Haesebrouck, F; Ducatelle, R; Taminau, B; Van Immerseel, F

    2015-09-01

    In broiler chickens, feed additives, including prebiotics, are widely used to improve gut health and to stimulate performance. Xylo-oligosaccharides (XOS) are hydrolytic degradation products of arabinoxylans that can be fermented by the gut microbiota. In the current study, we aimed to analyze the prebiotic properties of XOS when added to the broiler diet. Administration of XOS to chickens, in addition to a wheat-rye-based diet, significantly improved the feed conversion ratio. XOS significantly increased villus length in the ileum. It also significantly increased numbers of lactobacilli in the colon and Clostridium cluster XIVa in the ceca. Moreover, the number of gene copies encoding the key bacterial enzyme for butyrate production, butyryl-coenzyme A (butyryl-CoA):acetate CoA transferase, was significantly increased in the ceca of chickens administered XOS. In this group of chickens, at the species level, Lactobacillus crispatus and Anaerostipes butyraticus were significantly increased in abundance in the colon and cecum, respectively. In vitro fermentation of XOS revealed cross-feeding between L. crispatus and A. butyraticus. Lactate, produced by L. crispatus during XOS fermentation, was utilized by the butyrate-producing Anaerostipes species. These data show the beneficial effects of XOS on broiler performance when added to the feed, which potentially can be explained by stimulation of butyrate-producing bacteria through cross-feeding of lactate and subsequent effects of butyrate on gastrointestinal function. PMID:26092452

  8. Lipid-Linked Oligosaccharides in Membranes Sample Conformations That Facilitate Binding to Oligosaccharyltransferase

    PubMed Central

    Kern, Nathan R.; Lee, Hui Sun; Wu, Emilia L.; Park, Soohyung; Vanommeslaeghe, Kenno; MacKerell, Alexander D.; Klauda, Jeffery B.; Jo, Sunhwan; Im, Wonpil

    2014-01-01

    Lipid-linked oligosaccharides (LLOs) are the substrates of oligosaccharyltransferase (OST), the enzyme that catalyzes the en bloc transfer of the oligosaccharide onto the acceptor asparagine of nascent proteins during the process of N-glycosylation. To explore LLOs’ preferred location, orientation, structure, and dynamics in membrane bilayers of three different lipid types (dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, and dioleoylphosphatidylcholine), we have modeled and simulated both eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol) and bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol) LLOs, which are composed of an isoprenoid moiety and an oligosaccharide, linked by pyrophosphate. The simulations show no strong impact of different bilayer hydrophobic thicknesses on the overall orientation, structure, and dynamics of the isoprenoid moiety and the oligosaccharide. The pyrophosphate group stays in the bilayer head group region. The isoprenoid moiety shows high flexibility inside the bilayer hydrophobic core, suggesting its potential role as a tentacle to search for OST. The oligosaccharide conformation and dynamics are similar to those in solution, but there are preferred interactions between the oligosaccharide and the bilayer interface, which leads to LLO sugar orientations parallel to the bilayer surface. Molecular docking of the bacterial LLO to a bacterial OST suggests that such orientations can enhance binding of LLOs to OST. PMID:25418169

  9. Lactodifucotetraose, a human milk oligosaccharide, attenuates platelet function and inflammatory cytokine release.

    PubMed

    Newburg, David S; Tanritanir, Ayse C; Chakrabarti, Subrata

    2016-07-01

    Human milk strongly quenches inflammatory processes in vitro, and breastfed infants have lower incidence of inflammatory diseases than those fed artificially. Platelets from neonates, in contrast to those from adults, are less responsive to platelet agonists such as collagen, thrombin, ADP, and epinephrine. Breastfed infants absorb oligosaccharides intact from the human milk in their gut to the circulation. This study was to determine whether these oligosaccharides can attenuate platelet function and platelet secretion of pro-inflammatory proteins, and to identify the active component. The natural mixture of oligosaccharides from human milk and pure individual human milk oligosaccharides were tested for their ability to modulate responses of platelets isolated from human blood following exposure to thrombin, ADP, and collagen. Human milk and the natural mixture of human milk oligosaccharides inhibited platelet release of inflammatory proteins. Of the purified human milk oligosaccharides tested, only lactodifucotetraose (LDFT) significantly inhibited thrombin induced release of the pro-inflammatory proteins RANTES and sCD40L. LDFT also inhibited platelet adhesion to a collagen-coated surface, as well as platelet aggregation induced by ADP or collagen. These data indicate that LDFT may help modulate hemostasis by suppressing platelet-induced inflammatory processes in breastfed infants. This activity suggests further study of LDFT for its potential as a therapeutic agent in infants and adults. PMID:26743063

  10. A comparative study of free oligosaccharides in the milk of domestic animals.

    PubMed

    Albrecht, Simone; Lane, Jonathan A; Mariño, Karina; Al Busadah, Khalid A; Carrington, Stephen D; Hickey, Rita M; Rudd, Pauline M

    2014-04-14

    The present study was conducted to obtain a comprehensive overview of oligosaccharides present in the milk of a variety of important domestic animals including cows, goats, sheep, pigs, horses and dromedary camels. Using an analytical workflow that included ultra-performance liquid chromatography-hydrophilic interaction liquid chromatography with fluorescence detection coupled to quadrupole time-of-flight MS, detailed oligosaccharide libraries were established. The partial or full characterisation of the neutral/fucosylated, phosphorylated and sialylated structures was facilitated by sequencing with linkage- and sugar-specific exoglycosidases. Relative peak quantification of the 2-aminobenzamide-labelled oligosaccharides provided additional information. Milk from domestic animals contained a much larger variety of complex oligosaccharides than was previously assumed, and thirteen of these structures have been identified previously in human milk. The direct comparison of the oligosaccharide mixtures reflects their role in the postnatal maturation of different types of gastrointestinal systems, which, in this way, are prepared for certain post-weaning diets. The potential value of animal milk for the commercial extraction of oligosaccharides to be used in human and animal health is highlighted. PMID:24635885

  11. Structure, oligosaccharide structures, and posttranslationally modified sites of the nicotinic acetylcholine receptor.

    PubMed Central

    Poulter, L; Earnest, J P; Stroud, R M; Burlingame, A L

    1989-01-01

    Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californica contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor. Asparagine-143 of the alpha subunit (in consensus numbering) is shown to be glycosylated with high-mannose oligosaccharide. Asparagine-453 of the gamma subunit is not glycosylated, a fact that bears on the question of the orientations of putative transmembranous helices M3, MA, and M4. The structures of the six major acetylcholine receptor oligosaccharides are determined: the major components (70%) are of the high-mannose type, with bi-, tri-, and tetraantennary complex oligosaccharides making up approximately equal to 22 mol% of the total carbohydrate. This application of a multichannel array detector mass spectrometer provided a breakthrough in sensitivity that allowed us to identify the site of attachment of, and the sequence of, oligosaccharides on a 300-kDa membrane protein from only 5 pmol of the isolated oligosaccharide. Images PMID:2771948

  12. Quantification of neutral human milk oligosaccharides by graphitic carbon HPLC with tandem mass spectrometry

    PubMed Central

    Bao, Yuanwu; Chen, Ceng; Newburg, David S.

    2012-01-01

    Defining the biologic roles of human milk oligosaccharides (HMOS) requires an efficient, simple, reliable, and robust analytical method for simultaneous quantification of oligosaccharide profiles from multiple samples. The HMOS fraction of milk is a complex mixture of polar, highly branched, isomeric structures that contain no intrinsic facile chromophore, making their resolution and quantification challenging. A liquid chromatography-mass spectrometry (LC-MS) method was devised to resolve and quantify 11 major neutral oligosaccharides of human milk simultaneously. Crude HMOS fractions are reduced, resolved by porous graphitic carbon HPLC with a water/acetonitrile gradient, detected by mass spectrometric specific ion monitoring, and quantified. The HPLC separates isomers of identical molecular weights allowing 11 peaks to be fully resolved and quantified by monitoring mass to charge (m/z) ratios of the deprotonated negative ions. The standard curves for each of the 11 oligosaccharides is linear from 0.078 or 0.156 to 20 μg/mL (R2 > 0.998). Precision (CV) ranges from 1% to 9%. Accuracy is from 86% to 104%. This analytical technique provides sensitive, precise, accurate quantification for each of the 11 milk oligosaccharides and allows measurement of differences in milk oligosaccharide patterns between individuals and at different stages of lactation. PMID:23068043

  13. Oligosaccharides in several Philippine indigenous food legumes: determination, localization and removal.

    PubMed

    Revilleza, M J; Mendoza, E M; Raymundo, L C

    1990-01-01

    The oligosaccharide profile of raw mature seeds of seven different legumes indigenous to the Philippines was measured in 70% ethanol extracts of the seeds by thin layer chromatography using HPTLC plates and quantified by a densitometer. Based on the results, the legumes could be ranked according to decreasing oligosaccharide content or flatulence potential as follows: Sam-samping (Clitoria ternatea) greater than hyacinth bean (Dolichos lablab) greater than sabawel (Mucuna pruriens) greater than lima bean (Phaseolus lunatus) greater than swordbean (Canavalia gladiata) greater than rice bean (Vigna umbellata) greater than jack bean (Canavalia ensiformis). Sam-samping had 4.79% total oligosaccharides and hyacinth bean or batao, 3.66%. A jack bean accession had 1.79% oligosaccharides. Simple processing methods were tested to detoxify the oligosaccharides. Soaking the batao seeds had no effect while boiling even resulted in a net 23-31% increase in the levels of raffinose, stachyose and verbascose. On the other hand, two min of dry roasting resulted in complete removal of oligosaccharides whereas germination resulted in about 30-40% decrease after 1 and 2 days, respectively. PMID:2345736

  14. Sequencing of oligosaccharides using enzyme array digestion with electrochemical and fluorescent detections

    SciTech Connect

    Sun, M.; Lee, C.S.

    1997-12-31

    The objective of this study is to develop a rapid and sensitive method for oligosaccharide sequencing. The oligosaccharides are subjected to the enzyme array digestion with exoglycosidases of known and well-defined specificities. The enzyme array method involves the division of oligosaccharide sample into aliquots, and the incubation of each aliquot with a precisely defined mixture of exoglycosidases. In the enzyme array method, the presence of a specific linkage anywhere in the oligosaccharide is determined by the inability of an enzyme mixture lacking a given enzyme to cleave that linkage ( a stop point) and the ability of the other enzymes to cleave the linkage up to that point. The direct quantification of released monosaccharides from the enzyme array can be achieved by using pulsed amperometric detection (PAD) or by fluorescent derivatization with a fluorophoric agent. The measured monosaccharide concentrations in combination with the enzyme array analysis provide detail characterization of oligosaccharides with their sugar composition, configuration, and linkage information, The released monosaccharides are further quantified by anion exchange chromatography and capillary electrophoresis for the comparison with the results obtained from PAD and fluorescence measurements. Our enzyme array-electrochemical (or fluorescent) detection method does not require any separation procedure and any prior labeling of oligosaccharide and have several practical advantages over the current carbohydrate sequencing techniques including simplicity, speed, and the ability to use small amounts of starting material.

  15. S-Nitrosothiol-modified nitric oxide-releasing chitosan oligosaccharides as antibacterial agents.

    PubMed

    Lu, Yuan; Shah, Anand; Hunter, Rebecca A; Soto, Robert J; Schoenfisch, Mark H

    2015-01-01

    S-Nitrosothiol-modified chitosan oligosaccharides were synthesized by reaction with 2-iminothiolane hydrochloride and 3-acetamido-4,4-dimethylthietan-2-one, followed by thiol nitrosation. The resulting nitric oxide (NO)-releasing chitosan oligosaccharides stored ∼0.3μmol NO mg(-1) chitosan. Both the chemical structure of the nitrosothiol (i.e. primary and tertiary) and the use of ascorbic acid as a trigger for NO donor decomposition were used to control the NO-release kinetics. With ascorbic acid, the S-nitrosothiol-modified chitosan oligosaccharides elicited a 4-log reduction in Pseudomonas aeruginosa viability. Confocal microscopy indicated that the primary S-nitrosothiol-modified chitosan oligosaccharides associated more with the bacteria relative to the tertiary S-nitrosothiol system. The primary S-nitrosothiol-modified chitosan oligosaccharides elicited minimal toxicity towards L929 mouse fibroblast cells at the concentration necessary for a 4-log reduction in bacterial viability, further demonstrating the potential of S-nitrosothiol-modified chitosan oligosaccharides as NO-release therapeutics. PMID:25449913

  16. Preparation of κ-carra-oligosaccharides with microwave assisted acid hydrolysis method

    NASA Astrophysics Data System (ADS)

    Li, Guangsheng; Zhao, Xia; Lv, Youjing; Li, Miaomiao; Yu, Guangli

    2015-04-01

    A rapid method of microwave assisted acid hydrolysis was established to prepare κ-carra-oligosaccharides. The optimal hydrolysis condition was determined by an orthogonal test. The degree of polymerization (DP) of oligosaccharides was detected by high performance thin layer chromatography (HPTLC) and polyacrylamide gel electrophoresis (PAGE). Considering the results of HPTLC and PAGE, the optimum condition of microwave assisted acid hydrolysis was determined. The concentration of κ-carrageenan was 5 mg mL-1; the reaction solution was adjusted to pH 3 with diluted hydrochloric acid; the solution was hydrolyzed under microwave irradiation at 100 for 15 °C min. Oligosaccharides were separated by a Superdex 30 column (2.6 cm × 90 cm) using AKTA Purifier UPC100 and detected with an online refractive index detector. Each fraction was characterized by electrospray ionization mass spectrometry (ESI-MS). The data showed that odd-numbered κ-carra-oligosaccharides with DP ranging from 3 to 21 could be obtained with this method, and the structures of the oligosaccharides were consistent with those obtained by traditional mild acid hydrolysis. The new method was more convenient, efficient and environment-friendly than traditional mild acid hydrolysis. Our results provided a useful reference for the preparation of oligosaccharides from other polysaccharides.

  17. Mass spectrometric detection of multiple extended series of neutral highly fucosylated N-acetyllactosamine oligosaccharides in human milk

    NASA Astrophysics Data System (ADS)

    Pfenninger, Anja; Chan, Shiu-Yung; Karas, Michael; Finke, Berndt; Stahl, Bernd; Costello, Catherine E.

    2008-12-01

    Complex mixtures of high-molecular weight fractions of pooled neutral human milk oligosaccharides (obtained via gel permeation chromatography) have been investigated. The subfractions were each permethylated and analyzed by high-resolution mass spectrometry, using matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, in order to investigate their oligosaccharide compositions. The obtained spectra reveal that human milk contains more complex neutral oligosaccharides than have been described previously; the data show that these oligosaccharides can be highly fucosylated, and that their poly-N-acetyllactosamine cores are substituted with up to 10 fucose residues on an oligosaccharide that has 7-N-acetyllactosamine units. This is the first report of the existence in human milk of this large range of highly fucosylated oligosaccharides which possess novel, potentially immunologically active structures.

  18. Synthesis of galacto-oligosaccharides by β-galactosidase from Aspergillus oryzae using partially dissolved and supersaturated solution of lactose.

    PubMed

    Vera, Carlos; Guerrero, Cecilia; Conejeros, Raúl; Illanes, Andrés

    2012-03-10

    The effect of enzyme to substrate ratio, initial lactose concentration and temperature has been studied for the kinetically controlled reaction of lactose transgalactosylation with Aspergillus oryzae β-galactosidase, to produce prebiotic galacto-oligosaccharides (GOS). Enzyme to substrate ratio had no significant effect on maximum yield and specific productivity. Galacto-oligosaccharide syntheses at very high lactose concentrations (40, 50 and 60%, w/w, lactose monohydrate) were evaluated at different temperatures (40, 47.5 and 55°C). Within these ranges, lactose could be found as a supersaturated solution or a heterogeneous system with precipitated lactose, resulting in significant effect on GOS synthesis. An increase in initial lactose concentration produced a slight increase in maximum yield as long as lactose remained dissolved. Increase in temperature produced a slight decrease in maximum yield and an increase in specific productivity when supersaturation of lactose occurred during reaction. Highest yield of 29 g GOS/100 g lactose added was obtained at a lactose monohydrate initial concentration of 50% (w/w) and 47.5°C. Highest specific productivity of 0.38 g GOSh(-1) mg enzyme(-1) was obtained at lactose monohydrate initial concentration of 40% (w/w) and 55°C, where a maximum yield of 27 g GOS/100 g lactose added was reached. This reflects the complex interplay between temperature and initial lactose concentration on the reaction of synthesis. When lactose precipitation occurred, values of yields and specific productivities lower than 22 g GOS/100 g lactose added and 0.03 gGOSh(-1) mg enzyme(-1) were obtained, respectively. PMID:22305174

  19. Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

    SciTech Connect

    Jones, Meredith B.; Tomiya, Noboru; Betenbaugh, Michael J.; Krag, Sharon S.

    2010-04-23

    Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man{sub 5}GlcNAc{sub 2}-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man{sub 9}GlcNAc{sub 2}-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man{sub 5}GlcNAc{sub 2}-PP-Dol through Glc{sub 1}Man{sub 9}GlcNAc{sub 2}-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

  20. Biomimetic oligosaccharide and peptide surfactant polymers designed for cardiovascular biomaterials

    NASA Astrophysics Data System (ADS)

    Ruegsegger, Mark Andrew

    A common problem associated with cardiovascular devices is surface induced thrombosis initiated by the rapid, non-specific adsorption of plasma proteins onto the biomaterial surface. Control of the initial protein adsorption is crucial to achieve the desired longevity of the implanted biomaterial. The cell membrane glycocalyx acts as a non-thrombogenic interface through passive (dense oligosaccharide structures) and active (ligand/receptor interactions) mechanisms. This thesis is designed to investigate biomimicry of the cell glycocalyx to minimize non-specific protein adsorption and promote specific ligand/receptor interactions. Biomimetic macromolecules were designed through the molecular-scale engineering of polymer surfactants, utilizing a poly(vinyl amine) (PVAm) backbone to which hydrophilic (dextran, maltose, peptide) and hydrophobic alkyl (hexanoyl or hexanal) chains are simultaneously attached. The structure was controlled through the molar feed ratio of hydrophobic-to-hydrophilic groups, which also provided control of the solution and surface-active properties. To mimic passive properties, a series of oligomaltose surfactants were synthesized with increasing saccharide length (n = 2, 7, 15 where n is number of glucose units) to investigate the effect of coating height on protein adsorption. The surfactants were characterized by infra red (IR) and nuclear magnetic resonance (NMR) spectroscopies for structural properties and atomic force microscopy (AFM) and contact angle goniometry for surface activity. Protein adsorption under dynamic flow (5 dyn/cm2) was reduced by 85%--95% over the bare hydrophobic substrate; platelet adhesion dropped by ˜80% compared to glass. Peptide ligands were incorporated into the oligosaccharide surfactant to promote functional activity of the passive coating. The surfactants were synthesized to contain 0%, 25%, 50%, 75%, and 100% peptide ligand density and were stable on hydrophobic surfaces. The peptide surface density was

  1. Characterisation of separated end hyaluronan oligosaccharides from leech hyaluronidase and evaluation of angiogenesis.

    PubMed

    Lv, Mengxian; Wang, Miao; Cai, Weiwei; Hao, Wenxing; Yuan, Panhong; Kang, Zhen

    2016-05-20

    Hyaluronan oligosaccharides (o-HAs), especially saturated o-HAs, have attracted intensive attention due to their potential applications in medical treatments. In this study, the hydrolysis process of leech hyaluronidase (LHase) towards the hyaluronan was investigated by HPLC and HPLC/ESI-MS. The proportions of hyaluronan tetrasaccharide (HA4) with hexasaccharide (HA6), end products, were illustrated to have a relationship with the amount of LHase. Higher yield of HA4 was achieved with higher activity of LHase. After optimisation of the packing resin and operation parameters (balanced pH, elution concentration, elution volume and elution flow rate), the highly pure HA4 and HA6 were efficiently separated and prepared by combining ion exchange Q-Sepharose Fast Flow and size exclusion column chromatography. Compared with o-HAs (average Mr of 4000 Da), HA4 and HA6 were demonstrated to show higher activity for promoting angiogenesis, which was similar with the corresponding HA4 and HA6 produced by bovine testicular hyaluronidase. The pure HA4 and HA6 that prepared from LHase will attract intensive studies and be used in potential applications in near future. PMID:26917404

  2. Microwave superheated water and dilute alkali extraction of brewers' spent grain arabinoxylans and arabinoxylo-oligosaccharides.

    PubMed

    Coelho, Elisabete; Rocha, M Angélica M; Saraiva, Jorge A; Coimbra, Manuel A

    2014-01-01

    Microwave superheated water extractions (MWE) were performed to evaluate the feasibility of this technology for quantitative recovery of the arabinoxylans (AX) or arabinoxylo-oligosaccharides (AXOS) from brewers' spent grain (BSG). The AX+AXOS yield increased with the increase of the temperature in the range from 140 to 210 °C during 2 min. The higher temperatures promoted depolymerisation, debranching, and deesterification of the polysaccharides, with formation of brown products. The conditions that promote a compromise between the yield and the structure obtained, minimizing the thermal degradation of the fractions extracted by MWE are the following: (1) 140 °C, to remove the residual starch mixed with β-glucans; (2) Suspension of the residue left in water and treated at 180 °C; (3) suspension of the residue in 0.1 M KOH and treated at 180 °C. Using this sequential procedure, it was possible to extract 62% of BSG AX+AXOS, presenting degrees of polymerization ranging between 7 and 24 xylose residues, and a degree of phenolic acids esterification between 5 and 21%. The structural variability obtained by MWE allows defining specific types of compounds for different applications and uses depending on the extraction conditions used. PMID:24274525

  3. Phloem loading in Verbascum phoeniceum L. depends on the synthesis of raffinose-family oligosaccharides

    PubMed Central

    McCaskill, Ashlee; Turgeon, Robert

    2007-01-01

    Phloem loading is the initial step in photoassimilate export and the one that creates the driving force for mass flow. It has been proposed that loading occurs symplastically in species that translocate carbohydrate primarily as raffinose family oligosaccharides (RFOs). In these plants, dense fields of plasmodesmata connect bundle sheath cells to specialized companion cells (intermediary cells) in the minor veins. According to the polymer trap model, advanced as a mechanism of symplastic loading, sucrose from the mesophyll diffuses into intermediary cells and is converted there to RFOs. This process keeps the sucrose concentration low and, because of the larger size of the RFOs, prevents back diffusion. To test this model, the RFO pathway was down-regulated in Verbascum phoeniceum L. by suppressing the synthesis of galactinol synthase (GAS), which catalyzes the first committed step in RFO production. Two GAS genes (VpGAS1 and VpGAS2) were cloned and shown to be expressed in intermediary cells. Simultaneous RNAi suppression of both genes resulted in pronounced inhibition of RFO synthesis. Phloem transport was negatively affected, as evidenced by the accumulation of carbohydrate in the lamina and the reduced capacity of leaves to export sugars during a prolonged dark period. In plants with severe down-regulation, additional symptoms of reduced export were obvious, including impaired growth, leaf chlorosis, and necrosis and curling of leaf margins. PMID:18048337

  4. Human Milk Oligosaccharides (HMOS): Structure, Function, and Enzyme-Catalyzed Synthesis.

    PubMed

    Chen, Xi

    2015-01-01

    The important roles played by human milk oligosaccharides (HMOS), the third major component of human milk, in the health of breast-fed infants have been increasingly recognized, as the structures of more than 100 different HMOS have now been elucidated. Despite the recognition of the various functions of HMOS as prebiotics, antiadhesive antimicrobials, and immunomodulators, the roles and the applications of individual HMOS species are less clear. This is mainly due to the limited accessibility to large amounts of individual HMOS in their pure forms. Current advances in the development of enzymatic, chemoenzymatic, whole-cell, and living-cell systems allow for the production of a growing number of HMOS in increasing amounts. This effort will greatly facilitate the elucidation of the important roles of HMOS and allow exploration into the applications of HMOS both as individual compounds and as mixtures of defined structures with desired functions. The structures, functions, and enzyme-catalyzed synthesis of HMOS are briefly surveyed to provide a general picture about the current progress on these aspects. Future efforts should be devoted to elucidating the structures of more complex HMOS, synthesizing more complex HMOS including those with branched structures, and developing HMOS-based or HMOS-inspired prebiotics, additives, and therapeutics. PMID:26613816

  5. Specific sizes of hyaluronan oligosaccharides stimulate fibroblast migration and excisional wound repair.

    PubMed

    Tolg, Cornelia; Telmer, Patrick; Turley, Eva

    2014-01-01

    The extracellular matrix polysaccharide hyaluronan (HA) plays a key role in both fibrotic and regenerative tissue repair. Accumulation of high molecular weight HA is typical of regenerative repair, which is associated with minimal inflammation and fibrosis, while fragmentation of HA is typical of postnatal wounds, which heal in the presence of inflammation and transient fibrosis. It is generally considered that HA oligosaccharides and fragments of a wide size range support these processes of adult, fibrotic wound repair yet the consequences of sized HA fragments/oligosaccharides to each repair stage is not well characterized. Here, we compared the effects of native HA, HA oligosaccharide mixtures and individual sizes (4-10 mer oligosaccharides, 5 and, 40 kDa) of HA oligosaccharides and fragments, on fibroblast migration in scratch wound assays and on excisional skin wound repair in vivo. We confirm that 4-10 mer mixtures significantly stimulated scratch wound repair and further report that only the 6 and 8 mer oligosaccharides in this mixture are responsible for this effect. The HA 6 mer promoted wound closure, accumulation of wound M1 and M2 macrophages and the M2 cytokine TGFβ1, but did not increase myofibroblast differentiation. The effect of 6 mer HA on wound closure required both RHAMM and CD44 expression. In contrast, The 40 kDa HA fragment inhibited wound closure, increased the number of wound macrophages but had no effect on TGFβ1 accumulation or subsequent fibrosis. These results show that specific sizes of HA polymer have unique effects on postnatal wound repair. The ability of 6 mer HA to promote wound closure and inflammation resolution without increased myofibroblast differentiation suggests that this HA oligosaccharide could be useful for treatment of delayed or inefficient wound repair where minimal fibrosis is advantageous. PMID:24551108

  6. Engineering a thermostable Halothermothrix orenii β-glucosidase for improved galacto-oligosaccharide synthesis.

    PubMed

    Hassan, Noor; Geiger, Barbara; Gandini, Rosaria; Patel, Bharat K C; Kittl, Roman; Haltrich, Dietmar; Nguyen, Thu-Ha; Divne, Christina; Tan, Tien Chye

    2016-04-01

    Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining β-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a β-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k cat, but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k cat). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with ~10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs. PMID:26621798

  7. Potential of novel dextran oligosaccharides as prebiotics for obesity management through in vitro experimentation.

    PubMed

    Sarbini, Shahrul R; Kolida, Sofia; Deaville, Eddie R; Gibson, Glenn R; Rastall, Robert A

    2014-10-28

    The energy-salvaging capacity of the gut microbiota from dietary ingredients has been proposed as a contributing factor for the development of obesity. This knowledge generated interest in the use of non-digestible dietary ingredients such as prebiotics to manipulate host energy homeostasis. In the present study, the in vitro response of obese human faecal microbiota to novel oligosaccharides was investigated. Dextrans of various molecular weights and degrees of branching were fermented with the faecal microbiota of healthy obese adults in pH-controlled batch cultures. Changes in bacterial populations were monitored using fluorescent in situ hybridisation and SCFA concentrations were analysed by HPLC. The rate of gas production and total volume of gas produced were also determined. In general, the novel dextrans and inulin increased the counts of bifidobacteria. Some of the dextrans were able to alter the composition of the obese human microbiota by increasing the counts of Bacteroides-Prevotella and decreasing those of Faecalibacterium prausnitzii and Ruminococcus bromii/R. flavefaciens. Considerable increases in SCFA concentrations were observed in response to all substrates. Gas production rates were similar during the fermentation of all dextrans, but significantly lower than those during the fermentation of inulin. Lower total gas production and shorter time to attain maximal gas production were observed during the fermentation of the linear 1 kDa dextran than during the fermentation of the other dextrans. The efficacy of bifidobacteria to ferment dextrans relied on the molecular weight and not on the degree of branching. In conclusion, there are no differences in the profiles between the obese and lean human faecal fermentations of dextrans. PMID:25196744

  8. OMICS-rooted studies of milk proteins, oligosaccharides and lipids.

    PubMed

    Casado, Begoña; Affolter, Michael; Kussmann, Martin

    2009-12-01

    Milk has co-evolved with mammals and mankind to nourish their offspring and is a biological fluid of unique complexity and richness. It contains all necessary nutrients for the growth and development of the newborn. Structure and function of biomolecules in milk such as the macronutrients (glyco-) proteins, lipids, and oligosaccharides are central topics in nutritional research. Omics disciplines such as proteomics, glycomics, glycoproteomics, and lipidomics enable comprehensive analysis of these biomolecule components in food science and industry. Mass spectrometry has largely expanded our knowledge on these milk bioactives as it enables identification, quantification and characterization of milk proteins, carbohydrates, and lipids. In this article, we describe the biological importance of milk macronutrients and review the application of proteomics, glycomics, glycoproteomics, and lipidomics to the analysis of milk. Proteomics is a central platform among the Omics tools that have more recently been adapted and applied to nutrition and health research in order to deliver biomarkers for health and comfort as well as to discover beneficial food bioactives. PMID:19793547

  9. Galacto-oligosaccharides attenuate renal injury with microbiota modification.

    PubMed

    Furuse, Satoshi U; Ohse, Takamoto; Jo-Watanabe, Airi; Shigehisa, Akira; Kawakami, Koji; Matsuki, Takahiro; Chonan, Osamu; Nangaku, Masaomi

    2014-07-01

    Tubulointerstitial injury is central to the progression of end-stage renal disease. Recent studies have revealed that one of the most investigated uremic toxins, indoxyl sulfate (IS), caused tubulointerstitial injury through oxidative stress and endoplasmic reticulum (ER) stress. Because indole, the precursor of IS, is synthesized from dietary tryptophan by the gut microbiota, we hypothesized that the intervention targeting the gut microbiota in kidney disease with galacto-oligosaccharides (GOS) would attenuate renal injury. After 2 weeks of GOS administration for 5/6 nephrectomized (Nx) or sham-operated (Sham) rats, cecal indole and serum IS were measured, renal injury was evaluated, and the effects of GOS on the gut microbiota were examined using pyrosequencing methods. Cecal indole and serum IS were significantly decreased and renal injury was improved with decreased infiltrating macrophages in GOS-treated Nx rats. The expression levels of ER stress markers and apoptosis were significantly increased in the Nx rats and decreased with GOS. The microbiota analysis indicated that GOS significantly increased three bacterial families and decreased five families in the Nx rats. In addition, the analysis also revealed that the bacterial family Clostridiaceae was significantly increased in the Nx rats compared with the Sham rats and decreased with GOS. Taken altogether, our data show that GOS decreased cecal indole and serum IS, attenuated renal injury, and modified the gut microbiota in the Nx rats, and that the gut microbiota were altered in kidney disease. GOS could be a novel therapeutic agent to protect against renal injury. PMID:24994892

  10. Oligosaccharides Affect Performance and Gut Development of Broiler Chickens

    PubMed Central

    Ao, Z.; Choct, M.

    2013-01-01

    The effects of oligosaccharide supplementation on the growth performance, flock uniformity and GIT development of broiler chickens were investigated. Four diets, one negative control, one positive control supplemented with zinc-bacitracin, and two test diets supplemented with mannoligosaccharide (MOS) and fructooligosaccharide (FOS), were used for the experiment. Birds given MOS or FOS had improved body weight (BW) and feed efficiency (FCR), compared to those fed the negative control diet during the 35-d trial period. The effect on FCR became less apparent when the birds got older. FOS and MOS supplementation reduced the pancreas weight as a percentage of BW, with an effect similar to that of the antibiotic, at 35 d of age. Birds given MOS tended to have a heavier bursa (p = 0.164) and lower spleen/bursa weight ratio (p = 0.102) at 35 d of age. MOS and Zn-bacitracin showed a clear improvement on flock uniformity, compared to FOS. The mortality rate was not affected by FOS or MOS. PMID:25049713

  11. Comparison of milk oligosaccharides between goats with and without the genetic ability to synthesize αs1-casein.

    PubMed

    Meyrand, M; Dallas, D C; Caillat, H; Bouvier, F; Martin, P; Barile, D

    2013-07-01

    Milk oligosaccharides (OS)-free complex carbohydrates-confer unique health benefits to the nursing neonate. Though human digestive enzymes cannot degrade these sugars, they provide nourishment to specific commensal microbes and act as decoys to prevent the adhesion of pathogenic micro-organisms to gastrointestinal cells. At present, the limited quantities of human milk oligosaccharides (HMO) impede research on these molecules and their potential applications in functional food formulations. Considerable progress has been made in the study of OS structures; however, the synthetic pathways leading to their synthesis in the mammary gland are poorly understood. Recent studies show that complex OS with fucose and N-acetyl neuraminic acid (key structural elements of HMO bioactivity) exist in goat milk. Polymorphisms in the CSN1S1 locus, which is responsible for synthesis of αs1-casein, affect lipid and casein micelle structure in goat milk. The present study sought to determine whether CSN1S1 polymorphisms also influence goat milk oligosaccharide (GMO) production and secretion. The GMO compositions of thirty-two goat milk samples, half of which were from genotype A/A (αs1-casein producers) and half from genotype O/O (αs1-casein non-producers), were determined with nanoflow liquid chromatography high-accuracy mass spectrometry. This study represents the most exhaustive characterization of GMO to date. A systematic and comprehensive GMO library was created, consolidating information available in the literature with the new findings. Nearly 30 GMO, 11 of which were novel, were confirmed via tandem mass spectrometric analyses. Six fucosylated OS were identified; 4 of these matched HMO compositions and three were identified for the first time in goat milk. Importantly, multivariate statistical analysis demonstrated that the OS profiles of the A/A and O/O genotype milks could be discriminated by the fucosylated OS. Quantitative analysis revealed that the goat milk samples

  12. Analysis of oligosaccharide sequences of trace Caulophyllum robustum saponins by direct infusion multiple-stage tandem mass spectrometry.

    PubMed

    Xia, Yong-Gang; Liang, Jun; Li, Guo-Yu; Yang, Bing-You; Kuang, Hai-Xue

    2015-08-10

    The saponins in Caulophyllum robustum have not yet been fully characterized. Furthermore these saponins are often present in trace amounts and are structurally complex. Here, a simple direct infusion electrospray ion trap multiple-stage tandem mass spectrometry (DI-ESI-IT-MS(n)) method was described for the characterization of trace C. robustum saponins. Eight reference saponins from the C. robustum hairy root were investigated by DI-ESI-IT-MS(n) in positive ion mode. Some fragmentation approaches were proposed through analysis of the [M+Na](+) ions: (1) preferential cleavage of the C-28 ester glycosidic bond to provide complementary [Y0α+Na](+) and [Bα+Na](+) ions for bidesmosidic saponins; (2) diagnostically neutral loss of CO2 from free carboxyl groups at C-28 for monodesmosidic saponins; and (3) the ion intensity ratio between [C2β+Na](+) and [B2β+Na](+), which is sensitive to the structural differences between the two isomeric β-sugar chains (Glc → (2)Ara and Glc → (3)Ara). The DI-ESI-IT-MS(n) method was successfully used for the analysis of trace C. robustum saponins with [M+Na](+) ions at m/z 1745.6, 1729.5, 1583.7, 1567.7, 1421.7 and 1405.7. This article highlights the discovery and identification of complex α- and β-oligosaccharide moieties in Caulophyllum saponins by glycosidic product ions along with cross ring cleavage product ions. Five oligosaccharide moieties were unambiguously or tentatively identified as Rha → (4)Glc → (6)Glc → (4)Rha → (4)Glc → (6)Glc, Glc → (4)Glc → (6)Glc → (4)Rha → (4)Glc → (6)Glc, Rha → Glc → Glc (Glc) → (2,3)Ara, Glc → Glc (Glc) → (2,3)Ara and Glc (Glc) → (2,3)Ara. Accuracy of the analytical procedure was demonstrated by structural identification of two saponins isolated using 1D and 2D-NMR spectroscopy. The DI-ESI-IT-MS(n) method facilitates rapid discovery and analysis of trace Caulophyllum saponins and is a powerful and practical tool for structural characterization of complex

  13. Effects of oligosaccharides in a soybean meal-based diet on fermentative and immune responses in broiler chicks challenged with Eimeria acervulina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fermentable oligosaccharides, particularly those found in soybean meal (SBM), may modulate fermentation in the ceca, thus affecting intestinal immune responses to intestinal pathogens. We hypothesized that fermentable oligosaccharides found in SBM would positively impact cecal fermentation and inte...

  14. Oligosaccharide Binding Proteins from Bifidobacterium longum subsp. infantis Reveal a Preference for Host Glycans

    PubMed Central

    Garrido, Daniel; Kim, Jae Han; German, J. Bruce; Raybould, Helen E.; Mills, David A.

    2011-01-01

    Bifidobacterium longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota, and it has been characterized by its foraging capacity for human milk oligosaccharides (HMO). Its genome sequence revealed an overabundance of the Family 1 of solute binding proteins (F1SBPs), part of ABC transporters and associated with the import of oligosaccharides. In this study we have used the Mammalian Glycan Array to determine the specific affinities of these proteins. This was correlated with binding protein expression induced by different prebiotics including HMO. Half of the F1SBPs in B. infantis were determined to bind mammalian oligosaccharides. Their affinities included different blood group structures and mucin oligosaccharides. Related to HMO, other proteins were specific for oligomers of lacto-N-biose (LNB) and polylactosamines with different degrees of fucosylation. Growth on HMO induced the expression of specific binding proteins that import HMO isomers, but also bind blood group and mucin oligosaccharides, suggesting coregulated transport mechanisms. The prebiotic inulin induced other family 1 binding proteins with affinity for intestinal glycans. Most of the host glycan F1SBPs in B. infantis do not have homologs in other bifidobacteria. Finally, some of these proteins were found to be adherent to intestinal epithelial cells in vitro. In conclusion, this study represents further evidence for the particular adaptations of B. infantis to the infant gut environment, and helps to understand the molecular mechanisms involved in this process. PMID:21423604

  15. Structure elucidation of two novel yak milk oligosaccharides and their DFT studies

    NASA Astrophysics Data System (ADS)

    Singh, Ashish Kumar; Ranjan, Ashok Kr.; Srivastava, Gaurav; Deepak, Desh

    2016-03-01

    Milk is a primary dynamic biological fluid responsible for development of neonates. Besides the other regular constituents it have oligosaccharides in it which are responsible for antitumor, anticancer, antigenic and immunostimulant activities. In our endeavor to find biologically active novel oligosaccharides, yak milk was taken, which is a rich source of oligosaccharide and its milk is used as antihypertensive, antioxidative and heart strengthening agent in folk medicine. For this purpose yak milk was processed by method of Kobata and Ginsburg followed by gel filtration HPLC and CC which resulted in the isolation of two novel milk oligosaccharides namely (I) Grunniose and (II) Vakose. The structure of purified milk oligosaccharides were elucidated with the help of chemical degradation, chemical transformation, spectroscopic techniques like NMR (1H, 13C and 2D-NMR), structure reporter group theory and mass spectrometry. The optimized geometry of compound Grunniose and Vakose, at B3LYP method and 6-311 + G basis set on Gaussian 09 program, show that the compound Grunniose is lower in energy as compared to compound Vakose.

  16. [Study on intestinal absorption features of oligosaccharides in Morinda officinalis How. with sigle-pass perfusion].

    PubMed

    Deng, Shao-Dong; Zhang, Peng; Lin, Li; Xiao, Feng-Xia; Lin, Jing-Ran

    2015-01-01

    To study the in situ intestinal absorption of five oligosaccharides contained in Morinda officinalis How. (sucrose, kestose, nystose, 1F-Fructofuranosyinystose and Bajijiasu). The absorption of the five oligosaccharides in small intestine (duodenum, jejunum and ileum) and colon of rats and their contents were investigated by using in situ single-pass perfusion model and HPLC-ELSD. The effects of drug concentration, pH in perfusate and P-glycoprotein inhibitor on the intestinal absorption were investigated to define the intestinal absorption mechanism of the five oligosaccharides in rats. According to the results, all of the five oligosaccharides were absorbed in the whole intestine, and their absorption rates were affected by the pH of the perfusion solution, drug concentration and intestinal segments. Verapamil Hydrochloride could significantly increase the absorptive amount of sucrose and Bajijiasu, suggesting sucrose and Bajijiasu are P-gp's substrate. The five oligosaccharides are absorbed mainly through passive diffusion in the intestinal segments, without saturated absorption. They are absorbed well in all intestines and mainly in duodenum and jejunum. PMID:25993803

  17. Effect of the intramolecular hydrogen bond on the spectral and optical properties in chitosan oligosaccharide

    NASA Astrophysics Data System (ADS)

    Li, Xin; Yang, Mengshi; Shi, Xiao; Chu, Xiuxiang; Chen, Liang; Wu, Qiang; Wang, Yueyue

    2015-05-01

    The geometric structures, hydrogen bond types, IR spectra and nonlinear optical properties of chitosan oligosaccharide (degree of polymerization 2-5) are studied by density-functional theory (DFT) at B3LYP/6-31+G(d) level. We have analyzed the statistics of relationship between IR spectra and bond lengths, and angles of amino, hydroxyl. The results show that: (1) the active groups C3-OH, C6-OH and -NH2 can form intramolecular hydrogen bond in chitosan oligosaccharide; (2) the IR spectra of three active groups have size effect in growth process, however, its IR intensity increases significantly and IR frequencies are red shifted obviously when the active hydroxyl form hydrogen bonds, because the bond length of active hydroxyl becomes longer; (3) the effect of hydrogen bond on intensity and frequency of the three vibration mode of amino is the main factor and complication. The paper also provides the nonlinear optical properties of chitosan oligosaccharide. The reason why hydrogen bond can make an appreciable difference to IR spectra, and the nonlinear optical properties of chitosan oligosaccharide are discussed. This research has important significance in the characterization of chitosan oligosaccharide, the properties of chitosan material and hydrogen bond by infrared spectroscopy.

  18. Neutral and acidic milk oligosaccharides of the striped skunk (Mephitidae: Mephitis mephitis).

    PubMed

    Taufik, Epi; Sekii, Nobuhiro; Senda, Akitsugu; Fukuda, Kenji; Saito, Tadao; Eisert, Regina; Oftedal, Olav T; Urashima, Tadasu

    2013-07-01

    The biological significance of the tremendous variation in proportions of oligosaccharides and lactose among mammalian milks is poorly understood. We investigated milk oligosaccharides of the striped skunk (Mephitis mephitis) and compared these results to other species of the clade Mustelida. Individual oligosaccharides were identified by proton nuclear magnetic resonance spectroscopy. In the striped skunk, six oligosaccharides were identified: isoglobotriose, 2'-fucosyllactose, A-tetrasaccharide, Galili pentasaccharide, 3'-sialyllactose and monosialyl monogalactosyl lacto-N-neohexaose. Four of these have been found in related Mustelida and the other two in more distantly related carnivorans. The neutral and acidic oligosaccharides derive from three core structures: lactose (Gal(β1-4)Glc), lacto-N-neotetraose (Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc) and lacto-N-neohexaose (Gal(β1-4)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc). PMID:23607515

  19. Effect of esterified oligosaccharides on the formation and stability of oil-in-water emulsions.

    PubMed

    Udomrati, Sunsanee; Khalid, Nauman; Gohtani, Shoichi; Nakajima, Mitsutoshi; Neves, Marcos A; Uemura, Kunihiko; Kobayashi, Isao

    2016-06-01

    Hydrophobically modified oligosaccharides were prepared by an enzyme-catalyzed reaction of maltodextrin/xylo-oligosaccharide and palmitic acid. Maltodextrin with dextrose equivalent (DE) of 16 palmitate (DE16_P) and 9 palmitate (DE9_P), as well as xylo-oligosaccharide palmitate (Xylo_P), were used. The effect of the concentration (10-50% (w/w)) and type of esterified oligosaccharides on the Sauter mean diameter and droplet-size distribution, the rate of coalescence (Kc), and the creaming properties of O/W emulsions were investigated. Esterified oligosaccharides (EO) adsorbed to the surface of the oil droplets. EO formed polydisperse O/W emulsions with particle sizes between 12 and 70μm, depended on concentration of EO. The Sauter mean diameter, Kc, and the creaming index decreased markedly, with increasing concentration of EO. The type of ester minimally affected the Sauter mean diameter at each ester concentration. DE9_P inhibited coalescence and creaming more efficiently than other EO, mainly due to the higher viscosity of the continuous phase. PMID:27083342

  20. Composition and antioxidant activity of water-soluble oligosaccharides from Hericium erinaceus.

    PubMed

    Hou, Yiling; Ding, Xiang; Hou, Wanru

    2015-05-01

    Oligosaccharide are carbohydrate molecules, comprising repeating units joined together by glycosidic bonds. In recent years, an increasing number of oligosaccharides have been reported to exhibit various biological activities, including antitumor, immune-stimulation and antioxidation effects. In the present study, crude water‑soluble oligosaccharides were extracted from the fruiting bodies of Hericium erinaceus with water and then successively purified by diethylaminoethyl‑cellulose 52 and Sephadex G‑100 column chromatography, yielding one major oligosaccharide fraction: Hericium erinaceus oligosaccharide (HEO‑A). The structural features of HEO‑A were investigated by a combination of monosaccharide component analysis by thin layer chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy, scanning electron microscopy and high‑performance gel permeation chromatography. The results indicated that HEO‑A was composed of D‑xylose and D‑glucose, and the average molecular size was ~1,877 Da. The antioxidant activity of HEO‑A was evaluated using three biochemical methods to determine the scavenging activity of HEO‑A on 1,1‑diphenyl‑2‑picrylhydrazyl, hydrogen peroxide and 2,2'‑azino‑bis(3‑ethylbenzthiazoline‑6‑sufonic acid) diammonium radicals. The results indicated that HEO‑A may serve as an effective healthcare food and source of natural antioxidant compounds. PMID:25529054

  1. Select human milk oligosaccharides directly modulate peripheral blood mononuclear cells isolated from 10-d-old pigs.

    PubMed

    Comstock, Sarah S; Wang, Mei; Hester, Shelly N; Li, Min; Donovan, Sharon M

    2014-03-14

    Infant formulas lack the complex mixture of oligosaccharides found in human milk. These human milk oligosaccharides (HMO) may be pivotal to the development of the neonatal immune system. Few comprehensive analyses of the effects of HMO on immune cells from neonates have been undertaken. Herein, the direct effects of HMO on immune cells were analysed ex vivo. Peripheral blood mononuclear cells (PBMC) isolated from 10-d-old sow-reared (SR) or colostrum-deprived formula-fed (FF) pigs were stimulated for 72 h with single HMO, mixtures of single HMO or a complex mixture of HMO isolated from human milk (iHMO). T-cell phenotype, cytokine production and proliferation were measured by flow cytometry, immunoassay and [³H]thymidine incorporation, respectively. Stimulation with HMO had direct effects on PBMC. For instance, cells stimulated with iHMO produced more IL-10 than unstimulated cells, and cells stimulated with fucosylated HMO tended to proliferate less than unstimulated cells. Additionally, co-stimulation with HMO mixtures or single HMO altered PBMC responses to phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) stimulation. Compared with PBMC stimulated with PHA alone, cells co-stimulated with iHMO and PHA proliferated more and had fewer detectable CD4⁺CD8⁺ T cells. Compared with PBMC stimulated by LPS alone, cells co-stimulated with a mixture of sialylated HMO and LPS proliferated more and tended to have fewer detectable CD4⁺ T cells. Differences in the baseline responses of PBMC isolated from the SR or FF pigs were observed. In summary, HMO directly affected PBMC populations and functions. Additionally, ex vivo measurements of PBMC phenotype, cytokine production and proliferation were influenced by the neonate's diet. PMID:24131853

  2. Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells

    SciTech Connect

    Davidson, D.J.; Fraser, M.J.; Castellino, F.J. )

    1990-06-12

    A comparison has been made between the Asn{sup 289}-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, the authors find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate and truncated high-mannose oligosaccharide. Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. The data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, the authors conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.

  3. Hydrophilic interaction liquid chromatography for the separation, purification, and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz.

    PubMed

    Liang, Tu; Fu, Qing; Li, Fangbing; Zhou, Wei; Xin, Huaxia; Wang, Hui; Jin, Yu; Liang, Xinmiao

    2015-08-01

    A systematic strategy based on hydrophilic interaction liquid chromatography was developed for the separation, purification and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz. Methods with enough hydrophilicity and selectivity were utilized to resolve the problems encountered in the separation of oligosaccharides such as low retention, low resolution and poor solubility. The raffinose family oligosaccharides in L. lucidus Turcz. were isolated using solid-phase extraction followed by hydrophilic interaction liquid chromatography at semi-preparative scale to obtain standards of stachyose, verbascose and ajugose. Utilizing the obtained oligosaccharides as standards, a quantitative determination method was developed, validated and applied for the content determination of raffinose family oligosaccharides both in the aerial and root parts of L. lucidus Turcz. There were no oligosaccharides in the aerial parts, while in the root parts, the total content was 686.5 mg/g with the average distribution: raffinose 66.5 mg/g, stachyose 289.0 mg/g, verbascose 212.4 mg/g, and ajugose 118.6 mg/g. The result provided the potential of roots of L. lucidus Turcz. as new raffinose family oligosaccharides sources for functional food. Moreover, since the present systematic strategy is efficient, sensitive and robust, separation, purification and quantification of oligosaccharides by hydrophilic interaction liquid chromatography seems to be possible. PMID:26011699

  4. Understanding sugar yield loss and enzyme inhibition due to oligosaccharides accumulation during high solids-loading enzymatic hydrolisis.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During enzymatic hydrolysis of biomass, polysaccharides are cleaved by glycosyl hydrolases to soluble oligosaccharides and further hydrolyzed by ß-glucosidase, ß-xylosidase and other enzymes to monomeric sugars. However, not all oligosaccharides can be fully hydrolyzed and they may accumulate to 18-...

  5. Oligosaccharide binding to barley alpha-amylase 1.

    PubMed

    Robert, Xavier; Haser, Richard; Mori, Haruhide; Svensson, Birte; Aghajari, Nushin

    2005-09-23

    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers." PMID:16030022

  6. Expeditious oligosaccharide synthesis via selective, semi-orthogonal, and orthogonal activation

    PubMed Central

    Kaeothip, Sophon; Demchenko, Alexei V.

    2011-01-01

    Traditional strategies for oligosaccharide synthesis often require extensive protecting and/or leaving group manipulations between each glycosylation step, thereby increasing the total number of synthetic steps while decreasing the efficiency of the synthesis. In contrast, expeditious strategies allow for the rapid chemical synthesis of complex carbohydrates by minimizing extraneous chemical manipulations. Oligosaccharide synthesis by selective activation of one leaving group over another is one such expeditious strategy. Herein, the significant improvements that have recently emerged in the area of the selective activation are discussed. The development of orthogonal strategy further expands the scope of the selective activation methodology. Surveyed in this article, are representative examples wherein these excellent innovations have been applied to the synthesis of various oligosaccharide sequences. PMID:21663897

  7. Formation of Lipid-Bound Oligosaccharides Containing Mannose. Their Role in Glycoprotein Synthesis

    PubMed Central

    Behrens, Nicolas H.; Carminatti, Hector; Staneloni, Roberto J.; Leloir, Luis F.; Cantarella, Ana I.

    1973-01-01

    Incubation of GDP-[14C]mannose with liver microsomes and magnesium ions led to the formation of dolichol monophosphate mannose and of other acidlabile compounds that contain oligosaccharides. Formation of these compounds was enhanced by addition of an acceptor lipid in the same fractions of DEAE-cellulose chromatography where bound dolichol is found. Alkaline treatment of the oligosaccharides, obtained by acid methanolysis, followed by paper electrophoresis, gave rise to the formation of two positively charged substances believed to be formed by deacetylation of hexosamine residues. Incubation of the oligosaccharide-containing compounds with liver microsomes and manganese ions resulted in a transfer to endogenous protein. The role of dolichol derivatives in glycoprotein synthesis is discussed. PMID:4519632

  8. Tumor cell surface beta 1-6 branched oligosaccharides and lung metastasis.

    PubMed

    Lu, Y; Pelling, J C; Chaney, W G

    1994-01-01

    NIH3T3 cells transfected with an activated Ha-ras oncogene were treated with L-PHA, the leukoagglutinin from red kidney beans. Cell lines resistant to L-PHA-mediated cytotoxicity were isolated and found to contain reduced levels of L-PHA-binding oligosaccharides. The levels of N-acetylglucosaminyltransferase V, the enzyme responsible for the initiation of the beta 1-6 branch, were reduced in L-PHA-resistant cells. Tumorigenicity in nude mice was unchanged by the change in oligosaccharide expression, but the ability to form lung tumors after intravenous injection was significantly reduced. These results demonstrate that the ability of NIH3T3 cells transfected with an activated Ha-ras oncogene to form lung tumors after intravenous injection into nude mice is reduced in all six L-PHA selected cell lines containing a reduction in beta 1-6 branched Asn-linked oligosaccharides. PMID:8287620

  9. Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis of hyaluronan oligosaccharides

    PubMed Central

    Sakai, Shinobu; Hirano, Kana; Toyoda, Hidenao; Linhardt, Robert J.; Toida, Toshihiko

    2014-01-01

    A new method is presented for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronan (HA) with bacterial hyaluronidase (E.C. 4.2.2.1, from Streptomyces hyalurolyticus) using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS). Mixtures containing HA oligosaccharides of tetrasaccharide (4-mer)–34-mer were analyzed using this method. The carboxyl groups of the glucuronate residues in the prepared HA oligomers, were modified as the acidic form (—COOH), sodium salts (—COONa), organic ammonium salts, or methylesters before MALDI-TOFMS measurement. Among these samples, the methylester form of glucuronate residues in HA oligosaccharides, prepared by methylation using trimethylsilyl diazomethane, afforded high sensitivity for spectra. This simple modification method for carboxyl group methylation of acidic polysaccharides [Hirano et al., Carbohydr. Res., 340, (2005) 2297–2304] provides samples suitable for MALDI-TOF mass spectrometric analysis throughout a significantly enhanced range of masses. PMID:17543609

  10. Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis.

    PubMed Central

    Rice, K G; Rottink, M K; Linhardt, R J

    1987-01-01

    Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE). The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation. Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size. High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c. and were used to help characterize the gradient PAGE separation. Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively. The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed. Images Fig. 3. Fig. 5. Fig. 7. PMID:3446173

  11. Isolation, structure elucidation and DFT study on two novel oligosaccharides from yak milk

    NASA Astrophysics Data System (ADS)

    Singh, Meenakshi; Kumar, Alok; Srivastava, Gaurav; Deepak, Desh; Singh, M. P. V. V.

    2016-08-01

    Two novel oligosaccharides were isolated from yak milk. The milk was processed by the method of Kobata and Ginsberg involving deproteination, centrifugation and lyophilization followed by gel filtrate chromatography acetylation and silica gel column chromatography of derivatized oligosaccharides while their homogeneity was confirmed by HPLC. The structures of these isolated oligosaccharides were elucidated by chemical transformation, chemical degradation, 1H, 13C NMR, 2D NMR (COSY, TOCSY and HSQC) and mass spectrometry. The geometry of compound A (Bosiose) and B (Bovisose) have been optimized at B3LYP method and 6-311 + G(d,p) basis set. The difference between the energies of A and B is 1.269 a.u. or 796.309 kcal/mol.

  12. Mechanisms involved in enhancement of the expression and function of aggrecanases by hyaluronan oligosaccharides

    PubMed Central

    Ariyoshi, Wataru; Takahashi, Nobunori; Hida, Daisuke; Knudson, Cheryl B.; Knudson, Warren

    2011-01-01

    Objective Small hyaluronan (HA) oligosaccharides serve as competitive receptor antagonists to displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and induces stimulation of genes involved in matrix degradation such as matrix metalloproteinases as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2. The objective of this study was to determine changes in the expression and function of aggrecanases after disruption of chondrocyte CD44-HA interactions. Methods Bovine articular chondrocytes or bovine cartilage tissue were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in aggrecanase were monitored by real time reverse transcriptase-polymerase chain reaction and western blot analysis of ADAMTS4, ADAMTS5 and aggrecan proteolytic fragments. To test the interactions between ADAMTS4 and MT4-MMP, protein lysates purified from stimulated chondrocytes were subjected to co-immunoprecipitation. Results Disruption of chondrocyte CD44-HA interactions with HA oligosaccharides induced the transcription of ADAMTS4 and ADAMTS5 in time- and dose-dependent manner. The association of GPI-anchored MT4-MMP with ADAMTS4 was also induced in articular chondrocytes by HA oligosaccharides. Inhibition of the NF-κB pathway blocked HA oligosaccharides-mediated stimulation of aggrecanases. Conclusions Disruptive changes in chondrocyte-matrix interactions by HA oligosaccharides induce matrix degradation and elevate aggrecanases via the activation of the NF-κB signaling pathway. PMID:21905012

  13. Simultaneous analysis of monosaccharides and oligosaccharides by high-performance liquid chromatography with postcolumn fluorescence derivatization.

    PubMed

    Kakita, Hirotaka; Kamishima, Hiroshi; Komiya, Katsuo; Kato, Yoshio

    2002-06-28

    To develop a fluorimetric HPLC technique for the simultaneous microanalysis of reducing mono- and oligosaccharides, the technique of linear gradient elution was introduced into the postcolumn fluorimetric detemination system of reducing saccharides with benzamidine. Fluorescence measurement was performed at 288 nm for excitation and 470 nm for emission and an optimization study for this postcolumn fluorescence derivatization carried out. Under optimum conditions, the detection limits of D-glucose and maltohexaose were 1.78 and 2.59 pmol, respectively. The present method was successfully applied to saccharide analysis and should prove useful for automated simultaneous microanalysis of reducing mono- and oligosaccharides in foods. PMID:12186393

  14. Anomeric Reactivity-Based One-Pot Synthesis of Heparin-Like Oligosaccharides

    PubMed Central

    Polat, Tülay

    2008-01-01

    A highly efficient one-pot methodology is described for the synthesis of heparin and heparan sulfate oligosaccharides utilizing thioglycosides with well defined reactivity as building blocks. l-idopyranosyl and d-glucopyranosyl thioglycosides 5 and 10 were used as donors due to low reactivity of uronic acids as the glycosyl donors in the one-pot synthesis. The formation of uronic acids by a selective oxidation at C-6 was performed after assembly of the oligosaccharides. The efficiency of this strategy with the flexibility for sulfate incorporation was demonstrated in the representative synthesis of disaccharides 17, 18, tetrasaccharide 23 and pentasaccharide 26. PMID:17914818

  15. Cooperative Interactions of Oligosaccharide and Peptide Moieties of a Glycopeptide Derived from IgE with Galectin-9.

    PubMed

    Nakakita, Shin-Ichi; Itoh, Aiko; Nakakita, Yukari; Nonaka, Yasuhiro; Ogawa, Takashi; Nakamura, Takanori; Nishi, Nozomu

    2016-01-01

    We previously showed that galectin-9 suppresses degranulation of mast cells through protein-glycan interaction with IgE. To elucidate the mechanism of the interaction in detail, we focused on identification and structural analysis of IgE glycans responsible for the galectin-9-induced suppression using mouse monoclonal IgE (TIB-141). TIB-141 in combination with the antigen induced degranulation of RBL-2H3 cells, which was almost completely inhibited by human and mouse galectin-9. Sequential digestion of TIB-141 with lysyl endopeptidase and trypsin resulted in the identification of a glycopeptide (H-Lys13-Try3; 48 amino acid residues) with a single N-linked oligosaccharide near the N terminus capable of neutralizing the effect of galectin-9 and another glycopeptide with two N-linked oligosaccharides (H-Lys13-Try1; 16 amino acid residues) having lower activity. Enzymatic elimination of the oligosaccharide chain from H-Lys13-Try3 and H-Lys13-Try1 completely abolished the activity. Removal of the C-terminal 38 amino acid residues of H-Lys13-Try3 with glutamyl endopeptidase, however, also resulted in loss of the activity. We determined the structures of N-linked oligosaccharides of H-Lys13-Try1. The galectin-9-binding fraction of pyridylaminated oligosaccharides contained asialo- and monosialylated bi/tri-antennary complex type oligosaccharides with a core fucose residue. The structures of the oligosaccharides were consistent with the sugar-binding specificity of galectin-9, whereas the nonbinding fraction contained monosialylated and disialylated biantennary complex type oligosaccharides with a core fucose residue. Although the oligosaccharides linked to H-Lys13-Try3 could not be fully characterized, these results indicate the possibility that cooperative binding of oligosaccharide and neighboring polypeptide structures of TIB-141 to galectin-9 affects the overall affinity and specificity of the IgE-lectin interaction. PMID:26582205

  16. Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae β-Galactosidase.

    PubMed

    Cardelle-Cobas, Alejandra; Olano, Agustin; Irazoqui, Gabriela; Giacomini, Cecilia; Batista-Viera, Francisco; Corzo, Nieves; Corzo-Martínez, Marta

    2016-01-01

    β-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP) ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae β-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with β-galactosidase immobilized onto a glutaraldehyde-agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP ≥ 3 were 50 °C, pH 6.5, 450 g/L of lactulose, and 8 U/mL of enzyme, reaching yields of ca. 50% (w/v) of total OsLu and ca. 20% (w/v) of OsLu with DP 3, being 6'-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60 °C, pH 4.5, 450 g/L of lactulose and 8 U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae β-galactosidase immobilized on a support of glutaraldehyde-agarose constitutes an efficient and cost-effective alternative to the use of soluble β-galactosidases for the synthesis of prebiotic OsLu mixtures. PMID:27014684

  17. Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae β-Galactosidase

    PubMed Central

    Cardelle-Cobas, Alejandra; Olano, Agustin; Irazoqui, Gabriela; Giacomini, Cecilia; Batista-Viera, Francisco; Corzo, Nieves; Corzo-Martínez, Marta

    2016-01-01

    β-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP) ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae β-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with β-galactosidase immobilized onto a glutaraldehyde–agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP ≥ 3 were 50 °C, pH 6.5, 450 g/L of lactulose, and 8 U/mL of enzyme, reaching yields of ca. 50% (w/v) of total OsLu and ca. 20% (w/v) of OsLu with DP 3, being 6′-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60 °C, pH 4.5, 450 g/L of lactulose and 8 U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae β-galactosidase immobilized on a support of glutaraldehyde–agarose constitutes an efficient and cost-effective alternative to the use of soluble β-galactosidases for the synthesis of prebiotic OsLu mixtures. PMID:27014684

  18. Lacto N Tetraose, Fucosylation, and Secretor Status are Highly Variable in Human Milk Oligosaccharides From Women Delivering Preterm

    PubMed Central

    De Leoz, Maria Lorna A.; Gaerlan, Stephanie C.; Strum, John S.; Dimapasoc, Lauren M.; Mirmiran, Majid; Tancredi, Daniel J.; Smilowitz, Jennifer T.; Kalanetra, Karen M.; Mills, David A.; German, J. Bruce; Lebrilla, Carlito B.; Underwood, Mark A.

    2012-01-01

    Breast milk is the ideal nutrition for term infants but must be supplemented to provide adequate growth for most premature infants. Human milk oligosaccharides (HMOs) are remarkably abundant and diverse in breast milk and yet provide no nutritive value to the infant. HMOs appear to have at least two major functions: prebiotic activity (stimulation of the growth of commensal bacteria in the gut) and protection against pathogens. Investigations of HMOs in milk from women delivering preterm have been limited. We present the first detailed mass spectrometric analysis of the fucosylation and sialylation in HMOs in serial specimens of milk from fifteen women delivering preterm and seven women delivering at term using nano-high performance liquid chromatography chip/time-of-flight mass spectrometry. A mixed-effects model with Levene’s test was used for the statistical analyses. We find that lacto-N-tetraose, a core HMO, is both more abundant and more highly variable in the milk of women delivering preterm. Furthermore, fucosylation in preterm milk is not as well regulated as in term milk, resulting in higher within and between mother variation in women delivering preterm vs. term. Of particular clinical interest, the α1,2-linked fucosylated oligosaccharide 2′-fucosyllactose, an indicator of secretor status, is not consistently present across lactation of several mothers that delivered preterm. The immaturity of HMO production does not appear to resolve over the time of lactation and may have relevance to the susceptibility of premature infants to necrotizing enterocolitis, late onset sepsis, and related neurodevelopmental impairments. PMID:22900748

  19. Prebiotic Oligosaccharides: Comparative Evaluation Using In Vitro Cultures of Infants' Fecal Microbiomes

    PubMed Central

    Stiverson, J.; Williams, T.; Chen, J.; Adams, S.; Hustead, D.; Price, P.; Guerrieri, J.; Deacon, J.

    2014-01-01

    The objective of this study was to systematically assess the bifidogenic effect of three commonly used prebiotic products using in vitro cultures of infant fecal samples. Fresh stool samples collected from six term infants, each exclusively fed human milk (n = 3) or infant formula (n = 3), at 28 days of age were used as inocula. The following prebiotic products were added at concentrations applicable to infant formula: Vivinal GOS 15 (containing 28.5% galacto-oligosaccharide [GOS]) at 7.2 g/liter, Beneo HP (99.5% long-chain inulin [IN]) at 0.8 g/liter, Beneo Synergy 1 (enriched oligofructose and inulin [OF-IN]) at 4 g/liter, and a combination of Vivinal GOS 15 (7.2 g/liter) and Beneo HP (0.8 g/liter) (GOS-IN). The growth of total bacteria, Bifidobacterium, Bacteroides, Bifidobacterium longum, and Escherichia coli was quantified using specific quantitative PCR (qPCR). Bifidobacterium was also enumerated on selective Beerens agar plates, with representative colonies identified by sequencing of their 16S rRNA genes. Volatile fatty acids (VFA) and pH in the cultures were also determined. Irrespective of the feeding methods, the GOS product, either alone or in combination with Beneo HP, resulted in substantially higher growth of total bifidobacteria, and much of this growth was attributed to growth of B. longum. Beneo Synergy 1 also increased the abundance of total bifidobacteria and B. longum. Corresponding to the increases in these two bacterial groups, acetic acid concentrations were higher, while there was a trend of lower E. coli levels and pH. The lower pH and higher acetic acid concentration might be directly responsible for the lower E. coli population. At the concentrations studied, the GOS product was more bifidogenic and potent in inhibiting E. coli than the other products tested. These results suggest that supplementation of infant formula with GOS may increase intestinal bifidobacteria and benefit infant health. PMID:25239906

  20. Arabinoxylan Oligosaccharide Hydrolysis by Family 43 and 51 Glycosidases from Lactobacillus brevis DSM 20054

    PubMed Central

    Hell, Johannes; Lorenz, Cindy; Böhmdorfer, Stefan; Rosenau, Thomas; Kneifel, Wolfgang

    2013-01-01

    Due to their potential prebiotic properties, arabinoxylan-derived oligosaccharides [(A)XOS] are of great interest as functional food and feed ingredients. While the (A)XOS metabolism of Bifidobacteriaceae has been extensively studied, information regarding lactic acid bacteria (LAB) is still limited in this context. The aim of the present study was to fill this important gap by characterizing candidate (A)XOS hydrolyzing glycoside hydrolases (GHs) identified in the genome of Lactobacillus brevis DSM 20054. Two putative GH family 43 xylosidases (XynB1 and XynB2) and a GH family 43 arabinofuranosidase (Abf3) were heterologously expressed and characterized. While the function of XynB1 remains unclear, XynB2 could efficiently hydrolyze xylooligosaccharides. Abf3 displayed high specific activity for arabinobiose but could not release arabinose from an (A)XOS preparation. However, two previously reported GH 51 arabinofuranosidases from Lb. brevis were able to specifically remove α-1,3-linked arabinofuranosyl residues from arabino-xylooligosaccharides (AXHm3 specificity). These results imply that Lb. brevis is at least genetically equipped with functional enzymes in order to hydrolyze the depolymerization products of (arabino)xylans and arabinans. The distribution of related genes in Lactobacillales genomes indicates that GH 43 and, especially, GH 51 glycosidase genes are rare among LAB and mainly occur in obligately heterofermentative Lactobacillus spp., Pediococcus spp., members of the Leuconostoc/Weissella branch, and Enterococcus spp. Apart from the prebiotic viewpoint, this information also adds new perspectives on the carbohydrate (i.e., pentose-oligomer) metabolism of LAB species involved in the fermentation of hemicellulose-containing substrates. PMID:23995921

  1. Role of xylo-oligosaccharides in protection against salinity-induced adversities in Chinese cabbage.

    PubMed

    Chen, Weiwei; Guo, Chen; Hussain, Saddam; Zhu, Bingxin; Deng, Fang; Xue, Yan; Geng, Mingjian; Wu, Lishu

    2016-01-01

    Soil salinity is a stringent abiotic constraint limiting crop growth and productivity. The present study was carried out to appraise the role of xylo-oligosaccharides (XOSs) in improving the salinity tolerance of Chinese cabbage. Salinity stress (0.5% NaCl solution) and four levels (0, 40, 80, 120 mg L(-1)) of XOSs were imposed on 20-day-old plants cultured under controlled conditions. Salinity stress decreased the aboveground fresh biomass, photosynthesis, transpiration rate, stomatal conductance, internal CO2 concentration, water use efficiency, and chlorophyll contents but increased the stomatal limitation value of Chinese cabbage compared with control. Such physiological interferences, disturbances in plant water relations, and visually noticeable growth reductions in Chinese cabbage were significantly alleviated by the addition of XOSs under salinity stress. Under salinity stress, application of XOSs significantly enhanced the activities of enzymatic (superoxide dismutase, peroxidase, catalase) and non-enzymatic (ascorbate, carotene) antioxidants and reduced the malondialdehyde content in the leaves of Chinese cabbage. The XOS-applied plants under salinity stress also recorded higher soluble sugars, proline, and soluble protein content in their leaves. Exposure of salinity stress increased the ratio of Na(+)/K(+), Na(+)/Ca(2+), and Na(+)/Mg(2+) in shoot as well as root of Chinese cabbage, however, XOS application significantly reduced these ratios particularly in shoot. Lower levels of XOSs (40 or 80 mg L(-1)) were more effective for most of the studied attributes. The greater salinity tolerance and better growth in these treatments were related with enhanced antioxidative defense system, reduced lipid peroxidation, increased osmolyte accumulation, and maintenance of ionic balance. PMID:26358207

  2. Structure of a Sialo-Oligosaccharide from Glycophorin in Carp Red Blood Cell Membranes

    PubMed Central

    Aoki, Takahiko; Chimura, Kenji; Sugiura, Hikaru; Mizuno, Yasuko

    2014-01-01

    We isolated a high-purity carp glycophorin from carp erythrocyte membranes and prepared the oligosaccharide fraction from glycophorin by β-elimination [1]. The oligosaccharide fraction was separated into two components (P-1 and P-2) using a Glyco-Pak DEAE column. These O-linked oligosaccharides (P-1 and P-2) were composed of glucose, galactose, fucose, N-acetylgalactosamine and N-glycolylneuraminic acid (NeuGc). The P-1 and P-2 contained one and two NeuGc residues, respectively, and the P-1 exhibited bacteriostatic activity [1]. Using NMR and GC-MS, we determined that the structure of the bacteriostatic P-1 was NeuGcα2→6 (Fucα1→4) (Glcα1→3) Galβ1→4GalNAc-ol. This O-linked oligosaccharide was unique for a vertebrate with respect to the hexosamine and hexose linkages and its non-chain structure. PMID:25402951

  3. Is There a Role for Oligosaccharides in Seed Longevity? An Assessment of Intracellular Glass Stability1

    PubMed Central

    Buitink, Julia; Hemminga, Marcus A.; Hoekstra, Folkert A.

    2000-01-01

    We examined whether oligosaccharides extend seed longevity by increasing the intracellular glass stability. For that purpose, we used a spin probe technique to measure the molecular mobility and glass transition temperature of the cytoplasm of impatiens (Impatiens walleriana) and bell pepper (Capsicum annuum) seeds that were osmo-primed to change oligosaccharide content and longevity. Using saturation transfer electron paramagnetic resonance spectroscopy, we found that the rotational correlation time of the polar spin probe 3-carboxy-proxyl in the cytoplasm decreased, together with longevity, as a function of increasing seed water content, suggesting that longevity may indeed be regulated by cytoplasmic mobility. Osmo-priming of the seeds resulted in considerable decreases in longevity and oligosaccharide content, while the sucrose content increased. No difference in the glass transition temperature was found between control and primed impatiens seeds at the same temperature and water content. Similarly, there was no difference in the rotational motion of the spin probe in the cytoplasm between control and primed impatiens and bell pepper seeds. We therefore conclude that oligosaccharides in seeds do not affect the stability of the intracellular glassy state, and that the reduced longevity after priming is not the result of increased molecular mobility in the cytoplasm. PMID:10759518

  4. Digestive and physiological effects of a wheat bran extract, arabino-xylan-oligosaccharide, in breakfast cereal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We assessed whether a wheat bran extract containing arabino-xylan-oligosaccharide (AXOS) elicited a prebiotic effect and influenced other physiologic parameters when consumed in ready-to-eat cereal at two dose levels. This double-blind, randomized, controlled, crossover trial evaluated the effects o...

  5. In Vitro Fermentation of Oligosaccharides from Raffinose and Alternansucrase by Human Intestinal Bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The alternansucrases are enzymes involved in the synthesis of oligosaccharides with alternating glycosidic alpha-(1'3), alpha-(1'6) bonds by reactions where there is a donor of glucosyl residues (sucrose) and an acceptor. There are many carbohydrates that have been used as acceptors, including raff...

  6. A new family of oligosaccharides from the xyloglucan of Hymenaea courbaril L. (Leguminosae) cotyledons.

    PubMed

    Buckeridge, M S; Crombie, H J; Mendes, C J; Reid, J S; Gidley, M J; Vieira, C C

    1997-09-01

    The xyloglucan from cotyledons of Hymenaea courbaril was hydrolysed with endo-(1,4)-beta-D-glucanase (cellulase) and analysed by TLC and HPAEC. The limit digest was different from those obtained from xyloglucans of Tamarindus indica and Copaifera langsdorffii. On treatment with nasturtium beta-galactosidase, two main oligosaccharides were detected by TLC and HPAEC. Using a process of enzymatic sequencing involving alternate treatments with a pure xyloglucan oligosaccharide-specific alpha-xylosidase, and a pure beta-glucosidase, both from nasturtium, their structures were deduced to be XXXG and a new oligosaccharide XXXXG. These structures were confirmed by 1H NMR. The relative proportions of XXXG and XXXXG indicate that approximately half of the subunits in Hymenaea xyloglucan are based on the new oligosaccharides. In the native polymer the XXXXG subunits are likely to carry galactosyl substituents in varying proportions, since cellulase hydrolysates contained many bands which were converted to XXXXG on hydrolysis with nasturtium beta-galactosidase. Although no comparative studies on the physico-chemical properties of Hymenaea courbaril xyloglucan have yet been performed, our results indicate that this polymer is less interactive with iodine when compared with T. indica and C. langsdorffii xyloglucans, suggesting that changes in conformation may occur due to the presence of XXXXG. PMID:9352637

  7. Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies.

    PubMed

    Totten, Sarah M; Wu, Lauren D; Parker, Evan A; Davis, Jasmine C C; Hua, Serenus; Stroble, Carol; Ruhaak, L Renee; Smilowitz, Jennifer T; German, J Bruce; Lebrilla, Carlito B

    2014-12-01

    Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures. PMID:25358913

  8. The antioxidant effects of complexes of tilapia fish skin collagen and different marine oligosaccharides

    NASA Astrophysics Data System (ADS)

    Ren, Shuwen; Li, Jing; Guan, Huashi

    2010-12-01

    An excess of reactive oxygen species (ROS) leads to a variety of chronic health problems. As potent antioxidants, marine bioactive extracts containing oligosaccharides and peptides have been extensively studied. Recently, there is a growing interest in protein-polysaccharide complexes because of their potential uses in pharmaceutical and food industries. However, only few studies are available on the antioxidant activities of such complexes, in terms of their ROS scavenging capability. In this study, we combined different marine oligosaccharides (isolated and purified) with collagen peptides derived from tilapia fish skin, and evaluated the antioxidant activity of the marine peptide-oligosaccharide complexes vis-à-vis the activity of their original component molecules. Biochemical and cellular assays were performed to measure the scavenging effects on 1, 1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide radicals, and to evaluate the influences on the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the level of malondialdehyde (MDA) in UV-induced photoaging models. The results indicated that the antioxidant activities of all the complexes were stronger than those of their individual components. Among the 11 complexes tested, two complexes, namely MA1000+CP and κ-ca3000+CP, turned out to be highly effective antioxidants. Although the detailed mechanisms of this improved scavenging ability are not fully understood, this work provides insights into the design of highly efficient peptide-oligosaccharide complexes for potential applications in pharmaceutical, cosmetics and food industries.

  9. Effect of oligosaccharides on the adhesion of gut bacteria to human HT-29 cells.

    PubMed

    Altamimi, M; Abdelhay, O; Rastall, R A

    2016-06-01

    The influence of five oligosaccharides (cellobiose, stachyose, raffinose, lactulose and chito-oligosaccharides) on the adhesion of eight gut bacteria (Bifidobacterium bifidum ATCC 29521, Bacteroides thetaiotaomicron ATCC 29148D-5, Clostridium leptum ATCC 29065, Blautia coccoides ATCC 29236, Faecalibacterium prausnitzii ATCC 27766, Bacteroides fragilis ATCC 23745, Clostridium difficile ATCC 43255 and Lactobacillus casei ATCC 393) to mucous secreting and non-mucous secreting HT-29 human epithelial cells, was investigated. In pure culture, the bacteria showed variations in their ability to adhere to epithelial cells. The effect of oligosaccharides diminished adhesion and the presence of mucus played a major factor in adhesion, likely due to high adhesiveness to mucins present in the native human mucus layer covering the whole cell surface. However, clostridia displayed almost the same level of adhesion either with or without mucus being present. Bl. coccoides adhesion was decreased by stachyose and cellobiose in non-mucus-secreting cells in pure culture, while in mixed faecal culture cellobiose displayed the highest antiadhesive activity with an overall average of 65% inhibition amongst tested oligomers and lactulose displayed the lowest with an average of 47.4%. Bifidobacteria, Bacteroides, lactobacilli and clostridia were inhibited within the following ranges 47-78%, 32-65%, 11.7-58% and 64-85% respectively. This means that clostridia were the most strongly influenced members of the microflora amongst the bacterial groups tested in mixed culture. In conclusion, introducing oligosaccharides which are candidate prebiotics into pure or mixed cultures has affected bacterial adhesion. PMID:27018325

  10. Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research*

    PubMed Central

    Pedersen, Henriette L.; Fangel, Jonatan U.; McCleary, Barry; Ruzanski, Christian; Rydahl, Maja G.; Ralet, Marie-Christine; Farkas, Vladimir; von Schantz, Laura; Marcus, Susan E.; Andersen, Mathias C. F.; Field, Rob; Ohlin, Mats; Knox, J. Paul; Clausen, Mads H.; Willats, William G. T.

    2012-01-01

    Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes. PMID:22988248

  11. Lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells.

    PubMed

    Boomkamp, Stephanie D; Rountree, J S Shane; Neville, David C A; Dwek, Raymond A; Fleet, George W J; Butters, Terry D

    2010-04-01

    Sandhoff and Tay-Sachs disease are autosomal recessive GM2 gangliosidoses where a deficiency of lysosomal beta-hexosaminidase results in storage of glycoconjugates. Imino sugar (2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol) inhibition of beta-hexosaminidase in murine RAW264.7 macrophage-like cells led to lysosomal storage of glycoconjugates that were characterised structurally using fluorescence labelling of the free or glycolipid-derived oligosaccharides followed by HPLC and mass spectrometry. Stored glycoconjugates were confirmed as containing non-reducing GlcNAc or GalNAc residues resulting from the incomplete degradation of N-linked glycoprotein oligosaccharide and glycolipids, respectively. When substrate reduction therapeutics N-butyl-deoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ) were applied to the storage phenotype cells, an increase in glucosylated and galactosylated oligosaccharide species was observed due to endoplasmic reticulum alpha-glucosidases and lysosomal beta-galactosidase inhibition, respectively. Hexosaminidase inhibition triggered a tightly regulated cytokine-mediated inflammatory response that was normalised using imino sugars NB-DNJ and NB-DGJ, which restored the GM2 ganglioside storage burden but failed to reduce the levels of GA2 glycolipid or glycoprotein-derived N-linked oligosaccharides. Using a chemically induced gangliosidosis phenotype that can be modulated with substrate lowering drugs, the critical role of GM2 ganglioside in the progression of inflammatory disease is also demonstrated. PMID:20186478

  12. Sugar loss and enzyme inhibition due to oligosaccharides accumulation during high solids-loading enzymatic hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, ammonia fiber expansion: AFEX and extractive ammonia: EA). The methodology for large-scale separation of ...

  13. Type analysis of the oligosaccharide chains on microheterogeneous components of bovine pancreatic DNAase by the lectin-nitrocellulose sheet method.

    PubMed Central

    Kijimoto-Ochiai, S; Katagiri, Y U; Hatae, T; Okuyama, H

    1989-01-01

    The oligosaccharide chains of microheterogeneous bovine pancreatic DNAases were characterized by the lectin-nitrocellulose sheet method. The active fractions of the DNAases from column chromatography showed four major and several minor spots on a two-dimensional polyacrylamide gel. They were transferred on to nitrocellulose sheets and treated with glycosidases (neuraminidase, endo-beta-N-acetyl glucosaminidase H or F, or peptide N-glycosidase F) and treated with peroxidase-coupled lectins (concanavalin A, Ricinus communis agglutinin or wheat-germ agglutinin). From the results, the most probable oligosaccharide types were proposed to be as follows: the four major spots contained components which had high-mannose type or hybrid-type oligosaccharides, such as those susceptible to endo-beta-N-acetylglucosaminidase H. In addition, spot 1 contained a complex-type biantennary oligosaccharide without sialic acid and spot 3 contained a tri- or tetra-antennary complex-type oligosaccharide with sialic acid. The component corresponding to spot 2 had a hybrid-type oligosaccharide chain with a 'bisecting' acetylglucosamine, linked 1-4 to the beta-mannose residue of the trimannosyl core, and the component corresponding to spot 4 had a high-mannose-type oligosaccharide chain. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2920025

  14. Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free alpha-subunits

    SciTech Connect

    Gesundheit, N.; Gyves, P.W.; DeCherney, G.S.; Stannard, B.S.; Winston, R.L.; Weintraub, B.D.

    1989-06-01

    Mouse hemipituitaries in vitro secrete TSH, composed of an alpha-beta heterodimer, as well as excess (free) alpha-subunits. By dual metabolic labeling with (35S)sulfate and (3H)mannose, we have characterized oligosaccharides from secreted TSH alpha, TSH beta, and free alpha-subunits released from the apoprotein by enzymatic deglycosylation. Oligosaccharides from each subunit displayed a distinct anion exchange HPLC profile due to a specific pattern of sialylation and sulfation. Six species were obtained from TSH alpha (with two glycosylation sites), including neutral oligosaccharides as well as those with one or two negative charges. For TSH beta (with one glycosylation site) at least eight oligosaccharide species were noted, representing nearly every permutation of sialylation and sulfation; approximately 30% contained three or more negative charges. Analysis of (3H)mannose-labeled oligosaccharides on Concanavalin-A-agarose showed 85% binding for those from TSH alpha, 70% for free alpha, and 50% for those from TSH beta. These data demonstrate that oligosaccharides from secreted TSH beta were more sialylated and sulfated, consistent with a more complex branching pattern, than those from TSH alpha. Oligosaccharides from free alpha-subunit were more sialylated than those from TSH alpha, and the net negative charge was intermediate between those of TSH alpha and TSH beta. Although great microheterogeneity is present even at the single glycosylation site on the beta-subunit of secreted TSH, a pattern of sialylation and sulfation could be discerned.

  15. Structures of two novel, serologically nonrelated core oligosaccharides of Yokenella regensburgei lipopolysaccharides differing only by a single hexose substitution.

    PubMed

    Niedziela, Tomasz; Jachymek, Wojciech; Lukasiewicz, Jolanta; Maciejewska, Anna; Andersson, Rolf; Kenne, Lennart; Lugowski, Czeslaw

    2010-02-01

    Immunochemical analysis of the Yokenella regensburgei lipopolysaccharides (LPS) indicated the presence of the core oligosaccharide-related immunotypes among the investigated strains. The structure of the core oligosaccharide segment of the Y. regensburgei LPS has been investigated using chemical methods, mass spectrometry, and (1)H, (13)C NMR spectroscopy. It was concluded that the core oligosaccharides of the strains PCM 2476 and PCM 2477 are composed of an undecasaccharide. The combined data revealed two immunotypes of the core oligosaccharide recognized by antibodies against the whole bacterial cells. The structural differences between the core oligosaccharides are limited to the outermost terminal hexopyranose residue. In the core oligosaccharide of the strain PCM 2476, it was identified as alpha-d-Glcp and in that of the strain PCM 2477 as alpha-d-Galp. This subtle difference between the glycoforms of the LPS core appeared to be essential for formation of the epitopes recognized by the specific antibodies directed against the Y. regensburgei whole bacterial cells. The oligosaccharides are not substituted by phosphate groups. Instead, the carboxyl groups of Kdo and galacturonic acid residues present in the core provide the negative charges. The undecasaccharides represent a novel core type of bacterial LPS, which is characteristic for Y. regensburgei. PMID:19880424

  16. Action patterns of amylolytic enzymes as determined by the [1-14C]malto-oligosaccharide mapping method.

    PubMed

    Pazur, J H; Marchetti, N T

    1992-04-01

    A valuable technique for oligosaccharide mapping, utilizing radioactive malto-oligosaccharides, multiple-ascent p.c., and radioautography, has been developed for identifying the action patterns of the glucoamylase isozymes, alpha-amylases, beta-amylase, glucosyltransferase, and glucanosyltransferase. The glucoamylase isozymes act by multi-chain mechanisms on malto-oligosaccharides and most likely on starch and glycogen. The alpha-amylases act endo-wise and randomly hydrolyze alpha-(1----4)- but not alpha-(1----6)-glucosidic bonds. These amylases may act by single-chain and/or multi-chain mechanisms, depending on the number of hydrolytic attacks per single encounter of the enzyme and the substrate. The beta-amylases hydrolyze malto-oligosaccharides by a multi-chain mechanism. A fungal glucosyltransferase from Aspergillus niger transfers glucose units by a single-chain mechanism from maltose to glucosyl acceptors to yield new gluco-oligosaccharides with alpha-(1----4) and alpha-(1----6) linkages. A novel type of transferase isolated from Bacillus subtilis acts by a multi-chain mechanism and transfers segments of 2 to 5 glucose residues from malto-oligosaccharides to acceptor co-substrates. An alpha-amylase from the same organism removes maltotriose units from the non-reducing ends of oligosaccharides by a multi-chain mechanism. PMID:1379885

  17. Degradation of oligosaccharides in nonenzymatic browning by formation of alpha-dicarbonyl compounds via a "peeling off" mechanism.

    PubMed

    Hollnagel, A; Kroh, L W

    2000-12-01

    The formation of alpha-dicarbonyl-containing substances and Amadori rearrangement products was studied in the glycine-catalyzed (Maillard reaction) and uncatalyzed thermal degradation of glucose, maltose, and maltotriose using o-phenylenediamine as trapping agent. Various degradation products, especially alpha-dicarbonyl compounds, are formed from carbohydrates with differing degrees of polymerization during nonenzymatic browning. The different Amadori rearrangement products, isomerization products, and alpha-dicarbonyls produced by the used carbohydrates were quantified throughout the observed reaction time, and the relevance of the different degradation pathways is discussed. In the Maillard reaction (MR) the amino-catalyzed rearrangement with subsequent elimination of water predominated, giving rise to hexosuloses with alpha-dicarbonyl structure, whereas under caramelization conditions more sugar fragments with an alpha-dicarbonyl moiety were formed. For the MR of oligosaccharides a mechanism is proposed in which 1,4-dideoxyosone is formed as the predominating alpha-dicarbonyl in the quasi-water-free thermolysis of di- and trisaccharides in the presence of glycine. PMID:11312795

  18. Effects of Impurities in Alkali-Extracted Xylan on Its Enzymatic Hydrolysis to Produce Xylo-Oligosaccharides.

    PubMed

    Shen, Rui; Li, Hong-Qiang; Zhang, Jie; Xu, Jian

    2016-07-01

    As the second abundant natural carbohydrate, xylan is normally prepared through alkaline extraction and then used for xylo-oligosaccharides (XOS) production. However, the extracted xylan inevitably contains salt, ethanol, and pigment. In order to investigate the effects of these impurities on XOS production, the alkaline-extracted xylan with different kinds and concentrations of impurities was made and then hydrolyzed using alkaline xylanase (EC 3.2.1.8) to produce XOS. The results showed that a certain concentration of salt (NaCl) promoted the XOS production, while ethanol and pigment inhibited the enzymatic hydrolysis process significantly. The color value mainly ascribed to the phenolic compounds binding to xylan was a key restriction factor in the enzymatic hydrolysis later stage. Using optimal xylan sample (with 10 mg/mL NaCl, color value of 4.6 × 10(5), without ethanol) as substrate, the highest XOS yield of 58.58 % was obtained. As the substrate of XOS production, prepared xylan should contain colored materials and ethanol as less as possible, however, retains appropriate salt. PMID:26922729

  19. Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9

    SciTech Connect

    Wathen, M.W.; Aeed, P.A.; Elhammer, A.P. )

    1991-03-19

    The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with ({sup 3}H)glucosamine and infected wit a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Gal{beta}1-3GalNAc constituting 17 and 66% of the total number of structures respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked {alpha}1-6 to the asparagine-linked N-acetylglucosamine.

  20. Sequence-selective carbohydrate-DNA interaction: dimeric and monomeric forms of the calicheamicin oligosaccharide interfere with transcription factor function.

    PubMed Central

    Liu, C; Smith, B M; Ajito, K; Komatsu, H; Gomez-Paloma, L; Li, T; Theodorakis, E A; Nicolaou, K C; Vogt, P K

    1996-01-01

    The synthetic oligosaccharide moiety of the antibiotic calicheamicin and the head-to-head dimer of this oligosaccharide are known to bind to the minor groove of DNA in a sequence-selective manner preferring distinct target sequences. We tested these carbohydrates for their ability to interfere with transcription factor function. The oligosaccharides inhibit binding of transcription factors to DNA in a sequence-selective manner, probably by inducing a conformational change in DNA structure. They also interfere with transcription by polymerase II in vitro. The effective concentrations of the oligosaccharides for inhibition of transcription factor binding and for transcriptional inhibition are in the micromolar range. The dimer is a significantly more active inhibitor than is the monomer. Images Fig. 2 Fig. 3 PMID:8570664

  1. Generation and structural validation of a library of diverse xyloglucan-derived oligosaccharides, including an update on xyloglucan nomenclature.

    PubMed

    Tuomivaara, Sami T; Yaoi, Katsuro; O'Neill, Malcolm A; York, William S

    2015-01-30

    Xyloglucans are structurally complex plant cell wall polysaccharides that are involved in cell growth and expansion, energy metabolism, and signaling. Determining the structure-function relationships of xyloglucans would benefit from the availability of a comprehensive and structurally diverse collection of rigorously characterized xyloglucan oligosaccharides. Here, we present a workflow for the semi-preparative scale generation and purification of neutral and acidic xyloglucan oligosaccharides using a combination of enzymatic and chemical treatments and size-exclusion chromatography. Twenty-six of these oligosaccharides were purified to near homogeneity and their structures validated using a combination of matrix-assisted laser desorption/ionization mass spectrometry, high-performance anion exchange chromatography, and 1H nuclear magnetic resonance spectroscopy. Mass spectrometry and analytical chromatography were compared as methods for xyloglucan oligosaccharide quantification. 1H chemical shifts were assigned using two-dimensional correlation spectroscopy. A comprehensive update of the nomenclature describing xyloglucan side-chain structures is provided for reference. PMID:25497333

  2. A selected probiotic strain of Lactobacillus fermentum CM33 isolated from breast-fed infants as a potential source of β-galactosidase for prebiotic oligosaccharide synthesis.

    PubMed

    Sriphannam, Wattana; Lumyong, Saisamorn; Niumsap, Piyanuch; Ashida, Hisashi; Yamamoto, Kenji; Khanongnuch, Chartchai

    2012-02-01

    Lactic acid bacteria from healthy breast-fed infants were isolated and screened for β-galactosidase production in MRS broth. Among 49 isolates that exhibited the yellow clear zone on MRS agar supplemented with bromocresol blue, the isolate CM33 was selected as being the highest β-galactosidase producer and was identified as Lactobacillus fermentum based on its morphological characteristics and 16S rDNA nucleotide sequence. L. fermentum CM33 exhibited a good survival rate under the simulated stomach passage model, comparable to known probiotic strains L. gallinarum JCM2011 and L. agilis JCM1187. L. fermentum CM33 was antagonistic to pathogenic bacteria Listeria monocytogenes, Escherichia coli 0157:H7, Salmonella typhi, and Salmonella enteriditis, using the well diffusion method. In addition, the selected lactobacilli exhibited a high growth rate when cultivated in modified MRS containing commercial galactooligosaccharide (GOS) as a sole carbon source, as well as in glucose. A preliminary study on the enzymatic synthesis of oligosaccharide using crude β-galactosidase revealed the capability for oligosaccharide synthesis by the transgalactosylation activity. PMID:22367946

  3. Complete NMR assignment of a bisecting hybrid-type oligosaccharide transferred by Mucor hiemalis endo-β-N-acetylglucosaminidase.

    PubMed

    Yamanoi, Takashi; Oda, Yoshiki; Katsuraya, Kaname; Inazu, Toshiyuki; Yamamoto, Kenji

    2016-06-01

    This study describes the complete nuclear magnetic resonance (NMR) spectral assignment of a bisecting hybrid-type oligosaccharide 1, transferred by Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M). Through (1)H- and (13)C-NMR, DQF-COSY, HSQC, HMBC, TOCSY, and NOESY experiments, we determine the structure of the glycoside linkage formed by the Endo-M transglycosylation, i.e., the connection between GlcNAc and GlcNAc in oligosaccharide 1. PMID:27131291

  4. Applications of microencapsulated Bifidobacterium longum with Eleutherine americana in fresh milk tofu and pineapple juice.

    PubMed

    Phoem, Atchara N; Chanthachum, Suphitchaya; Voravuthikunchai, Supayang P

    2015-04-01

    Bifidobacterium longum was microencapsulated by extrusion technique and added in fresh milk tofu and pineapple juice. Microencapsulation of B. longum with Eleutherine americana extract, oligosaccharides extract, and commercial fructo-oligosaccharides was assessed for the bacterial survival after sequential exposure to simulated gastric and intestinal juices, and refrigeration storage. Microencapsulated B. longum with the extract and oligosaccharides extract in the food products showed better survival than free cells under adverse conditions. Sensory analysis demonstrated that the products containing co-encapsulated bacterial cells were more acceptable by consumers than free cells. Pineapple juice prepared with co-encapsulated cells had lower values for over acidification, compared with the juice with free cells added. This work suggested that microencapsulated B. longum with E. americana could enhance functional properties of fresh milk tofu and pineapple juice. PMID:25854832

  5. Applications of Microencapsulated Bifidobacterium Longum with Eleutherine Americana in Fresh Milk Tofu and Pineapple Juice

    PubMed Central

    Phoem, Atchara N.; Chanthachum, Suphitchaya; Voravuthikunchai, Supayang P.

    2015-01-01

    Bifidobacterium longum was microencapsulated by extrusion technique and added in fresh milk tofu and pineapple juice. Microencapsulation of B. longum with Eleutherine americana extract, oligosaccharides extract, and commercial fructo-oligosaccharides was assessed for the bacterial survival after sequential exposure to simulated gastric and intestinal juices, and refrigeration storage. Microencapsulated B. longum with the extract and oligosaccharides extract in the food products showed better survival than free cells under adverse conditions. Sensory analysis demonstrated that the products containing co-encapsulated bacterial cells were more acceptable by consumers than free cells. Pineapple juice prepared with co-encapsulated cells had lower values for over acidification, compared with the juice with free cells added. This work suggested that microencapsulated B. longum with E. americana could enhance functional properties of fresh milk tofu and pineapple juice. PMID:25854832

  6. Cello-oligosaccharide oxidation reveals differences between two lytic polysaccharide monooxygenases (family GH61) from Podospora anserina.

    PubMed

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M; Berrin, Jean-Guy; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis. PMID:23124232

  7. Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

    PubMed Central

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M.; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis. PMID:23124232

  8. Structural Characterization of Neutral Oligosaccharides by Laser-Enhanced In-Source Decay of MALDI-FTICR MS

    NASA Astrophysics Data System (ADS)

    Yang, Hongmei; Yu, Yingning; Song, Fengrui; Liu, Shuying

    2011-05-01

    MALDI in-source decay (ISD) technique described to date has proven to be a convenient and rapid method for sequencing purified peptides and proteins. However, the general ISD still can not produce adequate fragments for the detailed structural elucidation of oligosaccharides. In this study, an efficient and practical method termed the laser-enhanced ISD (LEISD) technique of MALDI-FTICR MS allows highly reliable and abundant fragmentation of the neutral oligosaccharides, which was attributed to the ultrahigh irradiation laser of mJ level. The yield of ISD fragmentation was evaluated under different laser powers for 7 neutral oligosaccharides using DHB as matrix. Better quality ISD spectra including fragment ions in low-mass region were obtained at higher laser power. Results from the LEISD of oligosaccharides demonstrated that a significantly better signal-to-noise ratio (S/N) and more structural information could be obtained in comparison to the conventional CID. It was also suggested that the valuable A ions derived from cross-ring cleavage of the linear oligosaccharides allowed the distinction among α(1 → 4)-, α(1 → 6)-, β(1 → 4)-, and β(1 → 3)-linked isobaric structures according to fragment types and intensities. In addition, ideal fragmentation ions observed by LEISD method facilitated the determination of the sequences and branched points of complex oligosaccharides from human milk.

  9. Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

    NASA Astrophysics Data System (ADS)

    Du, Yi; May, Kimberly; Xu, Wei; Liu, Hongcheng

    2012-07-01

    The presence of N-linked oligosaccharides in the CH2 domain has a significant impact on the structure, stability, and biological functions of recombinant monoclonal antibodies. The impact is also highly dependent on the specific oligosaccharide structures. The absence of core-fucose has been demonstrated to result in increased binding affinity to Fcγ receptors and, thus, enhanced antibody-dependent cellular cytotoxicity (ADCC). Therefore, a method that can specifically determine the level of oligosaccharides without the core-fucose (afucosylation) is highly desired. In the current study, recombinant monoclonal antibodies and tryptic peptides from the antibodies were digested using endoglycosidases F2 and H, which cleaves the glycosidic bond between the two primary GlcNAc residues. As a result, various oligosaccharides of either complex type or high mannose type that are commonly observed for recombinant monoclonal antibodies are converted to either GlcNAc residue only or GlcNAc with the core-fucose. The level of GlcNAc represents the sum of all afucosylated oligosaccharides, whereas the level of GlcNAc with the core-fucose represents the sum of all fucosylated oligosaccharides. LC-MS analysis of the enzymatically digested antibodies after reduction provided a quick estimate of the levels of afucosylation. An accurate determination of the level of afucosylation was obtained by LC-MS analysis of glycopeptides after trypsin digestion.

  10. Growth and anatomical parameters of adventitious roots formed on mung bean hypocotyls are correlated with galactoglucomannan oligosaccharides structure.

    PubMed

    Kollárová, K; Zelko, I; Henselová, M; Capek, P; Lišková, D

    2012-01-01

    The effect of galactoglucomannan oligosaccharides (GGMOs) compared with chemically modified oligosaccharides, GGMOs-g (with reduced number of D-galactose side chains) and GGMOs-r (with reduced reducing ends) on mung bean (Vigna radiata (L.) Wilczek) adventitious roots formation, elongation, and anatomical structure have been studied. All types of oligosaccharides influenced adventitious root formation in the same way: stimulation in the absence of exogenous auxin and inhibition in the presence of exogenous auxin. Both reactions are probably related with the presence/content of endogenous auxin in plant cuttings. However, the adventitious root length was inhibited by GGMOs both in the absence as well as in the presence of auxin (IBA or NAA), while GGMOs-g inhibition was significantly weaker compared with GGMOs. GGMOs-r were without significant difference on both processes, compared with GGMOs. GGMOs affected not only the adventitious root length but also their anatomy in dependence on the combination with certain type of auxin. The oligosaccharides influenced cortical cells division, which was reflected in the cortex area and in the root diameter. All processes followed were dependent on oligosaccharides chemical structure. The results suggest also that GGM-derived oligosaccharides may play an important role in adventitious roots elongation but not in their formation. PMID:22666154

  11. Characterization of the O- and N-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni

    SciTech Connect

    Nyame, A.K.

    1987-01-01

    The structures of the O- and N-linked oligosaccharides in glycoproteins synthesized by larval and adult schistosomes of Schistosoma mansoni have been investigated. Mechanically transformed schistosomula or adult schistosomes were incubated in media containing either (/sup 3/H)mannose, (/sup 3/H)glucosamine or (/sup 3/H)galactose for 48 and 24 hr, respectively, to radiolabel metabolically the oligosaccharide moieties of newly synthesized glycoproteins. Analyses of the radiolabeled glycoproteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and fluorography demonstrated that numerous glycoproteins from 48-hr old schistosomula and adult schistosomes were labeled by both the (/sup 3/H)mannose and (/sup 3/H)glucosamine precursors. The (/sup 3/H)galactose precursor was incorporated into numerous glycoproteins in adult schistosomes; however, few, if any, glycoproteins in schistosomula were labeled by this radioactive sugar precursor.

  12. Effect of Malate-oligosaccharide Solution on Antioxidant Capacity of Endurance Athletes.

    PubMed

    Qiang, Fu

    2015-01-01

    L-malate is an important intermediate on the process of metabolism; it plays an important role in generating mitochondria ATP both under aerobic and hypoxic condition. It is easy to be absorbed and come into mitochondrion through cell membrane and promote to produce energy in mitochondrion. The purpose of this investigation is to probe into the different influence malate ingestion on blood lactate and glucose kinetics during aerobic exercise athletes; at the same time, rats were used to study the effect of malate and oligosaccharide solution on the metabolism in muscle and liver. The supplement of malate-oligosaccharide solution may improve the level of antioxidants in vivo after exercise, and subsequently increase the total antioxidant capacity and decrease the level of lipid peroxidation. At the appropriate time sports drinks can add varying degrees of motion to extend time to fatigue enhance athletic ability, speed up the recovery process after exercise, reduce fatigue. PMID:26998183

  13. O-acetylated oligosaccharides from pectins of potato tuber cell walls.

    PubMed Central

    Ishii, T

    1997-01-01

    Acetylated trigalacturonides and rhamnogalacturonan I (RG-I)-derived oligosaccharides were isolated from a Driselase digest of potato tuber cell walls by ion-exchange and size-exclusion chromatography. The oligosaccharides were structurally characterized by fast atom bombardment-mass spectroscopy, nuclear magnetic resonance spectroscopy, and glycosyl-linkage composition analysis. One trigalacturonide contained a single acetyl group at O-3 of the reducing galacturonic acid residue. A second trigalacturonide contained two acetyl substituents, which were located on O-3 or O-4 of the nonreducing galacturonic acid residue and O-3 of the reducing galacturonic acid residue. RG-I backbone-derived oligomers had acetyl groups at O-2 of the galacturonic acid residues. Some of these galacturonic acid residues were O-acetylated at both O-2 and O-3 positions. Rhamnosyl residues of RG-I oligomers were not acetylated. PMID:9112775

  14. Bacteroides in the Infant Gut Consume Milk Oligosaccharides via Mucus-Utilization Pathways

    PubMed Central

    Marcobal, A.; Barboza, M.; Sonnenburg, E.D.; Pudlo, N.; Martens, E.C.; Desai, P.; Lebrilla, C.B.; Weimer, B.C.; Mills, D.A.; German, J.B.; Sonnenburg, J.L.

    2011-01-01

    Summary Newborns are colonized with an intestinal microbiota shortly after birth but the factors governing the retention and abundance of specific microbial lineages are unknown. Nursing infants consume human milk oligosaccharides (HMOs) that pass undigested to the distal gut where they may be digested by microbes. We determined that the prominent neonate gut residents, Bacteroides thetaiotaomicron and Bacteroides fragilis, induce the same genes during HMO consumption that are used to harvest host mucus glycans, which are structurally similar to HMOs. Lacto-N-neotetraose, a specific HMO component, selects for HMO-adapted species such as Bifidobacterium infantis, which cannot use mucus, and provides a selective advantage to B. infantis in vivo when bi-associated with B. thetaiotaomicron in the gnotobiotic mouse gut. This indicates that the complex oligosaccharide mixture within HMOs attracts both mutualistic mucus-adapted species and HMO-adapted bifidobacteria to the infant intestine that likely facilitate both milk and future solid food digestion. PMID:22036470

  15. Novel identification strategy for ground coffee adulteration based on UPLC-HRMS oligosaccharide profiling.

    PubMed

    Cai, Tie; Ting, Hu; Jin-Lan, Zhang

    2016-01-01

    Coffee is one of the most common and most valuable beverages. According to International Coffee Organization (ICO) reports, the adulteration of coffee for financial reasons is regarded as the most serious threat to the sustainable development of the coffee market. In this work, a novel strategy for adulteration identification in ground coffee was developed based on UPLC-HRMS oligosaccharide profiling. Along with integrated statistical analysis, 17 oligosaccharide composition were identified as markers for the identification of soybeans and rice in ground coffee. This strategy, validated by manual mixtures, optimized both the reliability and authority of adulteration identification. Rice and soybean adulterants present in ground coffee in amounts as low as 5% were identified and evaluated. Some commercial ground coffees were also successfully tested using this strategy. PMID:26213074

  16. Effect of Malate-oligosaccharide Solution on Antioxidant Capacity of Endurance Athletes

    PubMed Central

    Qiang, Fu

    2015-01-01

    L-malate is an important intermediate on the process of metabolism; it plays an important role in generating mitochondria ATP both under aerobic and hypoxic condition. It is easy to be absorbed and come into mitochondrion through cell membrane and promote to produce energy in mitochondrion. The purpose of this investigation is to probe into the different influence malate ingestion on blood lactate and glucose kinetics during aerobic exercise athletes; at the same time, rats were used to study the effect of malate and oligosaccharide solution on the metabolism in muscle and liver. The supplement of malate-oligosaccharide solution may improve the level of antioxidants in vivo after exercise, and subsequently increase the total antioxidant capacity and decrease the level of lipid peroxidation. At the appropriate time sports drinks can add varying degrees of motion to extend time to fatigue enhance athletic ability, speed up the recovery process after exercise, reduce fatigue. PMID:26998183

  17. Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases

    PubMed Central

    Díez-Municio, Marina; Herrero, Miguel; Olano, Agustín; Moreno, F Javier

    2014-01-01

    Prebiotic oligosaccharides are increasingly demanded within the Food Science domain because of the interesting healthy properties that these compounds may induce to the organism, thanks to their beneficial intestinal microbiota growth promotion ability. In this regard, the development of new efficient, convenient and affordable methods to obtain this class of compounds might expand even further their use as functional ingredients. This review presents an overview on the most recent interesting approaches to synthesize lactose-derived oligosaccharides with potential prebiotic activity paying special focus on the microbial glycoside hydrolases that can be effectively employed to obtain these prebiotic compounds. The most notable advantages of using lactose-derived carbohydrates such as lactosucrose, galactooligosaccharides from lactulose, lactulosucrose and 2-α-glucosyl-lactose are also described and commented. PMID:24690139

  18. Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human. beta. -hexosaminidase

    SciTech Connect

    O'Dowd, B.F.; Cumming, D.A.; Gravel, R.A.; Mahuran, D.

    1988-07-12

    Human ..beta..-hexosaminidase is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM/sub 2/ ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure ..cap alpha..(..beta../sub a/..beta../sub b/), and hexosaminidase B, 2(..beta../sub a/..beta../sub b/). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the ..beta../sub b/f chain, and one non concanavalin A binding glycopeptide, localized to the ..beta../sub a/ chain, were found associated with the ..beta..-subunit in both hexosaminidase A and hexosaminidase B. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. The unique glycopeptide associated with the ..beta../sub a/ chain contained a single GlcNAc residue. Thus all three mature polypeptides comprising the ..cap alpha.. and ..beta.. subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the ..cap alpha.. chain they found only one possible site for in vivo phosphorylation. In the ..beta.. it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an ..cap

  19. The Predominance of Type I Oligosaccharides Is a Feature Specific to Human Breast Milk123

    PubMed Central

    Urashima, Tadasu; Asakuma, Sadaki; Leo, Fiame; Fukuda, Kenji; Messer, Michael; Oftedal, Olav T.

    2012-01-01

    Human milk and colostrum contain ∼12–13 g/L and ∼22–24 g/L of oligosaccharides, respectively. The chemical structures of >100 human milk oligosaccharides (HMO) have been characterized to date. We determined the concentrations of 10 neutral and 9 acidic colostrum HMO collected during the first 3 d of lactation by using reverse phase HPLC after derivatization with 2-aminopyridine or 1-methyl-3-phenyl-5-pyrazolon. The predominant oligosaccharides were Fuc(α1-2)Gal(β1-4Glc (2′-FL), Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc (LNFP I), Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc (LNDFH I), and Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc (LNT), the concentration of each of which was ∼1–3 g/L. Because these HMO, other than 2′-FL, all contain the Lacto-N-biose type I structure [Gal(β1-3)GlcNAc], we conclude that HMO containing the type I structure predominate over those containing the N-acetyllactosamine type II structure [Gal(β1-4)GlcNAc]. This appears to be a feature that is specific to humans, because the milk and colostrum of other species, including apes and monkeys, either contain only type II oligosaccharides or type II predominate over type I. It is possible that type I HMO may have importance as substrates for beneficial bifidobacteria in breast-fed infants. The biological importance of type I HMO predominance warrants further study, both in relation to human health and to human evolution. PMID:22585927

  20. Characterization of the oligosaccharides of plasma sex hormone binding globulin from noncirrhotic alcoholic patients.

    PubMed

    Valladares, L; Erices, A; Lioi, X; Iturriaga, H

    2000-05-01

    In previous reports we have demonstrated high plasma levels of sex hormone-binding globulin (SHBG) in asymptomatic alcoholic men. In the present work the physicochemical properties of SHBG from plasma of noncirrhotic alcoholic patients have been further compared with SHBG of control subjects. Steroid binding to SHBG was similar for the two groups: alcoholic men, K(d) of 0.62 +/- 0.07 nM and control individuals, K(d) of 0.70 +/- 0.10 nM. The structure of oligosaccharides attached to SHBG from controls and alcoholic men were determined by using serial chromatography. Our data indicated that 7% of SHBG of control individuals was not retarded by the Con-A column, whereas approximately 30% of SHBG of alcoholic men eluted in the void volume of Con A. Approximately 46% of SHBG of alcoholics applied to Con A, possessed biantennary complex oligosaccharides, as indicated by the fact that it could be eluted with methyl-alpha-D-glucopyranoside and by its retention on wheat germ agglutinin; in contrast, when SHBG from control men was analyzed, approximately 51% was eluted with methyl-alpha-D-glucopyranoside. Approximately 9% of the biantennary complex oligosaccharides on SHBG of control men and none of those on SHBG from alcoholic men were fucosylated on the chitobiose core, as determined by chromatography on Lenn culinaris lectin. Galactosylated oligosaccharides were also present on the SHBG fraction as indicated by its interaction with Ricinus communis-I. Approximately 24% of SHBG of alcoholic men and 39% of those on SHBG from control individuals applied to Con-A were retained and could be eluted with methyl-alpha-D-mannopyranoside. Evidence based on the binding on mannoside-eluted SHBG to Con-A, wheat germ agglutinin, and R. communis-I indicated that at least the SHBG in this fraction, from alcoholics or controls, contained two glycosylation sites and that the sites were differentially glycosylated. PMID:10751639

  1. Specific Dietary Oligosaccharides Increase Th1 Responses in a Mouse Respiratory Syncytial Virus Infection Model

    PubMed Central

    Schijf, Marcel A.; Kruijsen, Debby; Bastiaans, Jacqueline; Coenjaerts, Frank E. J.; Garssen, Johan; van Bleek, Grada M.

    2012-01-01

    Breast feeding reduces the risk of developing severe respiratory syncytial virus (RSV) infections in infants. In addition to maternal antibodies, other immune-modulating factors in human milk contribute to this protection. Specific dietary prebiotic oligosaccharides, similar to oligosaccharides present in human milk, were evaluated in a C57BL/6 mouse RSV infection model. During primary RSV infection, increased numbers of RSV-specific CD4+ T cells producing gamma interferon (IFN-γ) were found in the lungs at days 8 to 10 postinfection in mice receiving diet containing short-chain galactooligosacharides, long-chain fructooligosaccharides, and pectin-derived acidic oligosaccharides (termed scGOS/lcFOS/pAOS). In a Th2-skewed formalin-inactivated (FI)-RSV vaccination model, the prebiotic diet reduced RSV-specific Th2 cytokine (interleukin-4 [IL-4], IL-5, and IL-13)-producing CD4+ T cells in the lung and the magnitude of airway eosinophilia at day 4 and 6 after infection. This was accompanied by a decreased influx of inflammatory dendritic cells (CD11b+/CD11c+) and increased numbers of IFN-γ-producing CD4+ and CD8+ T cells at day 8 after viral challenge. These findings suggest that specific dietary oligosaccharides can influence trafficking and/or effector functions of innate immune, CD4+, and CD8+ T cell subsets in the lungs of RSV-infected mice. In our models, scGOS/lcFOS/pAOS had no effect on weight but increased viral clearance in FI-RSV-vaccinated mice 8 days after infection. The increased systemic Th1 responses potentiated by scGOS/lcFOS/pAOS might contribute to an accelerated Th1/Th2 shift of the neonatal immune system, which might favor protective immunity against viral infections with a high attack rate in early infancy, such as RSV. PMID:22896622

  2. Specific dietary oligosaccharides increase Th1 responses in a mouse respiratory syncytial virus infection model.

    PubMed

    Schijf, Marcel A; Kruijsen, Debby; Bastiaans, Jacqueline; Coenjaerts, Frank E J; Garssen, Johan; van Bleek, Grada M; van't Land, Belinda

    2012-11-01

    Breast feeding reduces the risk of developing severe respiratory syncytial virus (RSV) infections in infants. In addition to maternal antibodies, other immune-modulating factors in human milk contribute to this protection. Specific dietary prebiotic oligosaccharides, similar to oligosaccharides present in human milk, were evaluated in a C57BL/6 mouse RSV infection model. During primary RSV infection, increased numbers of RSV-specific CD4(+) T cells producing gamma interferon (IFN-γ) were found in the lungs at days 8 to 10 postinfection in mice receiving diet containing short-chain galactooligosacharides, long-chain fructooligosaccharides, and pectin-derived acidic oligosaccharides (termed scGOS/lcFOS/pAOS). In a Th2-skewed formalin-inactivated (FI)-RSV vaccination model, the prebiotic diet reduced RSV-specific Th2 cytokine (interleukin-4 [IL-4], IL-5, and IL-13)-producing CD4(+) T cells in the lung and the magnitude of airway eosinophilia at day 4 and 6 after infection. This was accompanied by a decreased influx of inflammatory dendritic cells (CD11b(+)/CD11c(+)) and increased numbers of IFN-γ-producing CD4(+) and CD8(+) T cells at day 8 after viral challenge. These findings suggest that specific dietary oligosaccharides can influence trafficking and/or effector functions of innate immune, CD4(+), and CD8(+) T cell subsets in the lungs of RSV-infected mice. In our models, scGOS/lcFOS/pAOS had no effect on weight but increased viral clearance in FI-RSV-vaccinated mice 8 days after infection. The increased systemic Th1 responses potentiated by scGOS/lcFOS/pAOS might contribute to an accelerated Th1/Th2 shift of the neonatal immune system, which might favor protective immunity against viral infections with a high attack rate in early infancy, such as RSV. PMID:22896622

  3. Influence of Core Oligosaccharide of Lipopolysaccharide to Outer Membrane Behavior of Escherichia coli

    PubMed Central

    Wang, Zhou; Wang, Jianli; Ren, Ge; Li, Ye; Wang, Xiaoyuan

    2015-01-01

    Lipopolysaccharides, major molecules in the outer membrane of Gram-negative bacteria, play important roles on membrane integrity of the cell. However, how the core oligosaccharide of lipopolysaccharide affect the membrane behavior is not well understood. In this study, the relationship between the core oligosaccharide of lipopolysaccharide and the membrane behavior was investigated using a series of Escherichia coli mutants defective in genes to affect the biosynthesis of core oligosaccharide of lipopolysaccharide. Cell surface hydrophobicity, outer membrane permeability, biofilm formation and auto-aggregation of these mutant cells were compared. Compared to the wild type W3110, cell surface hydrophobicities of mutant ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaP, ΔwaaY and ΔwaaB were enhanced, outer membrane permeabilities of ΔwaaC, ΔwaaF, ΔwaaG and ΔwaaP were significantly increased, abilities of biofilm formation by ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaP, ΔwaaQ and ΔwaaY decreased, and auto-aggregation abilities of ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaU, ΔwaaP and ΔwaaY were strongly enhanced. These results give new insight into the influence of core oligosaccharide of lipopolysaccharide on bacterial cell membrane behavior. PMID:26023839

  4. Minimal oligosaccharide structures required for induction of immune responses against meningococcal immunotype L1, L2, and L3,7,9 lipopolysaccharides determined by using synthetic oligosaccharide-protein conjugates.

    PubMed Central

    Verheul, A F; Boons, G J; Van der Marel, G A; Van Boom, J H; Jennings, H J; Snippe, H; Verhoef, J; Hoogerhout, P; Poolman, J T

    1991-01-01

    The 12 types of meningococcal lipopolysaccharide (LPS) (immunotypes) contain immunotype-specific and cross-reactive epitopes situated on the oligosaccharide part of the LPS molecules. To identify useful cross-reactive epitopes and to determine minimal oligosaccharide structures required for the induction of an immune response against the most prevalent immunotypes, L1, L2, and L3,7,9, synthetic as well as native LPS-derived oligosaccharides were conjugated with tetanus toxoid. L3,7,9 phosphoethanolamine (PEA) group-containing oligosaccharide-tetanus toxoid conjugates evoked high immunoglobulin G (IgG) antibody levels in rabbits which were detected by an L2-, L3,7,9-, and, depending on the antiserum, L1-specific enzyme-linked immunosorbent assay (ELISA). Inhibition studies revealed that an identical antibody population was detected by L1 and L3,7,9 ELISA, indicating a similar tertiary structure of the inner core oligosaccharide of these two immunotypes. These antibodies recognize PEA group-containing epitopes present on the L1 and L3,7,9 LPS. An L2 PEA group-containing oligosaccharide-tetanus toxoid conjugate elicited L2- and L3,7,9-specific IgG antibodies, but in contrast with the L3,7,9 conjugates, no L1-specific IgG antibodies were evoked. These results indicate that L1 and L2 LPS do not contain cross-reactive epitopes, whereas both L2 and L3,7,9 LPS and L1 and L3,7,9 LPS possess common determinants. Three linear oligosaccharides and one branched oligosaccharide, representing partial structures of the inner core oligosacchardes of meningococcal LPS, were synthesized. Only the branched synthetic oligosaccharide-containing conjugate was able to induce and L1- and L3,7,9-specific immune response, whereas the linear oligosaccharide-protein conjugates evoked L2-specific immune responses. The branched oligosaccharide (beta-D-Glcp(1----4)-[L-alpha-D-Hepp(1----3)]-L-alpha-D-Hepp ) is therefore considered a minimal structure required for the induction of an immune

  5. Development of approaches to a third-generation carbohydrate-conjugate vaccine against Streptococcus pneumoniae: the search for optimal oligosaccharide ligands

    NASA Astrophysics Data System (ADS)

    Gening, M. L.; Kurbatova, E. A.; Tsvetkov, Yu E.; Nifantiev, N. E.

    2015-11-01

    The review addresses the application of synthetic oligosaccharides related to fragments of capsular polysaccharides from different serotypes of the bacterium Streptococcus pneumoniae for the design of third-generation pneumococcal conjugate vaccines. Special focus is given to characteristic features of the chemical structures of oligosaccharides required for the induction of the protective immune response when using synthetic glycoconjugate vaccines based on oligosaccharide ligands and carrier proteins. The bibliography includes 101 references.

  6. Annotation and structural elucidation of bovine milk oligosaccharides and determination of novel fucosylated structures

    PubMed Central

    Aldredge, Danielle L; Geronimo, Maria R; Hua, Serenus; Nwosu, Charles C; Lebrilla, Carlito B; Barile, Daniela

    2013-01-01

    Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high-potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of biotherapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structure-related specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomer-specific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass and structure to allow their rapid identification in future studies. PMID:23436288

  7. LC-MS n Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

    NASA Astrophysics Data System (ADS)

    Huang, Rongrong; Pomin, Vitor H.; Sharp, Joshua S.

    2011-09-01

    Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS n . The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS n fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS n experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS n methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.

  8. Antioxidant activity of oligosaccharide ester extracted from Polygala tenuifolia roots in senescence-accelerated mice.

    PubMed

    Liu, Ping; Hu, Yuan; Guo, Dai-Hong; Lu, Bao-Rong; Rahman, Khalid; Mu, Li-Hua; Wang, Dong-Xiao

    2010-07-01

    The constituents of the ethanol extract from the root of Polygala tenuifolia Willd. (Polygalaceae) were investigated for antioxidant activity in senescence-accelerated mice. Consequently, two relevant samples were obtained, a fraction separated by macroporous resin (YZ-OE), and a major pure crystal of 3,6'-disinapoyl sucrose (DISS). Based on HPLC-ESI-MS analysis, the most constituents in the YZ-OE fraction from the extract of P. tenuifolia were oligosaccharide esters. The antioxidant activities of these two samples were evaluated using the accelerated senescence-prone, short-lived mice (SAMP) in vivo. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were increased significantly in SAMP mice fed oligosaccharide esters (YZ-OE 50 mg/kg) and its constituents (DISS 50 mg/kg). However, the content of malondialdehyde (MDA) was increased in the blood and liver of SAMP mice. But when given YZ-OE, it could be decreased, by 44.3% and 47.5%, respectively, compared with the SAMP model. Results from the analyses indicated that the oligosaccharide esters (YZ-OE) from roots of P. tenuifolia had a high in vivo antioxidant activity. PMID:20645784

  9. Preparation and antioxidant activity of xanthan oligosaccharides derivatives with similar substituting degrees.

    PubMed

    Xiong, Xiaoying; Li, Ming; Xie, Jing; Xue, Bin; Sun, Tao

    2014-12-01

    Maleoyl xanthan oligosaccharides (XGOSMAs) and phthaloyl xanthan oligosaccharides (XGOSPAs) were prepared by reacting xanthan oligosaccharides with maleic anhydride and phthalic anhydride, respectively. The substituting degrees (DSs) of XGOSMAs and XGOSPAs were determined by a neutralization reaction. XGOAMA-1 (DS=0.30), XGOSPA (DS=0.31), XGOSMA-2 (DS=0.62) and XGOSPA-2 (DS=0.60) were selected for structural characterization and antioxidant activity evaluation. Their structural changes were confirmed by Fourier transform infrared spectra (FT-IR), and their molecular weights were determined with a gel permeation chromatography method (GPC). The pyruvate acid and reducing sugar contents were determined by ultraviolet spectrophotometry and the dinitrosalicylic acid method. The antioxidant activity was evaluated by the scavenging of the superoxide anion radical (O2(-)), hydroxyl radical (OH), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and determination of reducing power. The results indicated that XGOSPA exhibited higher antioxidant activity than XGOSMA with similar substituting degrees in all the above mentioned antioxidant evaluation systems, which may be related to the fact that phthaloyl group has a stronger electron-withdrawing effect than the maleoyl group. PMID:24996297

  10. An approach for separation and complete structural sequencing of heparin/heparan sulfate-like oligosaccharides.

    PubMed

    Huang, Rongrong; Liu, Jian; Sharp, Joshua S

    2013-06-18

    As members of the glycosaminoglycan (GAG) family, heparin and heparan sulfate (HS) are responsible for mediation of a wide range of essential biological actions, most of which are mediated by specific patterns of modifications of regions of these polysaccharides. To fully understand the regulation of HS modification and the biological function of HS through its interactions with protein ligands, it is essential to know the specific HS sequences present. However, the sequencing of mixtures of HS oligosaccharides presents major challenges due to the lability of the sulfate modifications, as well as difficulties in separating isomeric HS chains. Here, we apply a sequential chemical derivatization strategy involving permethylation, desulfation, and trideuteroperacetylation to label original sulfation sites with stable and hydrophobic trideuteroacetyl groups. The derivatization chemistry differentiates between all possible heparin/HS sequences solely by glycosidic bond cleavages, without the need to generate cross-ring cleavages. This derivatization strategy combined with LC-MS/MS analysis has been used to separate and sequence five synthetic HS-like oligosaccharides of sizes up to dodecasaccharide, as well as a highly sulfated Arixtra-like heptamer. This strategy offers a unique capability for the sequencing of microgram quantities of HS oligosaccharide mixtures by LC-MS/MS. PMID:23659663

  11. Granule-bound starch synthase I in isolated starch granules elongates malto-oligosaccharides processively.

    PubMed Central

    Denyer, K; Waite, D; Motawia, S; Møller, B L; Smith, A M

    1999-01-01

    Isoforms of starch synthase belonging to the granule-bound starch synthase I (GBSSI) class synthesize the amylose component of starch in plants. Other granule-bound isoforms of starch synthase, such as starch synthase II (SSII), are unable to synthesize amylose. The kinetic properties of GBSSI and SSII that are responsible for these functional differences have been investigated using starch granules from embryos of wild-type peas and rug5 and lam mutant peas, which contain, respectively, both GBSSI and SSII, GBSSI but not SSII and SSII but not GBSSI. We show that GBSSI in isolated granules elongates malto-oligosaccharides processively, adding more than one glucose molecule for each enzyme-glucan encounter. Granule-bound SSII can elongate malto-oligosaccharides, but has a lower affinity for these than GBSSI and does not elongate processively. As a result of these properties GBSSI synthesizes longer malto-oligosaccharides than SSII. The significance of these results with respect to the roles of GBSSI and SSII in vivo is discussed. PMID:10229673

  12. Modular Synthesis of Heparan Sulfate Oligosaccharides for Structure-Activity Relationship Studies

    PubMed Central

    Arungundram, Sailaja; Al-Mafraji, Kanar; Asong, Jinkeng; Leach, Franklin E.; Amster, I. Jonathan; Venot, Andre; Turnbull, Jeremy E.; Boons, Geert-Jan

    2010-01-01

    Although hundreds of heparan sulfate binding proteins have been identified, and implicated in a myriad of physiological and pathological processes, very little information is known about ligand requirements for binding and mediating biological activities by these proteins. This difficulty results from a lack of technology for establishing structure-activity-relationships, which in turn is due to the structural complexity of natural heparan sulfate (HS) and difficulties of preparing well-defined HS-oligosaccharides. To address this deficiency, we have developed a modular approach for the parallel combinatorial synthesis of HS oligosaccharides that utilizes a relatively small number of selectively protected disaccharide building blocks, which can easily be converted into glycosyl donors and acceptors. The utility of the modular building blocks has been demonstrated by the preparation of a library of twelve oligosaccharides, which has been employed to probe structural features of HS for inhibiting the protease, BACE-1. The complex variations in activity with structural changes support the view that important functional information is embedded in HS sequences. Furthermore, the most active derivative provides an attractive lead compound for the preparation of more potent compounds, which may find use as a therapeutic agent for Alzheimer's disease. PMID:19904943

  13. Potential anti-inflammatory and anti-infectious effects of human milk oligosaccharides.

    PubMed

    Kunz, C; Rudloff, S

    2008-01-01

    There is increasing evidence of the local effects within the gastro intestinal tract and the systemic functions of human milk oligosaccharides (HMO). In addition to the vast majority of in vitro data, animal studies underline the high potential of HMO to influence very different processes. HMO probably influence the composition of the gut microflora through effects on the growth of bifidus bacteria. Whether the concomitant low number of pathogenic microorganisms in breastfed infants is also caused by HMO is an intriguing question that still has yet to be proven. Due to the similarity of HMO to epithelial cell surface carbohydrates, an inhibitory effect on the adhesion of pathogens to the cell surface is most likely. If this could be shown in humans, HMO would provide a new way to prevent or treat certain infections. It would also indicate supplementing infant formula based on cow's milk with HMO, as those oligosaccharides are either not detectable or present only in low numbers in bovine milk. As some HMO can be absorbed and circulate in blood, systemic effects may also be influenced. Due to their similarities to selectin ligands, HMO have been tested in in vitro studies demonstrating their anti-inflammatory abilities. For example, it has been shown that sialic acid-containing oligosaccharides reduce the adhesion of leukocytes to endothelial cells, an indication for an immune regulatory effect of certain HMO. We cover these topics after a short introduction on the structures of HMO, with a particular emphasis on their blood group and secretor specificity. PMID:18183941

  14. Low anticoagulant heparin oligosaccharides as inhibitors of BACE-1, the Alzheimer's β-secretase.

    PubMed

    Zhang, Xiao; Zhao, Xiaoliang; Lang, Yinzhi; Li, Qinying; Liu, Xiaoxiao; Cai, Chao; Hao, Jiejie; Li, Guoyun; Yu, Guangli

    2016-10-20

    Heparin (HP) is a promising agent for anti-Alzheimer's disease (AD), but its anticoagulant activity limits its applications. So a low anticoagulant heparin (LAH) with anti-AD effect is needed. A novel LAH and heparan sulfate (HS) were purified from crude porcine intestinal heparin. Their structures were characterized by nuclear magnetic resonance and liquid chromatography-mass spectrometry. LAH had a relatively high degree of sulfation, but lower than that of HP. 3-O-Sulfated-containing glucosamine residues further confirmed the low anticoagulant activity of LAH. Sixteen oligosaccharides of LAH and HS were prepared and assigned. Evaluation of anti-BACE-1 activities suggested that their potencies were positively correlated with degree of sulfation and polymerization of oligosaccharides. Besides, LAH-derived hexa- to dodecasaccharides was promised to be administrated in vitro as BACE-1 inhibitors. This study presented ideal BACE-1 inhibitors, LAH-derived oligosaccharides, with virtually no anticoagulant activities, which were promised to be excellent leads for treatment of AD. PMID:27474542

  15. Structural analysis of the asparagine-linked oligosaccharides of cholinesterases. N-linked carbohydrates of cholinesterases

    SciTech Connect

    Saxena, A.; Doctor, B.P.

    1995-12-31

    Cholinesterases are serine esterases that hydrolyse choline esters faster than other substrates. They are highly glycosylated proteins with up to 24% of their molecular weight constituted of carbohydrates. Here we report the results of our studies on the glycosylation of fetal bovine serum acetylcholinesterase (FBS AChE) and horse serum butyrylcholinesterase (Eq BChE). Analysis of the monosaccharide content of the two enzymes indicated that Eq BChE contained 520 nmoles of monosaccharide/mg protein, as compared to 1290 nmoles/mg protein for Eq BChE. Both enzymes contained mannose, galactose, N-acetylglucosamine and sialic acid. Fucose was present in Eq BChE only. The structures of the two major oligosaccharides from FBS AChE and one major oligosaccharide from Eq BChE were determined and found to be very similar except that one of the oligosaccharides from FBS AChE contained a galactose alphal-3 galactose betal-4-determinant which has been identified as a potentially immunogenic determinant.

  16. Oligosaccharide-specific receptors for gangliosides in the central nervous system

    SciTech Connect

    Tiemeyer, M.J.

    1989-01-01

    Synthetic ganglioside-derivatized proteins were prepared, radiolabeled, and used as ligands to search for specific receptors on rat brain membranes. Chemical derivatization schemes were designed to covalently link gangliosides (specifically, G{sub T1b}) to bovine serum albumin (BSA) via their ceramide portions leaving the glycolipid oligosaccharides intact and limiting the ability of the ganglioside moiety to interact with brain membranes non-specifically by insertion or hydrophobic adsorption. Following characterization and tyrosine-radioiodination, {sup 125}I-(G{sub T1b}){sub 4} BSA (BSA derivatized with 4 G{sub T1b} moieties/protein molecule), revealed a high affinity and saturable binding site on rat brain membranes. Pretreatment of brain membranes with low concentrations of trypsin blocked binding, consistent with the presence of a proteinaceous ganglioside-receptor. The most potent lipid inhibitors of {sup 125}I-(G{sub T1b}){sub 4}BSA binding were the gangliosides G{sub T1b}, G{sub D1b}, and G{sub Q1b} which share common structural features in their oligosaccharide portions; maximal inhibitory potency required a full length gangliotetraose oligosaccharide core and {alpha}2-8 linked sialic acid.

  17. Human milk oligosaccharides: The role in the fine-tuning of innate immune responses.

    PubMed

    Kulinich, Anna; Liu, Li

    2016-09-01

    In order to secure the health of newborns over the period of immune immaturity during the first months of life, a mother provides her offspring with passive protection: bioactive molecules transferred through the placenta and breast milk. It is well known that human milk contains immunoglobulins (Ig), immune cells and diverse cytokines, which affect newborn directly or indirectly and contribute to the maturation of the immune system. However, in addition to the above-stated molecules, human milk oligosaccharides (HMOs), a complex mixture of free indigestible carbohydrates with multiple functions, play exceptional roles in the functioning of the infants' immune system. These biological molecules have been studied over decades, however, interest in HMOs does not seem to have abated. Although biological activities of oligosaccharides from human milk have been explicitly reviewed, information regarding the role of HMOs in inflammation remains rather fragmented. The purpose of this review is to compile existing knowledge about the role of certain species of HMOs, including fucosylated, galactosylated and sialylated oligosaccharides, and their signaling pathways in immunity and inflammation. The advances in applying this information to the treatment of diseases in infants as well as adults were also reviewed here. PMID:27448325

  18. Models based on ultraviolet spectroscopy, polyphenols, oligosaccharides and polysaccharides for prediction of wine astringency.

    PubMed

    Boulet, Jean-Claude; Trarieux, Corinne; Souquet, Jean-Marc; Ducasse, Maris-Agnés; Caillé, Soline; Samson, Alain; Williams, Pascale; Doco, Thierry; Cheynier, Véronique

    2016-01-01

    Astringency elicited by tannins is usually assessed by tasting. Alternative methods involving tannin precipitation have been proposed, but they remain time-consuming. Our goal was to propose a faster method and investigate the links between wine composition and astringency. Red wines covering a wide range of astringency intensities, assessed by sensory analysis, were selected. Prediction models based on multiple linear regression (MLR) were built using UV spectrophotometry (190-400 nm) and chemical analysis (enological analysis, polyphenols, oligosaccharides and polysaccharides). Astringency intensity was strongly correlated (R(2) = 0.825) with tannin precipitation by bovine serum albumin (BSA). Wine absorbances at 230 nm (A230) proved more suitable for astringency prediction (R(2) = 0.705) than A280 (R(2) = 0.56) or tannin concentration estimated by phloroglucinolysis (R(2) = 0.59). Three variable models built with A230, oligosaccharides and polysaccharides presented high R(2) and low errors of cross-validation. These models confirmed that polysaccharides decrease astringency perception and indicated a positive relationship between oligosaccharides and astringency. PMID:26212982

  19. Structure-reactivity relationship of Amadori rearrangement products compared to related ketoses.

    PubMed

    Kaufmann, Martin; Meissner, Philipp M; Pelke, Daniel; Mügge, Clemens; Kroh, Lothar W

    2016-06-16

    Structure-reactivity relationships of Amadori rearrangement products compared to their related ketoses were derived from multiple NMR spectroscopic techniques. Besides structure elucidation of six Amadori rearrangement products derived from d-glucose and d-galactose with l-alanine, l-phenylalanine and l-proline, especially quantitative (13)C selective saturation transfer NMR spectroscopy was applied to deduce information on isomeric systems. It could be shown exemplarily that the Amadori compound N-(1-deoxy-d-fructos-1-yl)-l-proline exhibits much higher isomerisation rates than d-fructose, which can be explained by C-1 substituent mediated intramolecular catalysis. In combination with a reduced carbonyl activity of Amadori compounds compared to their related ketoses which results in an increased acyclic keto isomer concentration, the results on isomerisation dynamics lead to a highly significant increased reactivity of Amadori compounds. This can be clearly seen, comparing approximated carbohydrate milieu stability time constants (ACuSTiC) which is 1 s for N-(1-deoxy-d-fructos-1-yl)-l-proline and 10 s for d-fructose at pD 4.20 ± 0.05 at 350 K. In addition, first NMR spectroscopic data are provided, which prove that α-pyranose of (amino acid substituted) d-fructose adopts both, (2)C5 and (5)C2 conformation. PMID:27152632

  20. Heparin oligosaccharides: inhibitors of the biological activity of bFGF on Caco-2 cells.

    PubMed Central

    Jayson, G. C.; Gallagher, J. T.

    1997-01-01

    A number of growth factors, including members of the fibroblast growth factor (FGF) family - hepatocyte growth factor, vascular endothelial growth factor and heparin-binding epidermal growth factor - are dependent on heparan sulphate (HS) for biological activity mediated through their high-affinity signal-transducing receptors. This obligate requirement for HS prompted the search for antagonists of HS function that could be used as anti-growth factor drugs for the treatment of cancer. Basic FGF (bFGF) was the focus of this study. Caco-2, a human colon carcinoma cell line, was adapted to growth in serum-free medium so that investigation of its growth factor requirements for growth and migration could be performed in defined conditions (Jayson GC, Evans GS, Pemberton PW, Lobley RW, Allen T 1994, Cancer Res, 54, 5718-5723). This cell line multiplied and moved in a dose-dependent manner in response to bFGF. Here, we show that the mitogenic response to bFGF is dependent on the presence of heparan sulphate. A library of heparin oligosaccharides with uniform composition but variable length was generated [general formula [IdoA(2S)-GlcNS(6S)n], and oligosaccharides of defined lengths were tested for their ability to inhibit the biological activity of bFGF. While intact heparin and heparin-derived fragments of 12 monosaccharide units did not affect bFGF-induced cell division or bFGF-induced cell migration, octasaccharides and decasaccharides potently inhibited the bFGF-induced growth and migration responses. In particular, octasaccharides completely inhibited these biological activities at 10 microg ml-, a clinically achievable and tolerable concentration. This study shows that the length of an oligosaccharide determines its ability to block the biological activity of bFGF. The observation that the biological activity of cell-surface heparan sulphate can be antagonized in this way in a human carcinoma cell line suggests that oligosaccharides should be investigated further as

  1. Phylogenetic and Functional Alterations in Bacterial Community Compositions in Broiler Ceca as a Result of Mannan Oligosaccharide Supplementation

    PubMed Central

    de Leeuw, Marcel; Penaud-Frézet, Stéphanie; Dimova, Diliana; Murphy, R. A.

    2015-01-01

    This study focused on identifying reproducible effects of dietary supplementation with a mannan oligosaccharide (MOS) on the broiler cecal bacterial community structure and function in a commercial production setting. Two separate trials, each with a control and a supplemented group, were carried out in the same commercial location and run concurrently. Approximately 10,000 birds from the same commercial hatchery were mirror imaged into each of four commercial broiler sheds and fed either a control or supplemented diet. Cecal contents were obtained on days 7, 21, and 35 posthatch from 12 randomly caught broilers from each group. Bacterial pyrosequencing was performed on all samples, with approximately 250,000 sequences obtained per treatment per time point. The predominant phyla identified at all three time points in both trials were Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Tenericutes, representing >99% of all sequences. MOS supplementation altered the bacterial community composition from 7 days supplementation through 35 days supplementation. Bacteroidetes appeared to be replacing Firmicutes as a result of supplementation, with the most noticeable effects after 35 days. The effects of supplementation were reproducible across both trials. PICRUSt was used to identify differences between the functional potentials of the bacterial communities as a result of MOS supplementation. Using level 3 KEGG ortholog function predictions, differences between control and supplemented groups were observed, with very strong segregation noted on day 35 posthatch in both trials. This indicated that alterations of bacterial communities as a result of MOS are likely to alter the functional capability of the cecum. PMID:25769823

  2. X-ray Crystallographic Analyses of Pig Pancreatic α-Amylase with Limit Dextrin, Oligosaccharide and α-Cyclodextrin†‡

    PubMed Central

    Larson, Steven B.; Day, John S.; McPherson, Alexander

    2010-01-01

    Further refinement of the model using maximum likelihood procedures and re-evaluation of the native electron density map has shown that crystals of pig pancreatic α-amylase, whose structure we reported more than fifteen years ago, in fact contain a substantial amount of carbohydrate. The carbohydrate fragments are the products of glycogen digestion carried out as an essential step of the protein's purification procedure. In particular, the substrate-binding cleft contains a limit dextrin of six glucose residues, one of which contains both α-(1,4) and α-(1,6) linkages to contiguous residues. The disaccharide in the original model, shared between two amylase molecules in the crystal lattice, but also occupying a portion of the substrate binding cleft, is now seen to be a tetrasaccharide. There are, in addition, several other probable monosaccharide binding sites. To these results we have further reviewed our X-ray diffraction analysis of α-amylase complexed with α-cyclodextrin. α-Amylase binds three cyclodextrin molecules. Glucose residues of two of the rings superimpose upon the limit dextrin and the tetrasaccharide. The limit dextrin superimposes in large part upon linear oligosaccharide inhibitors visualized by other investigators. By comprehensive integration of these complexes we have constructed a model for the binding of polysaccharides having the helical character known to be present in natural substrates such as starch and glycogen. PMID:20222716

  3. Phylogenetic and functional alterations in bacterial community compositions in broiler ceca as a result of mannan oligosaccharide supplementation.

    PubMed

    Corrigan, A; de Leeuw, Marcel; Penaud-Frézet, Stéphanie; Dimova, Diliana; Murphy, R A

    2015-05-15

    This study focused on identifying reproducible effects of dietary supplementation with a mannan oligosaccharide (MOS) on the broiler cecal bacterial community structure and function in a commercial production setting. Two separate trials, each with a control and a supplemented group, were carried out in the same commercial location and run concurrently. Approximately 10,000 birds from the same commercial hatchery were mirror imaged into each of four commercial broiler sheds and fed either a control or supplemented diet. Cecal contents were obtained on days 7, 21, and 35 posthatch from 12 randomly caught broilers from each group. Bacterial pyrosequencing was performed on all samples, with approximately 250,000 sequences obtained per treatment per time point. The predominant phyla identified at all three time points in both trials were Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Tenericutes, representing >99% of all sequences. MOS supplementation altered the bacterial community composition from 7 days supplementation through 35 days supplementation. Bacteroidetes appeared to be replacing Firmicutes as a result of supplementation, with the most noticeable effects after 35 days. The effects of supplementation were reproducible across both trials. PICRUSt was used to identify differences between the functional potentials of the bacterial communities as a result of MOS supplementation. Using level 3 KEGG ortholog function predictions, differences between control and supplemented groups were observed, with very strong segregation noted on day 35 posthatch in both trials. This indicated that alterations of bacterial communities as a result of MOS are likely to alter the functional capability of the cecum. PMID:25769823

  4. Chemical characterization of milk oligosaccharides of the eastern quoll (Dasyurus viverrinus).

    PubMed

    Urashima, Tadasu; Sun, Yiliang; Fukuda, Kenji; Hirayama, Kentaro; Taufik, Epi; Nakamura, Tadashi; Saito, Tadao; Merchant, Jim; Green, Brian; Messer, Michael

    2015-08-01

    Structural characterizations of marsupial milk oligosaccharides have been performed in four species to date: the tammar wallaby (Macropus eugenii), the red kangaroo (Macropus rufus), the koala (Phascolarctos cinereus) and the common brushtail possum (Trichosurus vulpecula). To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, the oligosaccharides in the carbohydrate fraction of eastern quoll milk were characterized in this study. Neutral and acidic oligosaccharides were separated and characterized by (1)H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the neutral oligosaccharides were Gal(β1-3)Gal(β1-4)Glc (3'-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3",3'-digalactosyllactose), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I), Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)Gal(β1-4)Glc (galactosyl lacto-N-novopentaose II), Gal(β1-3)[Gal(β1-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose III) and Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novooctaose). The structures of the acidic oligosaccharides detected are Neu5Ac(α2-3)Gal(β1-4)Glc (3'-sialyllactose), Gal(β1-3)(O-3-sulfate)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate a), Gal(β1-3)[Gal(β1-4)(O-3-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose c), Neu5Ac(α2-3) Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, and Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc with an α(2

  5. OLIGOSACCHARIDES IN CANE AND THEIR FORMATION ON CANE DETERIORATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cane deterioration in the field, factory storage pile, or during factory milling processes has become a major technical concern in recent years, especially in those areas where mechanical harvesting of billeted sugar cane has increased. Not all deterioration products advocated as cane deterioratio...

  6. Ethanol production using a newly isolated Saccharomyces cerevisiae strain directly assimilating intact inulin with a high degree of polymerization.

    PubMed

    Yang, Fan; Liu, Zhicheng; Dong, Weifeng; Zhu, Linghuan; Chen, Xiaoyi; Li, Xianzhen

    2014-01-01

    An inulin-degrading strain L610, which was competent to directly convert inulin into ethanol, was isolated and identified as a strain of Saccharomyces cerevisiae according to physiological and phylogenetic analysis. Ion chromatography results showed that isolate L610 could assimilate the intact inulin completely without acidic or enzymatic pretreatment in contrast to the previously reported strains of S. cerevisiae, which could only ferment the fructo-oligosaccharides with a degree of polymerization less than 15. Strain L610 yielded 37.2 g/L ethanol within 48 H at a shake flask level under the evaluated culture conditions (11% inulin, 0.4% yeast extract, and 0.05% MgSO4 at 30 °C and pH 6.0). The conversion efficiency of inulin-type sugar to ethanol was 60% of the theoretical ethanol yield. Strain L610 produced 40.0 g/L of ethanol when directly fermented in Jerusalem artichoke (Helianthus tuberosus L.) powder suspension within 24 H, which was higher than the reported data, 28.9 g/L, produced by S. cerevisiae KCCM 50549. PMID:24237352

  7. Evaluation of the effect of post-translational modification toward protein structure: Chemical synthesis of glycosyl crambins having either a high mannose-type or a complex-type oligosaccharide.

    PubMed

    Dedola, Simone; Izumi, Masayuki; Makimura, Yutaka; Ito, Yukishige; Kajihara, Yasuhiro

    2016-11-01

    Glycoproteins are assembled and folded in the endoplasmic reticulum (ER) and transported to the Golgi for further processing of their oligosaccharides. During these processes, two types of oligosaccharides are used: that is, high mannose-type oligosaccharide in the ER and complex-type oligosaccharide in the Golgi. We were interested to know how two different types of oligosaccharides could influence the folding pathway or the final three-dimensional structure of the glycoproteins. For this purpose, we synthesized a new glycosyl crambin having complex-type oligosaccharide and evaluated the folding process, the final protein structure analyzed by NMR, and compared the CD spectra with previously synthesized glycosyl crambin bearing high mannose-type oligosaccharides. From our analysis, we found that the two different oligosaccharides do not influence the folding pathway in vitro and the final structure of the small glycoproteins. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 446-452, 2016. PMID:26587964

  8. Structural basis for the interaction between human milk oligosaccharides and the bacterial lectin PA-IIL of Pseudomonas aeruginosa

    PubMed Central

    2005-01-01

    One of the mechanisms contributing to the protection by breast-feeding of the newborn against enteric diseases is related to the ability of human milk oligosaccharides to prevent the attachment of pathogenic bacteria to the duodenual epithelium. Indeed, a variety of fucosylated oligosaccharides, specific to human milk, form part of the innate immune system. In the present study, we demonstrate the specific blocking of PA-IIL, a fucose-binding lectin of the human pathogen Pseudomonas aeruginosa, by milk oligosaccharides. Two fucosylated epitopes, Lewis a and 3-fucosyl-lactose (Lewis x glucose analogue) bind to the lectin with dissociation constants of 2.2×10−7 M and 3.6×10−7 M respectively. Thermodynamic studies indicate that these interactions are dominated by enthalpy. The entropy contribution is slightly favourable when binding to fucose and to the highest-affinity ligand, Lewis a. The high-resolution X-ray structures of two complexes of PA-IIL with milk oligosaccharides allow the precise determination of the conformation of a trisaccharide and a pentasaccharide. The different types of interaction between the oligosaccharides and the protein involve not only hydrogen bonding, but also calcium- and water-bridged contacts, allowing a rationalization of the thermodynamic data. This study provides important structural information about compounds that could be of general application in new therapeutic strategies against bacterial infections. PMID:15790314

  9. Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat Thy-1.

    PubMed Central

    Parekh, R B; Tse, A G; Dwek, R A; Williams, A F; Rademacher, T W

    1987-01-01

    To examine the extent to which protein structure and tissue-type influence glycosylation, we have determined the oligosaccharide structures at each of the three glycosylation sites (Asn-23, 74 and 98) of the cell surface glycoprotein Thy-1 isolated from rat brain and thymus. The results show that there is tissue-specificity of glycosylation and that superimposed on this is a significant degree of site-specificity. On the basis of the site distribution of oligosaccharides, we find that no Thy-1 molecules are in common between the two tissues despite the amino acid sequences being identical. We suggest, therefore, that by controlling N-glycosylation a tissue creates an unique set of glycoforms (same polypeptide but with oligosaccharides that differ either in sequence or disposition). The structures at each of the three sites were also determined for the thymocyte Thy-1 that binds to lentil lectin (Thy-1 L+) and for that which does not (Thy-1 L-). Segregation of intact thymus Thy-1 into two distinct sets of glycoforms by lentil lectin was found to be due to the structures at site 74. Analysis of oligosaccharide structures at the 'passenger' sites (23 and 98) suggests that either Thy-1 L+ and Thy-1 L- molecules are made in different cell-types or that the biosynthesis of oligosaccharides at one site is influenced by the glycosylation at other sites. PMID:2886334

  10. Structures of the SER/THR linked variant oligosaccharides present in equine chorionic gonadotropin (eCG). beta. -subunit

    SciTech Connect

    Bahl, O.P.; Anumula, K.R.

    1986-05-01

    eCG ..beta..-subunit contains more than 50% carbohydrate and constitutes about 72% of the hormone. O-linked carbohydrate (85%) was separated from the N-linked (15%) by gel filtration of the endoproteinase Lys-C digest. Six O-linked carbohydrate units were released by NaOH/NaB/sup 3/H/sub 4/ treatment. Oligosaccharides were fractionated by gel filtration and paper chromatography. Several oligosaccharides were obtained ranging in size from a sialyl di-saccharide to megalosaccharide with about 50 sugar residues. Methylation analyses and tlc examination of the oligosaccharides after endo- and exoglycosidase digestions and nitrous acid deamination and Smith degradation revealed a core structure of Gal..beta..1-4 GlcNAc..beta..1-6 (Gal ..beta..1-3) GalNAcH/sub 2/ with poly-N-acetyllactosamine peripheral extensions. Nearly 50% of the oligosaccharides were large and were preferentially extended on 1,6 arm of the GalNAcH/sub 2/ by repeating N-acetyllactosamine units. Furthermore, these oligosaccharides contained branching 1,3,6-linked galactoses giving rise to tri, penta and multiantennary structures.

  11. A quantitative and comprehensive method to analyze human milk oligosaccharide structures in the urine and feces of infants

    PubMed Central

    De Leoz, Maria Lorna A.; Wu, Shuai; Strum, John S.; Niñonuevo, Milady R.; Gaerlan, Stephanie C.; Mirmiran, Majid; German, J. Bruce; Mills, David A.; Lebrilla, Carlito B.; Underwood, Mark A.

    2013-01-01

    Human milk oligosaccharides (HMOs), though non-nutritive to the infant, shape the intestinal microbiota and protect against pathogens during early growth and development. Infant formulas with added galacto-oligosaccharides have been developed to mimic the beneficial effects of HMOs. Premature infants have an immature immune system and a leaky gut and are thus highly susceptible to opportunistic infections. A method employing nanoflow liquid chromatography time-of-flight mass spectrometry (MS) is presented to simultaneously identify and quantify HMOs in the feces and urine of infants, of which 75 HMOs have previously been fully structurally elucidated. Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS was employed for high-resolution and rapid compositional profiling. To demonstrate this novel method, samples from mother-infant dyads as well as samples from infants receiving infant formula fortified with dietary galacto-oligosaccharides or probiotic bifidobacteria were analyzed. Ingested oligosaccharides are demonstrated in high abundance in the infant feces and urine. While the method was developed to examine specimens from preterm infants, it is of general utility and can be used to monitor oligosaccharide consumption and utilization in term infants, children and adults. This method may therefore provide diagnostic and therapeutic opportunities. PMID:23468138

  12. Structural studies on colanic acid, the common exopolysaccharide found in the Enterobacteriaceae, by partial acid hydrolysis. Oligosaccharides from colanic acid

    PubMed Central

    Sutherland, I. W.

    1969-01-01

    The exopolysaccharide slime colanic acid has been isolated from representative strains of Escherichia coli, Salmonella typhimurium and Aerobacter cloacae. Analysis showed that each polymer contained glucose, galactose, fucose and glucuronic acid, together with acetate and pyruvate. The molar proportions of these components were 1:1·8:1·9:1:1:1 approximately. On the basis of periodate oxidation of the natural and deacetylated polysaccharide, glucose is proposed as the site of the acetyl groups. The pyruvate is attached to galactose. Three neutral oligosaccharides and ten electrophoretically mobile oligosaccharides were isolated and partially characterized. Four of the fragments were esters of pyruvic acid. Most oligosaccharides were isolated from all three polysaccharide preparations. Three further oligosaccharides were isolated from carboxyl-reduced colanic acid and sodium borotritide was used to label the glucose derived from glucuronic acid in these fragments. One trisaccharide was obtained from periodate-oxidized polysaccharide. On the basis of these oligosaccharides a repeating hexasaccharide unit of the following structure is proposed: [Formula: see text] The significance of this structure in colanic acid biosynthesis is discussed. PMID:4311825

  13. Fermentation properties and potential prebiotic activity of Bimuno® galacto-oligosaccharide (65 % galacto-oligosaccharide content) on in vitro gut microbiota parameters.

    PubMed

    Grimaldi, Roberta; Swann, Jonathan R; Vulevic, Jelena; Gibson, Glenn R; Costabile, Adele

    2016-08-01

    Prebiotic oligosaccharides have the ability to generate important changes in the gut microbiota composition that may confer health benefits to the host. Reducing the impurities in prebiotic mixtures could expand their applications in food industries and improve their selectivity and prebiotic effect on the potential beneficial bacteria such as bifidobacteria and lactobacilli. This study aimed to determine the in vitro potential fermentation properties of a 65 % galacto-oligosaccharide (GOS) content Bimuno® GOS (B-GOS) on gut microbiota composition and their metabolites. Fermentation of 65 % B-GOS was compared with 52 % B-GOS in pH- and volume-controlled dose-response anaerobic batch culture experiments. In total, three different doses (1, 0·5 and 0·33 g equivalent to 0·1, 0·05 and 0·033 g/l) were tested. Changes in the gut microbiota during a time course were identified by fluorescence in situ hybridisation, whereas small molecular weight metabolomics profiles and SCFA were determined by 1H-NMR analysis and GC, respectively. The 65 % B-GOS showed positive modulation of the microbiota composition during the first 8 h of fermentation with all doses. Administration of the specific doses of B-GOS induced a significant increase in acetate as the major SCFA synthesised compared with propionate and butyrate concentrations, but there were no significant differences between substrates. The 65 % B-GOS in syrup format seems to have, in all the analysis, an efficient prebiotic effect. However, the applicability of such changes remains to be shown in an in vivo trial. PMID:27267934

  14. Chemical characterization of milk oligosaccharides of the common wombat (Vombatus ursinus).

    PubMed

    Hirayama, Kentaro; Taufik, Epi; Kikuchi, Megumi; Nakamura, Tadashi; Fukuda, Kenji; Saito, Tadao; Newgrain, Keith; Green, Brian; Messer, Michael; Urashima, Tadasu

    2016-09-01

    Previous structural characterizations of marsupial milk oligosaccharides have been performed in the tammar wallaby, red kangaroo, koala, common brushtail possum and the eastern quoll. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of wombat milk carbohydrate were characterized in this study. Neutral and acidic oligosaccharides were isolated from the carbohydrate fractions of two samples of milk of the common wombat and characterized by (1) H-nuclear magnetic resonance spectroscopy. The structures of six neutral saccharides were found to be Gal(β1-4)Glc (lactose), Gal(β1-3)Gal(β1-4)Glc (3'-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3',3"-digalactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I) and Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novooctaose), while those of six acidic saccharides were Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc. (sialyl 3'-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3',3"-digalactosyllactose), Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose c), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc,, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc and Gal(β1-3)Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc. In addition, small amounts of sulfated oligosaccharides but no oligosaccharides containing Neu5Gc or α(2-6) linked Neu5Ac were detected. PMID:26608481

  15. A rationally engineered yeast pyruvyltransferase Pvg1p introduces sialylation-like properties in neo-human-type complex oligosaccharide.

    PubMed

    Higuchi, Yujiro; Yoshinaga, Sho; Yoritsune, Ken-Ichi; Tateno, Hiroaki; Hirabayashi, Jun; Nakakita, Shin-Ichi; Kanekiyo, Miho; Kakuta, Yoshimitsu; Takegawa, Kaoru

    2016-01-01

    Pyruvylation onto the terminus of oligosaccharide, widely seen from prokaryote to eukaryote, confers negative charges on the cell surface and seems to be functionally similar to sialylation, which is found at the end of human-type complex oligosaccharide. However, detailed molecular mechanisms underlying pyruvylation have not been clarified well. Here, we first determined the crystal structure of fission yeast pyruvyltransferase Pvg1p at a resolution of 2.46 Å. Subsequently, by combining molecular modeling with mutational analysis of active site residues, we obtained a Pvg1p mutant (Pvg1p(H168C)) that efficiently transferred pyruvyl moiety onto a human-type complex glycopeptide. The resultant pyruvylated human-type complex glycopeptide recognized similar lectins on lectin arrays as the α2,6-sialyl glycopeptides. This newly-generated pyruvylation of human-type complex oligosaccharides would provide a novel method for glyco-bioengineering. PMID:27194449

  16. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the sensitive determination of hyaluronan oligosaccharides.

    PubMed

    Rothenhöfer, Martin; Grundmann, Marco; Bernhardt, Günther; Matysik, Frank-Michael; Buschauer, Armin

    2015-04-15

    High performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) was optimized for the analysis of oligosaccharides derived from the extracellular matrix component hyaluronan. Using this sensitive approach, the separation of oligosaccharides consisting of two (molecular weight ca. 0.8 kDa) up to 25-30 (molecular weight: ca. 9.5-11.4 kDa) disaccharide moieties was possible. Standard oligosaccharides (comprising 2-4 repetitive disaccharides) were detectable at very low amounts of 0.2-0.3 pmol (20-30 nM). Including 10 min of column equilibration, a complex mixture of low molecular weight hyaluronan can be analyzed within 40 min. The HPAEC method was successfully applied to the study of the size-dependency of both the action of bovine testicular hyaluronidase (BTH) and the precipitation of hyaluronan by cetyltrimethylammonium bromide (CTAB), a physicochemical reaction often used for the determination of hyaluronan and hyaluronidase activity. PMID:25768984

  17. A rationally engineered yeast pyruvyltransferase Pvg1p introduces sialylation-like properties in neo-human-type complex oligosaccharide

    PubMed Central

    Higuchi, Yujiro; Yoshinaga, Sho; Yoritsune, Ken-ichi; Tateno, Hiroaki; Hirabayashi, Jun; Nakakita, Shin-ichi; Kanekiyo, Miho; Kakuta, Yoshimitsu; Takegawa, Kaoru

    2016-01-01

    Pyruvylation onto the terminus of oligosaccharide, widely seen from prokaryote to eukaryote, confers negative charges on the cell surface and seems to be functionally similar to sialylation, which is found at the end of human-type complex oligosaccharide. However, detailed molecular mechanisms underlying pyruvylation have not been clarified well. Here, we first determined the crystal structure of fission yeast pyruvyltransferase Pvg1p at a resolution of 2.46 Å. Subsequently, by combining molecular modeling with mutational analysis of active site residues, we obtained a Pvg1p mutant (Pvg1pH168C) that efficiently transferred pyruvyl moiety onto a human-type complex glycopeptide. The resultant pyruvylated human-type complex glycopeptide recognized similar lectins on lectin arrays as the α2,6-sialyl glycopeptides. This newly-generated pyruvylation of human-type complex oligosaccharides would provide a novel method for glyco-bioengineering. PMID:27194449

  18. Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis.

    PubMed

    Zhao, Yunxue; Li, Jing; Wang, Jingjian; Xing, Yanli; Geng, Meiyu

    2007-06-01

    Malignant transformation is associated with changes in the glycosylation of cell surface proteins and lipids. In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. In the present study, we have assessed the relationship between cell surface oligosaccharides and the metastasis ability of mouse mammary tumor cell lines 67NR and 4TO7. The cell surface oligosaccharides have been analyzed using specific binding assays with some plant lectins and the metastasis ability has been studied using transwell migration and invasion assays. In addition, we investigated the role of terminal sialic acids in the metastatic potential (cell adhesion on fibronectin, cell migration and invasion) in the 4TO7 cells on treatment with neuraminidase. The cell lines used in study have different metastasis abilities in vivo - the 67NR form primary tumors, but no tumor cells are detectable in any distant tissues, while cells of the 4TO7 line are able to spread to lung. In vitro metastasis experiments have revealed higher ability of adhesion, cell migration and invasion in the 4TO7 cells than the 67NR cells. Specific lectins binding assays show that the 4TO7 cells expressed more high-mannose type, multi-antennary complex-type N-glycans, beta-1,6-GlcNAc-branching, alpha-2,6-linked sialic acids, N-acetylgalactosamine and galactosyl(beta-1,3)-N-acetylgalactosamine. Removal of sialic acids on treatment with neuraminidase decreases adhesion, but increases the migration and has shown no significant change in the invasion ability of the 4TO7 cells. The study suggests that the sialic acids are not crucial for the cell migration and invasion in the 4TO7 cells. The findings provide the new insights in understanding the role of cell surface oligosaccharides in cancer metastasis. PMID:17650582

  19. Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum.

    PubMed Central

    Verhoeven, A J; Neve, B P; Jansen, H

    1999-01-01

    Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of alpha1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8+/-2.1-fold higher (mean+/-S.D., n=7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3+/-0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose-heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7+/-0. 3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not alpha1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked. PMID:9854035

  20. N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

    SciTech Connect

    Clagett-Dame, M.; McKelvy, J.F. )

    1989-10-01

    The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-(125I)iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide.

  1. Reduced immunogenicity of beta-lactoglobulin by conjugation with acidic oligosaccharides.

    PubMed

    Hattori, Makoto; Miyakawa, Shunpei; Ohama, Yukie; Kawamura, Hiroyuki; Yoshida, Tadashi; To-o, Kenji; Kuriki, Takashi; Takahashi, Koji

    2004-07-14

    Bovine beta-lactoglobulin (beta-LG) was conjugated with the acidic oligosaccharides, alginic acid oligosaccharide (ALGO) and phosphoryl oligosaccharides (POs) by the Maillard reaction to reduce the immunogenicity of beta-LG. The molar ratios of beta-LG to ALGO and POs in the conjugates were 1:6 and 1:8. The carbohydrate-binding sites in the beta-LG-ALGO conjugate were partially identified to be (60)Lys, (77)Lys, (100)Lys, (138)Lys, and (141)Lys. The isoelectric point of each conjugate was lower than that of beta-LG. CD spectra indicated that the secondary structure of beta-LG was almost maintained after conjugation. The results of fluorescence studies indicated that the conformation around Trp had not changed in each conjugate and that the surface of each conjugate was covered with a saccharide chain. Structural analyses with monoclonal antibodies indicated that the conformation around (8)Lys-(19)Trp (beta-sheet, random coil, short helix) in the conjugates had changed, whereas the native structure was maintained around (15)Val-(29)Ile (beta-sheet) and (125)Thr-(135)Lys (alpha-helix). The beta-LG-ALGO and beta-LG-POs conjugates maintained 77 and 70% of the retinol binding activity of beta-LG. Conjugation with ALGO and POs substantially enhanced the thermal stability of beta-LG. The anti-beta-LG antibody response was markedly reduced after immunization with both conjugates in BALB/c, C57BL/6, and C3H/He mice. B cell epitopes of beta-LG and the conjugate recognized in these mice were determined with 15-mer multipin peptides, and the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, whereas the antibody response to each epitope was dramatically reduced. In particular, effective reduction of the antibody response was observed in the vicinity of the carbohydrate-binding sites. Conjugation of beta-LG with these acidic oligosaccharides was effective in reducing the immunogenicity of beta-LG. The conjugates obtained in this study are

  2. Chemical characterization of milk oligosaccharides of the common brushtail possum (Trichosurus vulpecula).

    PubMed

    Urashima, Tadasu; Fujita, Saori; Fukuda, Kenji; Nakamura, Tadashi; Saito, Tadao; Cowan, Phil; Messer, Michael

    2014-07-01

    Structural characterizations of marsupial milk oligosaccharides have been performed in only three species: the tammar wallaby, the red kangaroo and the koala. To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, 21 oligosaccharides of the milk carbohydrate fraction of the common brushtail possum were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of mid-lactation milk and characterized by (1)H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the 7 neutral oligosaccharides were Gal(β1-3)Gal(β1-4)Glc (3'-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3", 3'-digalactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I), Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)Gal(β1-4)Glc (galactosyl lacto-N-novopentaose II). The structures of the 14 acidic oligosaccharides detected were Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3'-galactosyllactose), Gal(β1-3)(O-3-sulfate)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate a) Gal(β1-3)[Gal(β1-4)(O-3-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)(-3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)[Gal(β1-4)(-3-O-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulphate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2

  3. Fermentation of soluble cello-oligosaccharides by yeasts

    SciTech Connect

    Lastick, S.M.; Spindler, D.D.; Grohmann, K.

    1983-02-01

    Yeast strains that ferment cellobiose were examined with respect to fermentation on soluble cellodextrin preparations. Hydrolysis of the fermentation products was followed using thin layer chromatography. Candida and Brettanomyces sp. hydrolyze cellobiose and, at a much lower rate, cellotriose, indicating the presence of ..beta..-glucosidase (EC 3.2.1.21) activity. Enzyme assays conducted on B. clausenii fermentations indicated that the ..beta..-glucosidase remained cell-associated during fermentation. Torulopsis sp. hydrolyzed all of the cello-oligo-saccharides, indicating exoglucanase (EC 3.2.1.91) activity. The exogluconanase, a glycoprotein with an apparent molecular weight of 8.4 x 10/sup 4/ daltons, is exported into the culture medium.

  4. Inaccuracy of AOAC method 2009.01 with amyloglucosidase for measuring non-digestible oligosaccharides and proposal for an improvement of the method.

    PubMed

    Tanabe, Kenichi; Nakamura, Sadako; Oku, Tsuneyuki

    2014-05-15

    We wished to clarify the inaccuracy of AOAC method 2009.01 for the measurement of non-digestible oligosaccharides and to propose an improved method using porcine intestinal enzymes. Amyloglucosidase used in AOAC method 2009.01 scarcely hydrolyses sucrose, palatinose and panose (which are readily digested by intestinal enzymes). Hence, oligosaccharides could not be measured accurately by AOAC method 2009.01. To confirm the inaccuracy of the method, we used porcine intestinal enzymes instead of amyloglucosidase. Using the improved method, fructooligosaccharide and galactooligosaccharide were measured accurately as non-digestible oligosaccharides, but sucrose, palatinose, panose and isomaltooligosaccharide were not. The improved method hydrolysed digestible oligosaccharides into monosaccharides. These results demonstrate that the inaccuracy of AOAC method 2009.01 for oligosaccharide measurement is due to incomplete hydrolysis by amyloglucosidase. We propose that amyloglucosidase should be replaced with porcine intestinal enzymes for such measurements. PMID:24423568

  5. Formation of protein-oligosaccharide conjugates by laccase and tyrosinase.

    PubMed

    Selinheimo, Emilia; Lampila, Piritta; Mattinen, Maija-Liisa; Buchert, Johanna

    2008-05-14

    Proteins and certain carbohydrates contain phenolic moieties, which are potential sites for modification of the function of the biopolymers. In this study, the capability of two different fungal oxidative enzymes, laccase from Trametes hirsuta (ThL) and tyrosinase from Trichoderma reesei (TrT), to catalyze formation of hetero-cross-linking between tyrosine side chains of alpha-casein and phenolic acids of hydrolyzed oat spelt xylan (hOSX) was studied. Formation of reaction products was followed by size exclusion chromatography (SEC), fluorescence spectroscopy, and SDS-PAGE, using specific staining methods for proteins and protein-carbohydrate conjugates. ThL and TrT were observed to differ significantly in their ability to catalyze the formation of protein-carbohydrate conjugates or the linking of the small molecular weight phenolic compounds to alpha-casein. The efficiency of these enzymes to directly cross-link protein also differed notably. TrT was able to cross-link alpha-casein more efficiently than ThL. ThL-catalyzed casein cross-linking was significantly enhanced by ferulic acid, p-coumaric acid, and also hOSX. The main reaction products by ThL appeared to be phenolic acid-bridged alpha-caseins. Indications of hetero-cross-link formation between alpha-casein and hOSX by both oxidative enzymes could be visualized by glycoprotein-specific staining in the SDS-PAGE analysis, although ThL was observed to be more effective in the heteroconjugate formation than TrT. PMID:18422326

  6. Galacto-oligosaccharides have prebiotic activity in a dynamic in vitro colon model using a (13)C-labeling technique.

    PubMed

    Maathuis, Annet J H; van den Heuvel, Ellen G; Schoterman, Margriet H C; Venema, Koen

    2012-07-01

    Galacto-oligosaccharides (GOS) are considered to be prebiotic, although the contribution of specific members of the microbiota to GOS fermentation and the exact microbial metabolites that are produced upon GOS fermentation are largely unknown. We aimed to determine this using uniformly (13)C-labeled GOS. The normal (control) medium and unlabeled or (13)C-labeled GOS was added to a dynamic, validated, in vitro model of the large-intestine containing an adult-type microbiota. Liquid-chromatography MS was used to measure the incorporation of (13)C label into metabolites. 16S-rRNA stable isotope probing coupled to a phylogenetic micro-array was used to determine label incorporation in microbial biomass. The primary members within the complex microbiota that were directly involved in GOS fermentation were shown to be Bifidobacterium longum, B. bifidum, B. catenulatum, Lactobacillus gasseri, and L. salivarius, in line with the prebiotic effect of GOS, although some other species incorporated (13)C label also. GOS fermentation led to an increase in acetate (+49%) and lactate (+23%) compared with the control. Total organic acid production was 8.50 and 7.52 mmol/g of carbohydrate fed for the GOS and control experiments, respectively. At the same time, the cumulative production of putrefactive metabolites (branched-chain fatty acids and ammonia) was reduced by 55%. Cross-feeding of metabolites from primary GOS fermenters to other members of the microbiota was observed. Our findings support a prebiotic role for GOS and its potential to act as a synbiotic in combination with certain probiotic strains. PMID:22623395

  7. Effects of yeast cell wall-derived mannan-oligosaccharides on jejunal gene expression in young broiler chickens.

    PubMed

    Xiao, R; Power, R F; Mallonee, D; Routt, K; Spangler, L; Pescatore, A J; Cantor, A H; Ao, T; Pierce, J L; Dawson, K A

    2012-07-01

    The use of mannan-oligosaccharides (MOS) as alternatives to antibiotic growth promoters (AGP) has gained in popularity in recent years due to regulatory restrictions of using AGP in food animal production. Benefits of MOS usage include improvement on animal performance, feed efficiency, and gastrointestinal health. The molecular mechanisms of these functions however are not clear. The goal of the current study was to use a transcriptomics approach to investigate the effects of MOS on the intestinal gene expression profile of young broilers and characterize biological gene pathways responsible for the actions of MOS. One hundred and twenty 1-d-old Cobb 500 broiler chicks were randomly divided into 2 groups and were fed either a standard wheat-soybean meal-based (control) diet or the same diet supplemented with 2.2 g/kg of MOS (Bio-Mos, Alltech, Nicholasville, KY) for 3 wk, followed by jejunal gene expression profiling analysis using chicken-specific Affymetrix microarrays. Results indicated that a total of 672 genes were differentially expressed (P < 0.01 and fold change >1.2) in the jejunum by MOS supplementation. Association analysis indicated that differentially expressed genes are involved in diverse biological functions including energy production, cell death, and protein translation. Expression of 77 protein synthesis-related genes was differentially regulated by MOS in the jejunum. Further pathway analysis indicated that 15 genes related to oxidative phosphorylation were upregulated in the jejunum, and expression of genes important in cellular stress response, such as peroxiredoxin 1, superoxide dismutase 1, and thioredoxin, were also increased by MOS. Differential expression of genes associated with cellular immune processes, including lysozyme, lumican, β 2-microglobin, apolipoprotein A-1, and fibronectin 1, were also observed in MOS-fed broilers. In summary, this study systematically identified biological functions and gene pathways that are important in

  8. LC-MS/MS analysis of permethylated free oligosaccharides and N-glycans derived from human, bovine, and goat milk samples.

    PubMed

    Dong, Xue; Zhou, Shiyue; Mechref, Yehia

    2016-06-01

    Oligosaccharides in milk not only provide nutrition to the infants but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat, and human milks using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat, and human milk samples (without isomeric consideration) were 11, 8, and 11, respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat, and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by porous graphitic carbon LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using porous graphitic carbon column. Permethylation of the glycan structures facilitated the interpretation of MS/MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers. PMID:26959529

  9. A randomised, double-blind, placebo controlled cross-over study to determine the gastrointestinal effects of consumption of arabinoxylan-oligosaccharides enriched bread in healthy volunteers

    PubMed Central

    2012-01-01

    Background Prebiotics are food ingredients, usually non-digestible oligosaccharides, that are selectively fermented by populations of beneficial gut bacteria. Endoxylanases, altering the naturally present cereal arabinoxylans, are commonly used in the bread industry to improve dough and bread characteristics. Recently, an in situ method has been developed to produce arabinoxylan-oligosaccharides (AXOS) at high levels in breads through the use of a thermophilic endoxylanase. AXOS have demonstrated potentially prebiotic properties in that they have been observed to lead to beneficial shifts in the microbiota in vitro and in murine, poultry and human studies. Methods A double-blind, placebo controlled human intervention study was undertaken with 40 healthy adult volunteers to assess the impact of consumption of breads with in situ produced AXOS (containing 2.2 g AXOS) compared to non-endoxylanase treated breads. Volatile fatty acid concentrations in faeces were assessed and fluorescence in situ hybridisation was used to assess changes in gut microbial groups. Secretory immunoglobulin A (sIgA) levels in saliva were also measured. Results Consumption of AXOS-enriched breads led to increased faecal butyrate and a trend for reduced iso-valerate and fatty acids associated with protein fermentation. Faecal levels of bifidobacteria increased following initial control breads and remained elevated throughout the study. Lactobacilli levels were elevated following both placebo and AXOS-breads. No changes in salivary secretory IgA levels were observed during the study. Furthermore, no adverse effects on gastrointestinal symptoms were reported during AXOS-bread intake. Conclusions AXOS-breads led to a potentially beneficial shift in fermentation end products and are well tolerated. PMID:22657950

  10. Functional Analysis of Family GH36 α-Galactosidases from Ruminococcus gnavus E1: Insights into the Metabolism of a Plant Oligosaccharide by a Human Gut Symbiont

    PubMed Central

    Cervera-Tison, M.; Tailford, L. E.; Fuell, C.; Bruel, L.; Sulzenbacher, G.; Henrissat, B.; Berrin, J. G.; Fons, M.; Giardina, T.

    2012-01-01

    Ruminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14–21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2. Although aga1 showed a very simple genetic organization, aga2 is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed in Escherichia coli showed strict specificity for α-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the α-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with α-(1,6) regioselectivity, leading to the formation of product structures up to [Hex]12 and [Hex]8, respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota. PMID:22923411

  11. Galactoglucomannan Oligosaccharides (GGMO) from a Molasses Byproduct of Pine (Pinus taeda) Fiberboard Production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    “Temulose” is the trade name for a water-soluble molasses produced on a large scale (300 - 400 tonnes per year) as a byproduct of the fiberboard industry. The feedstock for temulose is predominantly a single species of pine (Pinus taeda) grown and harvested in stands in southeastern Texas. Because...

  12. Switchgrass alkaline pretreatment, enzymatic saccharification, and fermentation with residual oligosaccharide product analysis by mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Switchgrass (SG) is a potential renewable biomass source for conversion to liquid biofuels. Efficient conversion requires effective strategies for pretreatment and enzymatic saccharification to fermentable sugars. Standard analysis of fermentation broth includes detection of monosaccharides and etha...

  13. Galactoglucomannan Oligosaccharides (GGMO) from a Molasses Byproduct of Pine (Pinus taeda) Fiberboard Production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    "Temulose" is the trade name for a water-soluble molasses produced on a large scale (300 - 400 tonnes per year) as a byproduct of the fiberboard industry. The feedstock for temulose is predominantly a single species of pine (Pinus taeda) grown and harvested in stands in south-eastern Texas. Becaus...

  14. Agricultural residue valorization using a hydrothermal process for second generation bioethanol and oligosaccharides production.

    PubMed

    Vargas, Fátima; Domínguez, Elena; Vila, Carlos; Rodríguez, Alejandro; Garrote, Gil

    2015-09-01

    In the present work, the hydrothermal valorization of an abundant agricultural residue has been studied in order to look for high added value applications by means of hydrothermal pretreatment followed by fed-batch simultaneous saccharification and fermentation, to obtain oligomers and sugars from autohydrolysis liquors and bioethanol from the solid phase. Non-isothermal autohydrolysis was applied to barley straw, leading to a solid phase with about a 90% of glucan and lignin and a liquid phase with up to 168 g kg(-1) raw material valuable hemicellulose-derived compounds. The solid phase showed a high enzymatic susceptibility (up to 95%). It was employed in the optimization study of the fed-batch simultaneous saccharification and fermentation, carried out at high solids loading, led up to 52 g ethanol/L (6.5% v/v). PMID:26000836

  15. Chemical depolymerization of switchgrass xylan with oligosaccharide product analysis by HPAEC-PAD and mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylan is a barrier to enzymatic hydrolysis of plant cell walls. It is well accepted that the xylan layer needs to be removed to efficiently hydrolyze cellulose and consequently pretreatment conditions are in part optimized for maximal xylan depolymerization or displacement. Xylan consists of a lon...

  16. Plant nuclear pore complex proteins are modified by novel oligosaccharides with terminal N-acetylglucosamine.

    PubMed Central

    Heese-Peck, A; Cole, R N; Borkhsenious, O N; Hart, G W; Raikhel, N V

    1995-01-01

    Only a few nuclear pore complex (NPC) proteins, mainly in vertebrates and yeast but none in plants, have been well characterized. As an initial step to identify plant NPC proteins, we examined whether NPC proteins from tobacco are modified by N-acetylglucosamine (GlcNAc). Using wheat germ agglutinin, a lectin that binds specifically to GlcNAc in plants, specific labeling was often found associated with or adjacent to NPCs. Nuclear proteins containing GlcNAc can be partially extracted by 0.5 M salt, as shown by a wheat germ agglutinin blot assay, and at least eight extracted proteins were modified by terminal GlcNAc, as determined by in vitro galactosyltransferase assays. Sugar analysis indicated that the plant glycans with terminal GlcNAc differ from the single O-linked GlcNAc of vertebrate NPC proteins in that they consist of oligosaccharides that are larger in size than five GlcNAc residues. Most of these appear to be bound to proteins via a hydroxyl group. This novel oligosaccharide modification may convey properties to the plant NPC that are different from those of vertebrate NPCs. PMID:8589629

  17. Glycan specificity of myelin-associated glycoprotein and sialoadhesin deduced from interactions with synthetic oligosaccharides.

    PubMed

    Strenge, K; Schauer, R; Bovin, N; Hasegawa, A; Ishida, H; Kiso, M; Kelm, S

    1998-12-01

    Myelin-associated glycoprotein (MAG) and sialoadhesin (Sn) bind to sialylated glycans on cell surfaces and are thought to be involved in cell-cell interactions. In order to investigate how the interactions of these proteins are influenced by the glycan structure, we compared the inhibitory potencies of different synthetic monovalent oligosaccharides and polyvalent polyacrylamide derivatives. Using oligosaccharides with modifications in the sialic acid, galactose or N-acetylglucosamine moieties, we could demonstrate that both MAG and Sn bind with high preference to alpha2,3-linked sialic acid and interact at least with the three terminal monosaccharide units. For MAG, contacts with even more distant monosaccharides are likely, since pentasaccharides are bound better than trisaccharides. Also, an additional sialic acid at position six of the third-terminal monosaccharide unit enhances binding to MAG, whereas it does not influence binding to Sn significantly. Modifications of the sialic acid glycerol side chain demonstrated that the hydroxy groups at positions 8 and 9 are required for binding to both proteins. Surprisingly, MAG binds 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid significantly better than N-acetylneuraminic acid, whereas Sn prefers the latter structure. These results indicate that the interactions of MAG and Sn are mainly with sialic acid and that additional contacts with the subterminal galactose and N-acetylglucosamine residues also contribute to the binding strength, although to a lesser degree. PMID:9874234

  18. Structural basis for the recognition of complex-type biantennary oligosaccharides by Pterocarpus angolensis lectin.

    PubMed

    Buts, Lieven; Garcia-Pino, Abel; Imberty, Anne; Amiot, Nicolas; Boons, Geert-Jan; Beeckmans, Sonia; Versées, Wim; Wyns, Lode; Loris, Remy

    2006-06-01

    The crystal structure of Pterocarpus angolensis lectin is determined in its ligand-free state, in complex with the fucosylated biantennary complex type decasaccharide NA2F, and in complex with a series of smaller oligosaccharide constituents of NA2F. These results together with thermodynamic binding data indicate that the complete oligosaccharide binding site of the lectin consists of five subsites allowing the specific recognition of the pentasaccharide GlcNAc beta(1-2)Man alpha(1-3)[GlcNAc beta(1-2)Man alpha(1-6)]Man. The mannose on the 1-6 arm occupies the monosaccharide binding site while the GlcNAc residue on this arm occupies a subsite that is almost identical to that of concanavalin A (con A). The core mannose and the GlcNAc beta(1-2)Man moiety on the 1-3 arm on the other hand occupy a series of subsites distinct from those of con A. PMID:16704415

  19. Microanalysis of oligosaccharide HS203 in beagle dog plasma by postcolumn fluorescence derivatization method.

    PubMed

    Sun, Shumeng; Zhao, Xia; Li, Guangsheng; Yu, Guangli; Xing, Xiaoxu; Zeng, Yangyang; Wu, Jian; Wang, Jianing

    2012-06-20

    A rapid and sensitive postcolumn fluorescence derivatization method was developed for microanalysis of antidiabetic oligosaccharide HS203 in beagle dog plasma. After plasma protein was removed by a simple and fast ultrafiltration method, chromatographic separation was performed on an Asahipak GS-320 HQ column with a mobile phase of 50 mmol/L phosphate buffer (pH 6.7) and acetonitrile (83/17, v/v). The column effluent was monitored by fluorescence detection at 249 nm (excitation) and 435 nm (emission) using guanidine hydrochloride as a postcolumn derivatizing reagent. A satisfactory resolution of the analyte was achieved and the limit of detection was found to be 4 ng (more sensitive than silver staining of HS203 in polyacrylamide gel electrophoresis). The method described above was successfully applied to a pharmacokinetic study of HS203 and to monitor blood glucose level simultaneously in beagle dog. It is also possible to be applied for microanalysis of other oligosaccharides in biological samples. PMID:24750771

  20. Purification of Derivatized Oligosaccharides by Solid Phase Extraction for Glycomic Analysis

    PubMed Central

    Zhang, Qiwei; Li, Henghui; Feng, Xiaojun; Liu, Bi-Feng; Liu, Xin

    2014-01-01

    Profiling of glycans released from proteins is very complex and important. To enhance the detection sensitivity, chemical derivatization is required for the analysis of carbohydrates. Due to the interference of excess reagents, a simple and reliable purification method is usually necessary for the derivatized oligosaccharides. Various SPE based methods have been applied for the clean-up process. To demonstrate the differences among these methods, seven types of self-packed SPE cartridges were systematically compared in this study. The optimized conditions were determined for each type of cartridge and it was found that microcrystalline cellulose was the most appropriate SPE material for the purification of derivatized oligosaccharide. Normal phase HPLC analysis of the derivatized maltoheptaose was realized with a detection limit of 0.12 pmol (S N−1 = 3) and a recovery over 70%. With the optimized SPE method, relative quantification analysis of N-glycans from model glycoproteins were carried out accurately and over 40 N-glycans from human serum samples were determined regardless of the isomers. Due to the high stability and sensitivity, microcrystalline cellulose cartridge showed potential applications in glycomics analysis. PMID:24705408

  1. Sulfated Oligosaccharides Mediate the Interaction between a Marine Red Alga and Its Green Algal Pathogenic Endophyte.

    PubMed Central

    Bouarab, K; Potin, P; Correa, J; Kloareg, B

    1999-01-01

    The endophytic green alga Acrochaete operculata completely colonizes the sporophytes of the red alga Chondrus crispus; however, it does not penetrate beyond the outer cell layers of the gametophytes. Given that the life cycle phases of C. crispus differ in the sulfation pattern of their extracellular matrix carrageenans, we investigated whether carra-geenan fragments could modulate parasite virulence. lambda-Carrageenan oligosaccharides induced release of H(2)O(2), stimulated protein synthesis, increased carrageenolytic activity, and induced specific polypeptides in the pathogen, resulting in a marked increase in pathogenicity. In contrast, kappa-carrageenan oligosaccharides did not induce a marked release of H(2)O(2) from A. operculata but hindered amino acid uptake and enhanced their recognition by the host, resulting in a reduced virulence. Moreover, C. crispus life cycle phases were shown to behave differently in their response to challenge with cell-free extracts of A. operculata. Gametophytes exhibited a large burst of H(2)O(2), whereas only low levels were released from the sporophytes. PMID:10488232

  2. Multiple neutral alkali halide attachments onto oligosaccharides in electrospray ionization mass spectrometry

    NASA Astrophysics Data System (ADS)

    Striegel, André M.; Timpa, Judy D.; Piotrowiak, Piotr; Cole, Richard B.

    1997-03-01

    Oligosaccharides perform essential functions in a variety of biological and agricultural processes. Recent approaches to characterization of these molecules by mass spectrometry have utilized mainly soft-ionization methods such as electrospray ionization (ESI) and thermospray (TS), as well as fast atom bombardment (FAB). The behavior of a series of maltooligosaccharides with [alpha]-(1 --> 4) linkages, maltose (G2) through maltoheptaose (G7), under ESI conditions, has been investigated here. The oligosaccharides were dissolved in N,N-dimethylacetamide containing lithium chloride (DMAc/LiCl) prior to analysis by ESI-MS. A highly unusual feature, evident in all mass spectra obtained using this solvent system, was the presence of multiple [`]neutral' salt attachments onto lithium adducts of the sugars. Resultant ions took the form of [Gx + Li + nLiCl+, where n may reach a value as high as eight. Compared to LiCl, the propensity for alkali halide attachment using other alkali chlorides or lithium halides was greatly reduced. An investigation of this phenomenon is presented in which the organic and inorganic portions of the employed solvent were systematically varied, and semi-empirical computer modeling was performed to better understand lithium coordination by the sugars.

  3. A novel CFTR disease-associated mutation causes addition of an extra N-linked oligosaccharide.

    PubMed

    Hämmerle, M M; Aleksandrov, A A; Chang, X B; Riordan, J R

    2000-11-01

    We have examined the influence of a novel missense mutation in the fourth extracytoplasmic loop (EL4) of CFTR detected in a patient with cystic fibrosis. This substitution (T908N) creates a consensus sequence (N X S/T) for addition of an N-linked oligosaccharide chain near the C-terminal end of EL4. Oligosaccharyl transferase generally does not have access to this consensus sequence if it is closer than about twelve amino acids from the membrane. However, the T908N site is used, even though it is within four residues of the predicted membrane interface and the oligosaccharide chain added binds calnexin, a resident chaperone of the ER membrane. The chloride channel activity of this variant CFTR is abnormal as evidenced by a reduced rate of (36)Cl(-) efflux and a noisy single channel open state. This may reflect some displacement of the membrane spanning sequence C-terminal of EL4 since it contains residues influencing the ion pore. PMID:11443282

  4. Expeditious Chemoenzymatic Synthesis of Homogeneous N-Glycoproteins Carrying Defined Oligosaccharide Ligands

    PubMed Central

    Ochiai, Hirofumi; Huang, Wei; Wang, Lai-Xi

    2009-01-01

    An efficient chemoenzymatic method for the construction of homogeneous N-glycoproteins was described that explores the transglycosylation activity of the endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) with synthetic sugar oxazolines as the donor substrates. First, an array of large oligosaccharide oxazolines were synthesized and evaluated as substrates for the Endo-A catalyzed transglycosylation using ribonuclease B as a model system. The experimental results showed that Endo-A could tolerate modifications at the outer mannose residues of the Man3GlcNAc-oxazoline core, thus allowing introduction of large oligosaccharide ligands into a protein and meanwhile preserves the natural, core N-pentasaccharide (Man3GlcNAc2) structure in the resulting glycoprotein upon transglycosylation. In addition to ligands for galectins and mannose-binding lectins, azido functionality could be readily introduced at the N-pentasaccharide (Man3GlcNAc2) core using azido-containing Man3GlcNAc oxazoline as the donor substrate. The introduction of azido functionality permits further site-specific modifications of the resulting glycoproteins, as demonstrated by the successful attachment of two copies of αGal epitopes to ribonuclease B. This study reveals a broad substrate specificity of Endo-A for transglycosylation, and the chemoenzymatic method described here points to a new avenue for a quick access to various homogeneous N-glycoproteins for structure-activity relationship studies and for biomedical applications. PMID:18803385

  5. Screening Oligosaccharide Libraries against Lectins Using the Proxy Protein Electrospray Ionization Mass Spectrometry Assay.

    PubMed

    Han, Ling; Shams-Ud-Doha, Km; Kitova, Elena N; Klassen, John S

    2016-08-16

    An electrospray ionization mass spectrometry (ESI-MS) assay for screening carbohydrate libraries against lectins is described. The assay is based on the proxy protein ESI-MS method, which combines direct ESI-MS protein-ligand binding measurements and competitive protein binding, to simultaneously detect and quantify protein-carbohydrate interactions. Specific interactions between components of the library and the target protein (PT) are identified from changes in the relative abundances (as measured by ESI-MS) of the carbohydrate complexes of a proxy protein (Pproxy), which binds to all components of the library with known affinity, upon addition of PT to the solution. The magnitude of the change in relative abundance of a given Pproxy-ligand complex provides a quantitative measure of the affinity of the corresponding PT-ligand interaction. A mathematical framework for the implementation of the method in the case of monovalent (single binding site) Pproxy and monovalent and multivalent (multiple equivalent and independent binding sites) PT is described. The application of the method to screen small libraries of oligosaccharides, on the basis of human histo-blood group antigens and milk oligosaccharides, against an N-terminal fragment of the family 51 carbohydrate-binding module, a fucose-binding lectin from Ralstonia solanacearum, and human norovirus VA387 P particle (24-mer of the protruding domain of the capsid protein), serves to demonstrate the reliability and versatility of the assay. PMID:27366913

  6. Sialylated Milk Oligosaccharides Promote Microbiota-Dependent Growth in Models of Infant Undernutrition.

    PubMed

    Charbonneau, Mark R; O'Donnell, David; Blanton, Laura V; Totten, Sarah M; Davis, Jasmine C C; Barratt, Michael J; Cheng, Jiye; Guruge, Janaki; Talcott, Michael; Bain, James R; Muehlbauer, Michael J; Ilkayeva, Olga; Wu, Chao; Struckmeyer, Tedd; Barile, Daniela; Mangani, Charles; Jorgensen, Josh; Fan, Yue-mei; Maleta, Kenneth; Dewey, Kathryn G; Ashorn, Per; Newgard, Christopher B; Lebrilla, Carlito; Mills, David A; Gordon, Jeffrey I

    2016-02-25

    Identifying interventions that more effectively promote healthy growth of children with undernutrition is a pressing global health goal. Analysis of human milk oligosaccharides (HMOs) from 6-month-postpartum mothers in two Malawian birth cohorts revealed that sialylated HMOs are significantly less abundant in those with severely stunted infants. To explore this association, we colonized young germ-free mice with a consortium of bacterial strains cultured from the fecal microbiota of a 6-month-old stunted Malawian infant and fed recipient animals a prototypic Malawian diet with or without purified sialylated bovine milk oligosaccharides (S-BMO). S-BMO produced a microbiota-dependent augmentation of lean body mass gain, changed bone morphology, and altered liver, muscle, and brain metabolism in ways indicative of a greater ability to utilize nutrients for anabolism. These effects were also documented in gnotobiotic piglets using the same consortium and Malawian diet. These preclinical models indicate a causal, microbiota-dependent relationship between S-BMO and growth promotion. PMID:26898329

  7. Evaluation of a Group A Streptococcus synthetic oligosaccharide as vaccine candidate.

    PubMed

    Kabanova, Anna; Margarit, Immaculada; Berti, Francesco; Romano, Maria R; Grandi, Guido; Bensi, Giuliano; Chiarot, Emiliano; Proietti, Daniela; Swennen, Erwin; Cappelletti, Emilia; Fontani, Paola; Casini, Daniele; Adamo, Roberto; Pinto, Vittoria; Skibinski, David; Capo, Sabrina; Buffi, Giada; Gallotta, Marilena; Christ, William J; Campbell, A Stewart; Pena, John; Seeberger, Peter H; Rappuoli, Rino; Costantino, Paolo

    2010-12-10

    Bacterial infections caused by Group A Streptococcus (GAS) are a serious health care concern that currently cannot be prevented by vaccination. The GAS cell-wall polysaccharide (GAS-PS) is an attractive vaccine candidate due to its constant expression pattern on different bacterial strains and protective properties of anti-GAS-PS antibodies. Here we report for the first time the immunoprotective efficacy of glycoconjugates with synthetic GAS oligosaccharides as compared to those containing the native GAS-PS. A series of hexa- and dodecasaccharides based on the GAS-PS structure were prepared by chemical synthesis and conjugated to CRM(197). When tested in mice, the conjugates containing the synthetic oligosaccharides conferred levels of immunoprotection comparable to those elicited by the native conjugate. Antisera from immunized rabbits promoted phagocytosis of encapsulated GAS strains. Furthermore we discuss variables that might correlate with glycoconjugate immunogenicity and demonstrate the potential of the synthetic approach that benefits from increased antigen purity and facilitated manufacturing. PMID:20870056

  8. Different action patterns of glucoamylases on branched gluco-oligosaccharides from amylopectin.

    PubMed

    Jonathan, M C; van Brussel, M; Scheffers, M S; Kabel, M A

    2016-06-01

    A bottleneck in enzymatic starch hydrolysis, like in biofuel industry, is relatively slow degradation of branched structures compared to linear ones. This research aimed to evaluate glucoamylases for their activity towards branched gluco-oligosaccharides. The activity of seven modified glucoamylases and two homologs was compared to that of a reference glucoamylase obtained from a commercial enzyme cocktail 'Distillase® SSF'. All enzymes were evaluated for their activity towards panose (glc(α1-6)glc(α1-4)glc), pullulan and a purified branched gluco-oligosaccharide with a degree of polymerisation of 5 (bDP5) identified as glc(α1-4)[glc(α1-4)glc(α1-6)]glc(α1-4)glc. The enzymes degraded bDP5 differently, which was mainly due to variation in their capability to cleave α-(1→6)-linked or the α-(1→4)-linked glucosyl residue at the non-reducing end of the branched glucosyl residue. By comparing the enzyme activity towards bDP5 with those towards panose and pullulan, it was suggested that the activity towards bDP5 could be estimated only when the activity towards both commercial substrates was evaluated. PMID:27083360

  9. Mannose, glucosamine and inositol monophosphate inhibit the effects of insulin on lipogenesis. Further evidence for a role for inositol phosphate-oligosaccharides in insulin action.

    PubMed Central

    Machicao, F; Mushack, J; Seffer, E; Ermel, B; Häring, H U

    1990-01-01

    The mechanism of insulin signalling is not yet understood in detail. Recently, a role for inositol phosphate (IP)-oligosaccharides as second messengers transmitting the insulin signal at the post-kinase level was proposed. To evaluate this hypothesis further, we studied whether IP-oligosaccharides isolated from 'haemodialysate' have insulin-like activity. We found that these compounds mimic, in a dose-dependent fashion, the following effects of insulin in adipocytes. (1) Lipogenesis. Incorporation of [3H]glucose into lipids (expressed in nmol/min per 10(6) cells): basal, 0.74 +/- 0.05; insulin (1 mu unit/ml), 4.43 +/- 0.21; IP-oligosaccharide (2 micrograms/ml), 4.07 +/- 0.19. (2) Inhibition of isoprenaline (isoproterenol) (1 microM)-stimulated cyclic AMP levels and lipolysis. Cyclic AMP (pmol/10(5) cells): basal 0.84 +/- 0.05; isoprenaline, 4.03 +/- 0.19; isoprenaline + insulin (200 mu units/ml), 2.06 +/- 0.7; isoprenaline + IP-oligosaccharides (2 micrograms/ml), 2.4 +/- 0.29. Inhibition of lipolysis (mumol of glycerol/mg of protein): isoprenaline (1 microM), 166 +/- 11; isoprenaline+insulin (150 mu units/ml), 53 +/- 3.5; isoprenaline+IP-oligosaccharides (2 micrograms/ml), 58 +/- 5. (3) Stimulation of 3-O-methylglucose transport; basal, 9 +/- 3%; insulin (1 mu unit/ml), 67 +/- 4%, IP-oligosaccharides (2 micrograms/ml), 54 +/- 2%. To identify the active molecules of the IP-oligosaccharide fraction, competition experiments were performed. IP-oligosaccharide effects on lipogenesis were blocked by inositol monophosphate, glucosamine and mannose. In contrast, these compounds did not inhibit IP-oligosaccharide effects on membrane-mediated functions (3-O-methylglucose transport, cyclic AMP levels, lipolysis). We also found that the effect of insulin on lipogenesis was blocked by mannose, glucosamine and inositol monophosphate, whereas the insulin effects on 3-O-methylglucose, cyclic AMP and lipolysis were unaffected. The following conclusions were reached. (1) IP-oligosaccharides

  10. Improved synthesis of lysine- and arginine-derived Amadori and Heyns products and in vitro measurement of their angiotensin I-converting enzyme inhibitory activity.

    PubMed

    Srinivas, Sudhanva M; Harohally, Nanishankar V

    2012-02-15

    The L-lysine- and L-arginine-derived Amadori and Heyns products consisting of N-(1-deoxy-d-fructos-1-yl)amino acid and N-(2-deoxy-d-glucos-2-yl)amino acid were prepared by reaction of d-fructose and d-glucose with l-lysine hydrochloride and l-arginine hydrochloride using commercial zinc powder as deprotonating reagent and also as catalyst precursor in a simple synthetic route in high yield. These compounds were screened for angiotensin I-converting enzyme (ACE) inhibitory activity using a high-throughput colorimetric assay (utilizing porcine kidney ACE). The IC(50) values fall in the range of 1030-1175 μM, with N(α)-(1-deoxy-d-fructos-1-yl)arginine showing the best IC(50) value (1030 ± 38 μM). This study demonstrates an improved synthetic method for simple Amadori and Heyns products and their moderate ACE inhibitor activity. PMID:22242891

  11. Effect of Leuconostoc mesenteroides NRRL B-512F Dextransucrase Carboxy-Terminal Deletions on Dextran and Oligosaccharide Synthesis

    PubMed Central

    Monchois, Vincent; Reverte, Augustin; Remaud-Simeon, Magali; Monsan, Pierre; Willemot, René-Marc

    1998-01-01

    Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose. In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis. Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats. In order to determine the role of the C-terminal region of DSR-S in dextran or oligosaccharide synthesis, four DSR-S genes with deletions at the 3′ end were constructed. The results showed that the C-terminal region modulated the initial velocity of dextran synthesis but that the Km for sucrose, the optimum pH, and the activation energy were all unaffected by the deletions. The C-terminal domain modulated the rate of oligosaccharide synthesis whatever acceptor molecule was used (a good acceptor molecule such as maltose or a poor acceptor molecule such as fructose). The C-terminal domain seemed to play no role in the catalytic process in dextran and oligosaccharide synthesis. In fact, it seems that the role of the C-terminal domain of DSR-S may be to facilitate the translation of dextran and oligosaccharides from the catalytic site. PMID:9572930

  12. Structural characterization of neutral oligosaccharides with blood-group A and H activity isolated from bovine submaxillary mucin.

    PubMed Central

    Savage, A V; D'Arcy, S M; Donoghue, C M

    1991-01-01

    In this study we investigated the structures of 11 neutral oligosaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by h.p.l.c. One hexa-, one penta-, three tetra-, four tri- and two di-saccharides containing core types 1, 2, 3 or 4 were obtained. We report their structures, determined by a combination of one- and two-dimensional 1H n.m.r. spectroscopy at 270 MHz and methylation analysis involving g.l.c.-m.s., along with their approximate molar ratios. Only three of these oligosaccharides have previously been reported in this source. Of the new oligosaccharides, one contains the blood-group-A antigenic determinant, two contain the blood-group-H type 2 determinant, while another contains the blood-group-H type 3 determinant. The oligosaccharide GlcNAc beta (1----6)[GlcNAc beta (1----3)]GalNAcol, although previously found as a core structure, has been isolated here as a novel trisaccharide. PMID:1718265

  13. Effect on Intestinal Growth of the Population of Cellobiose-Oligosaccharides Obtained by Enzymatic Reaction with Dextransucrase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although the synthesis of oligosaccharides obtained by reactions catalyzed by dextransucrase using sucrose as donor and different carbohydrates as acceptors has been widely studied, the effect of many of these carbohydrates in the growth of intestinal flora has not yet been evaluated. Such is the c...

  14. The effects of galactoglucomannan oligosaccharide-arabinoxylan (GGMO-AX) complex on Eimeria acervulina infection in broiler chicks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fermentable carbohydrates may enhance the ability of the gastrointestinal tract to defend against pathogenic infection. We hypothesized that a mannose-rich, galactoglucomannan oligosaccharide-arabinoxylan (GGMO-AX) complex would positively impact immune status and prevent weight loss resulting from...

  15. Inhibition by pectic oligosaccharides of the invasion of undifferentiated and differentiated Caco-2 cells by Campylobacter jejuni

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of pectic oligosaccharides (POS) to inhibit adherence to and invasion of undifferentiated (UC) and differentiated (DC) Caco-2 cells by Campylobacter jejuni (C. jejuni) was investigated. It was observed that both adherence and invasion were significantly higher in UC than in DC. POS (2.5 ...

  16. Interaction of Dictyostelium discoideum lysosomal enzymes with the mammalian phosphomannosyl receptor. The importance of oligosaccharides which contain phosphodiesters

    SciTech Connect

    Freeze, H.H.

    1985-07-25

    Mammalian cell lysosomal enzymes or phosphorylated oligosaccharides derived from them are endocytosed by a phosphomannosyl receptor (PMR) found on the surface of fibroblasts. Various studies suggest that 2 residues of Man-6-P in phosphomonoester linkage but not diester linkage (PDE) are essential for a high rate of uptake. The lysosomal enzymes of the slime mold Dictyostelium discoideum are also recognized by the PMR on these cells; however, none of the oligosaccharides from these enzymes contain 2 phosphomonoesters. Instead, most contain multiple sulfate esters and 2 residues of Man-6-P in an unusual PDE linkage. In this study the authors have tried to account for the unexpected highly efficient uptake of the slime mold enzymes. The results show that nearly all of the alpha-mannosidase molecules contain the oligosaccharides required for uptake. Competition of SVI-beta-glucosidase uptake by various carbohydrate-containing fractions indicates that the best inhibitors are those with 2 PDE, either with or without sulfate esters. Complete denaturation of SVI-labeled wild-type beta-glucosidase in sodium dodecyl sulfate/dithiothreitol also reduces its uptake by about 10-fold. Taken together, these results suggest that the interactions of multiple, weakly binding oligosaccharides, especially those with 2 PDE, are important for the high rate of uptake of the slime mold enzymes.

  17. INFLUENCE OF GLYCOSIDIC LINKAGES AND MOLECULAR WEIGHT ON THE FERMENTATION OF MALTOSE-BASED OLIGOSACCHARIDES BY GUT BACTERIA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A structure-function study was carried out to increase knowledge of how glycosidic linkages and molecular weight of carbohydrates contribute towards the selectivity of fermentation by gut bacteria. Oligosaccharides with maltose as the common carbohydrate source were used. Potentially prebiotic alt...

  18. The activation of fibroblast growth factors by heparin: synthesis, structure, and biological activity of heparin-like oligosaccharides.

    PubMed

    de Paz, J L; Angulo, J; Lassaletta, J M; Nieto, P M; Redondo-Horcajo, M; Lozano, R M; Giménez-Gallego, G; Martín-Lomas, M

    2001-09-01

    An effective strategy has been designed for the synthesis of oligosaccharides of different sizes structurally related to the regular region of heparin; this is illustrated by the preparation of hexasaccharide 1 and octasaccharide 2. This synthetic strategy provides the oligosaccharide sequence containing a D-glucosamine unit at the nonreducing end that is not available either by enzymatic or chemical degradation of heparin. It may permit, after slight modifications, the preparation of oligosaccharide fragments with different charge distribution as well. NMR spectroscopy and molecular dynamics simulations have shown that the overall structure of 1 in solution is a stable right-hand helix with four residues per turn. Hexasaccharide 1 and, most likely, octasaccharide 2 are, therefore, chemically well-defined structural models of naturally occurring heparin-like oligosaccharides for use in binding and biological activity studies. Both compounds 1 and 2 induce the mitogenic activity of acid fibroblast growth factor (FGF1), with the half-maximum activating concentration of 2 being equivalent to that of heparin. Sedimentation equilibrium analysis with compound 2 suggests that heparin-induced FGF1 dimerization is not an absolute requirement for biological activity. PMID:11828504

  19. Effects of Yeast Oligosaccharide Diet Supplements on Growth and Disease Resistance in Juvenile Nile Tilapia, Oreochromis niloticus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercially available yeast, and yeast subcomponents consisting mainly of beta-glucan or oligosaccharide feed additives, were added to diets of juvenile (12-18g) Nile tilapia (Oreochromis niloticus) at rates recommended by suppliers. Three experiments followed a basic protocol with varied rates of...

  20. Self-Assembly of Oligosaccharide-b-PMMA Block Copolymer Systems: Glyco-Nanoparticles and Their Degradation under UV Exposure.

    PubMed

    Zepon, Karine M; Otsuka, Issei; Bouilhac, Cécile; Muniz, Edvani C; Soldi, Valdir; Borsali, Redouane

    2016-05-10

    This paper discusses the self-assembly of oligosaccharide-containing block copolymer and the use of ultraviolet (UV) to obtain nanoporous glyco-nanoparticles by photodegradation of the synthetic polymer block. Those glyco-nanoparticles consisting of oligosaccharide-based shell and a photodegradable core domain were obtained from the self-assembly of maltoheptaose-block-poly(methyl methacrylate) (MH-b-PMMA48) using the nanoprecipitation protocol. MH-b-PMMA48 self-assembled into well-defined spherical micelles (major compound) with a hydrodynamic radius (Rh) of ca. 10 nm and also into large compound micellar aggregates (minor compound) with an Rh of ca. 65 nm. The oligosaccharide shells of these glyco-nanoparticles were cross-linked through the Michael-type addition of divinyl sulfone under dilute conditions to minimize the intermicellar cross-linking. The core domain photodegradation of the cross-linked glyco-nanoparticles was induced under exposure to 254 nm UV radiation, resulting in porous glyco-nanoparticles with an Rh of ca. 44 nm. The morphology of the cross-linked shell and the core photodegradation of these glyco-nanoparticles were characterized using static light scattering, dynamic light scattering, Fourier transform infrared spectroscopy, proton nuclear magnetic resonance, field-emission gun-scanning electron microscopy, and transmission electron microscopy. The innovative aspect of this approach concerns the fact that after removing the PMMA domains the porous nanoparticles are mostly composed of biocompatible and nontoxic oligosaccharides. PMID:27054350

  1. Ion exchange chromatographic separation and isolation of oligosaccharides of intact low-molecular-weight heparin for the determination of their anticoagulant and anti-inflammatory properties.

    PubMed

    Shastri, Madhur D; Johns, Cameron; Hutchinson, Joseph P; Khandagale, Manish; Patel, Rahul P

    2013-07-01

    It is well known that enoxaparin, a widely used anticoagulant and low-molecular-weight heparin containing a large number of oligosaccharides, possesses anti-inflammatory activity. Whilst enoxaparin has shown promising results in various inflammatory disorders, some of its oligosaccharides have anti-inflammatory properties and others increase the risk of bleeding due to their anticoagulant effects. The aim of this study was to develop an effective ion exchange chromatographic (IC) technique which allows the separation, isolation and, consequently, the identification of different oligosaccharides of enoxaparin with or without anticoagulant activity. The developed method utilises a semi-preparative CarboPac PA100 (9 × 250 mm) ion exchange column with sodium chloride gradient elution and UV detection at 232 nm. The method successfully resolved enoxaparin into more than 30 different peaks. IC-derived oligosaccharides with high, moderate, low or no anticoagulant activity were identified using an anti-factor Xa assay. The anti-inflammatory activity of selected oligosaccharides was investigated using the Griess assay. Using this technique, the oligosaccharides of enoxaparin with low or no anticoagulant activity, whilst exhibiting significant anti-inflammatory activity, could be fractionated. This technique can provide a platform to identify the oligosaccharides which are devoid of significant anticoagulant activity and are responsible for the therapeutic effects of enoxaparin that have been observed in various inflammatory conditions. PMID:23712644

  2. Exposure of Bifidobacterium longum subsp. infantis to Milk Oligosaccharides Increases Adhesion to Epithelial Cells and Induces a Substantial Transcriptional Response

    PubMed Central

    Kavanaugh, Devon W.; O’Callaghan, John; Buttó, Ludovica F.; Slattery, Helen; Lane, Jonathan; Clyne, Marguerite; Kane, Marian; Joshi, Lokesh; Hickey, Rita M.

    2013-01-01

    In this study, we tested the hypothesis that milk oligosaccharides may contribute not only to selective growth of bifidobacteria, but also to their specific adhesive ability. Human milk oligosaccharides (3′sialyllactose and 6′sialyllactose) and a commercial prebiotic (Beneo Orafti P95; oligofructose) were assayed for their ability to promote adhesion of Bifidobacterium longum subsp. infantis ATCC 15697 to HT-29 and Caco-2 human intestinal cells. Treatment with the commercial prebiotic or 3′sialyllactose did not enhance adhesion. However, treatment with 6′sialyllactose resulted in increased adhesion (4.7 fold), while treatment with a mixture of 3′- and 6′-sialyllactose substantially increased adhesion (9.8 fold) to HT-29 intestinal cells. Microarray analyses were subsequently employed to investigate the transcriptional response of B. longum subsp. infantis to the different oligosaccharide treatments. This data correlated strongly with the observed changes in adhesion to HT-29 cells. The combination of 3′- and 6′-sialyllactose resulted in the greatest response at the genetic level (both in diversity and magnitude) followed by 6′sialyllactose, and 3′sialyllactose alone. The microarray data was further validated by means of real-time PCR. The current findings suggest that the increased adherence phenotype of Bifidobacterium longum subsp. infantis resulting from exposure to milk oligosaccharides is multi-faceted, involving transcription factors, chaperone proteins, adhesion-related proteins, and a glycoside hydrolase. This study gives additional insight into the role of milk oligosaccharides within the human intestine and the molecular mechanisms underpinning host-microbe interactions. PMID:23805302

  3. Comparison of biological activities of human antithrombins with high-mannose or complex-type nonfucosylated N-linked oligosaccharides.

    PubMed

    Yamada, Tsuyoshi; Kanda, Yutaka; Takayama, Makoto; Hashimoto, Akitoshi; Sugihara, Tsutomu; Satoh-Kubota, Ai; Suzuki-Takanami, Eri; Yano, Keiichi; Iida, Shigeru; Satoh, Mitsuo

    2016-05-01

    The structure of the N-linked oligosaccharides attached to antithrombin (AT) has been shown to affect its anticoagulant activity and pharmacokinetics. Human AT has biantennary complex-type oligosaccharides with the unique feature of lacking a core fucose, which affects its biological activities by changing its heparin-binding affinity. In human plasma, AT circulates as a mixture of the α-form bearing four oligosaccharides and the β-form lacking an oligosaccharide at Asn135. However, it remains unclear how the immature high-mannose-type oligosaccharides produced by mammalian cells affect biological activities of AT. Here, we succeeded in directly comparing the activities between the high-mannose and complex types. Interestingly, although there were no substantial differences in thrombin inhibitory activity, the high-mannose type showed higher heparin-binding affinity. The anticoagulant activities were increased by heparin and correlated with the heparin-binding affinity, resulting in the strongest anticoagulant activity being displayed in the β-form with the high-mannose type. In pharmacokinetic profiling, the high-mannose type showed a much shorter plasma half-life than the complex type. The β-form was found to have a prolonged plasma half-life compared with the α-form for the high-mannose type; conversely, the α-form showed a longer half-life than the β-form for the complex-type. The present study highlights that AT physiological activities are strictly controlled not only by a core fucose at the reducing end but also by the high-mannose-type structures at the nonreducing end. The β-form with the immature high-mannose type appears to function as a more potent anticoagulant than the AT typically found in human plasma, once it emerges in the blood. PMID:26747427

  4. Suppression of amyloid beta A11 antibody immunoreactivity by vitamin C: possible role of heparan sulfate oligosaccharides derived from glypican-1 by ascorbate-induced, nitric oxide (NO)-catalyzed degradation.

    PubMed

    Cheng, Fang; Cappai, Roberto; Ciccotosto, Giuseppe D; Svensson, Gabriel; Multhaup, Gerd; Fransson, Lars-Åke; Mani, Katrin

    2011-08-01

    Amyloid β (Aβ) is generated from the copper- and heparan sulfate (HS)-binding amyloid precursor protein (APP) by proteolytic processing. APP supports S-nitrosylation of the HS proteoglycan glypican-1 (Gpc-1). In the presence of ascorbate, there is NO-catalyzed release of anhydromannose (anMan)-containing oligosaccharides from Gpc-1-nitrosothiol. We investigated whether these oligosaccharides interact with Aβ during APP processing and plaque formation. anMan immunoreactivity was detected in amyloid plaques of Alzheimer (AD) and APP transgenic (Tg2576) mouse brains by immunofluorescence microscopy. APP/APP degradation products detected by antibodies to the C terminus of APP, but not Aβ oligomers detected by the anti-Aβ A11 antibody, colocalized with anMan immunoreactivity in Tg2576 fibroblasts. A 50-55-kDa anionic, sodium dodecyl sulfate-stable, anMan- and Aβ-immunoreactive species was obtained from Tg2576 fibroblasts using immunoprecipitation with anti-APP (C terminus). anMan-containing HS oligo- and disaccharide preparations modulated or suppressed A11 immunoreactivity and oligomerization of Aβ42 peptide in an in vitro assay. A11 immunoreactivity increased in Tg2576 fibroblasts when Gpc-1 autoprocessing was inhibited by 3-β[2(diethylamino)ethoxy]androst-5-en-17-one (U18666A) and decreased when Gpc-1 autoprocessing was stimulated by ascorbate. Neither overexpression of Gpc-1 in Tg2576 fibroblasts nor addition of copper ion and NO donor to hippocampal slices from 3xTg-AD mice affected A11 immunoreactivity levels. However, A11 immunoreactivity was greatly suppressed by the subsequent addition of ascorbate. We speculate that temporary interaction between the Aβ domain and small, anMan-containing oligosaccharides may preclude formation of toxic Aβ oligomers. A portion of the oligosaccharides are co-secreted with the Aβ peptides and deposited in plaques. These results support the notion that an inadequate supply of vitamin C could contribute to late onset AD

  5. Transglycosylation properties of maltodextrin glucosidase (MalZ) from Escherichia coli and its application for synthesis of a nigerose-containing oligosaccharide

    SciTech Connect

    Song, Kyung-Mo; Shim, Jae-Hoon; Park, Jong-Tae; Kim, Sung-Hee; Kim, Young-Wan; Boos, Winfried; Park, Kwan-Hwa

    2010-06-18

    The transglycosylation reaction of maltodextrin glucosidase (MalZ) cloned and purified from Escherichia coli K12 was characterized and applied to the synthesis of branched oligosaccharides. Purified MalZ preferentially catalyzed the hydrolysis of maltodextrin, {gamma}-cyclodextrin (CD), and cycloamylose (CA). In addition, when the enzyme was incubated with 5% maltotriose (G3), a series of transfer products were produced. The resulting major transfer products, annotated as T1, T2, and T3, were purified and their structures were determined by TLC, MALDI-TOF/MS, {sup 13}C NMR, and enzymatic analysis. T1 was identified as a novel compound, maltosyl {alpha}-1,3-maltose, whereas T2 and T3 were determined to be isopanose and maltosyl-{alpha}-1,6-maltose, respectively. These results indicated that MalZ transferred sugar moiety mainly to C-3 or C-6-OH of glucose of the acceptor molecule. To obtain highly concentrated transfer products, the enzyme was reacted with 10% liquefied cornstarch, and then glucose and maltose were removed by immobilized yeast. The T1 content of the resulting reaction mixture reached 9.0%. The mixture of T1 containing a nigerose moiety can have an immunopotentiating effect on the human body and may be a potential functional sugar stuff.

  6. Enzymatic Decoration of Prebiotic Galacto-oligosaccharides (Vivinal GOS) with Sialic Acid Using Trypanosoma cruzi trans-Sialidase and Two Bovine Sialoglycoconjugates as Donor Substrates.

    PubMed

    Wilbrink, Maarten H; ten Kate, Geert A; Sanders, Peter; Gerwig, Gerrit J; van Leeuwen, Sander S; Sallomons, Erik; Klarenbeek, Bert; Hage, Johannes A; van Vuure, Carine A; Dijkhuizen, Lubbert; Kamerling, Johannis P

    2015-07-01

    Decoration of prebiotic galacto-oligosaccharides (GOS) with sialic acid yields mixtures of GOS and sialylated GOS (Sia-GOS), novel products that are expected to have both prebiotic and antiadhesive functionalities. The recombinantly produced trans-sialidase enzyme from Trypanosoma cruzi (TcTS), an enzyme with the ability to transfer (α2-3)-linked sialic acid from sialogalactoglycans to asialogalactoglycans, was employed to catalyze this sialylation. As sialic acid acceptor substrates, Vivinal GOS and derived fractions of specific degree of polymerization were taken. As sialic acid donor substrates, bovine κ-casein-derived glycomacropeptide [>99% N-acetylneuraminic acid (Neu5Ac); <1% N-glycolylneuraminic acid (Neu5Gc)] and bovine blood plasma glycoprotein mixture (45% Neu5Ac; 55% Neu5Gc) were selected, yielding potential food and feed products, respectively. High-pH anion-exchange chromatography, matrix-assisted laser-desorption ionization time-of-flight mass spectrometry, and nuclear magnetic resonance spectroscopy were used for product analysis. PMID:26044147

  7. Hyaluronan Oligosaccharides Induce MMP-1 and -3 via Transcriptional Activation of NF-κB and p38 MAPK in Rheumatoid Synovial Fibroblasts

    PubMed Central

    Hanabayashi, Masahiro; Takahashi, Nobunori; Sobue, Yasumori; Hirabara, Shinya; Ishiguro, Naoki; Kojima, Toshihisa

    2016-01-01

    Objective To explore the effect of hyaluronan oligosaccharides (HAoligos) on interactions between HA and its principal receptor, CD44, in rheumatoid synovial fibroblasts (RSFs) and matrix metalloproteinase (MMP) production. Methods RSFs were isolated from rheumatoid synovial tissue. HA distribution was visualized by immunocytochemistry. MMP-1 and MMP-3 induction was analyzed by real-time RT-PCR and immunoblotting. The interaction between HAoligos and their MMP-producing receptors was tested by blocking with anti-CD44 and anti-Toll-like receptor 4 (TLR-4). Phosphorylation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) was analyzed by immunoblotting. Results Endogenous HA decreased after treatment with HAoligos, while MMP-1 and MMP-3 expression increased in a dose-dependent manner. Pretreatment with anti-CD44 or anti-TLR-4 antibody significantly reduced the effect of HAoligos on MMP-1 and MMP-3 mRNA expression. NF-κB and p38 MAPK phosphorylation was enhanced by HAoligos pretreated with anti-TLR-4, and HAoligo-induced MMP production was blocked with an inhibitor of NF-κB and p38 MAPK pathways. Conclusions