Analysis and Prediction of Myristoylation Sites Using the mRMR Method, the IFS Method and an Extreme Learning Machine Algorithm.

PubMed

Wang, ShaoPeng; Zhang, Yu-Hang; Huang, GuoHua; Chen, Lei; Cai, Yu-Dong

2017-01-01

Myristoylation is an important hydrophobic post-translational modification that is covalently bound to the amino group of Gly residues on the N-terminus of proteins. The many diverse functions of myristoylation on proteins, such as membrane targeting, signal pathway regulation and apoptosis, are largely due to the lipid modification, whereas abnormal or irregular myristoylation on proteins can lead to several pathological changes in the cell. To better understand the function of myristoylated sites and to correctly identify them in protein sequences, this study conducted a novel computational investigation on identifying myristoylation sites in protein sequences. A training dataset with 196 positive and 84 negative peptide segments were obtained. Four types of features derived from the peptide segments following the myristoylation sites were used to specify myristoylatedand non-myristoylated sites. Then, feature selection methods including maximum relevance and minimum redundancy (mRMR), incremental feature selection (IFS), and a machine learning algorithm (extreme learning machine method) were adopted to extract optimal features for the algorithm to identify myristoylation sites in protein sequences, thereby building an optimal prediction model. As a result, 41 key features were extracted and used to build an optimal prediction model. The effectiveness of the optimal prediction model was further validated by its performance on a test dataset. Furthermore, detailed analyses were also performed on the extracted 41 features to gain insight into the mechanism of myristoylation modification. This study provided a new computational method for identifying myristoylation sites in protein sequences. We believe that it can be a useful tool to predict myristoylation sites from protein sequences. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Optimization of Protein Extraction and Two-Dimensional Electrophoresis Protocols for Oil Palm Leaf.

PubMed

Daim, Leona Daniela Jeffery; Ooi, Tony Eng Keong; Yusof, Hirzun Mohd; Majid, Nazia Abdul; Karsani, Saiful Anuar Bin

2015-08-01

Oil palm (Elaeis guineensis) is an important economic crop cultivated for its nutritional palm oil. A significant amount of effort has been undertaken to understand oil palm growth and physiology at the molecular level, particularly in genomics and transcriptomics. Recently, proteomics studies have begun to garner interest. However, this effort is impeded by technical challenges. Plant sample preparation for proteomics analysis is plagued with technical challenges due to the presence of polysaccharides, secondary metabolites and other interfering compounds. Although protein extraction methods for plant tissues exist, none work universally on all sample types. Therefore, this study aims to compare and optimize different protein extraction protocols for use with two-dimensional gel electrophoresis of young and mature leaves from the oil palm. Four protein extraction methods were evaluated: phenol-guanidine isothiocyanate, trichloroacetic acid-acetone precipitation, sucrose and trichloroacetic acid-acetone-phenol. Of these four protocols, the trichloroacetic acid-acetone-phenol method was found to give the highest resolution and most reproducible gel. The results from this study can be used in sample preparations of oil palm tissue for proteomics work.

Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry

PubMed Central

El-Rami, Fadi; Nelson, Kristina; Xu, Ping

2017-01-01

Streptococcus sanguinis is a commensal and early colonizer of oral cavity as well as an opportunistic pathogen of infectious endocarditis. Extracting the soluble proteome of this bacterium provides deep insights about the physiological dynamic changes under different growth and stress conditions, thus defining “proteomic signatures” as targets for therapeutic intervention. In this protocol, we describe an experimentally verified approach to extract maximal cytoplasmic proteins from Streptococcus sanguinis SK36 strain. A combination of procedures was adopted that broke the thick cell wall barrier and minimized denaturation of the intracellular proteome, using optimized buffers and a sonication step. Extracted proteome was quantitated using Pierce BCA Protein Quantitation assay and protein bands were macroscopically assessed by Coomassie Blue staining. Finally, a high resolution detection of the extracted proteins was conducted through Synapt G2Si mass spectrometer, followed by label-free relative quantification via Progenesis QI. In conclusion, this pipeline for proteomic extraction and analysis of soluble proteins provides a fundamental tool in deciphering the biological complexity of Streptococcus sanguinis. PMID:29152022

Utilization of non-conventional systems for conversion of biomass to food components: Recovery optimization and characterizations of algal proteins and lipids

NASA Technical Reports Server (NTRS)

Karel, M.; Nakhost, Z.

1986-01-01

Protein isolate obtained from green algae (Scenedesmus obliquus) cultivated under controlled conditions was characterized. Molecular weight determination of fractionated algal proteins using SDS-polyacrylamide gel electrophoresis revealed a wide spectrum of molecular weights ranging from 15,000 to 220,000. Isoelectric points of dissociated proteins were in the range of 3.95 to 6.20. Amino acid composition of protein isolate compared favorably with FAO standards. High content of essential amino acids leucine, valine, phenylalanine and lysine makes algal protein isolate a high quality component of closed environment life support system (CELSS) diets. To optimize the removal of algal lipids and pigments supercritical carbon dioxide extraction (with and without ethanol as a co-solvent) was used. Addition of ethanol to supercritical CO2 resulted in more efficient removal of algal lipids and produced protein isolate with a good yield and protein recovery. The protein isolate extracted by the above mixture had an improved water solubility.

Application of Box-Behnken experimental design to optimize the extraction of insecticidal Cry1Ac from soil.

PubMed

Li, Yan-Liang; Fang, Zhi-Xiang; You, Jing

2013-02-20

A validated method for analyzing Cry proteins is a premise to study the fate and ecological effects of contaminants associated with genetically engineered Bacillus thuringiensis crops. The current study has optimized the extraction method to analyze Cry1Ac protein in soil using a response surface methodology with a three-level-three-factor Box-Behnken experimental design (BBD). The optimum extraction conditions were at 21 °C and 630 rpm for 2 h. Regression analysis showed a good fit of the experimental data to the second-order polynomial model with a coefficient of determination of 0.96. The method was sensitive and precise with a method detection limit of 0.8 ng/g dry weight and relative standard deviations at 7.3%. Finally, the established method was applied for analyzing Cry1Ac protein residues in field-collected soil samples. Trace amounts of Cry1Ac protein were detected in the soils where transgenic crops have been planted for 8 and 12 years.

Protein extraction and gel-based separation methods to analyze responses to pathogens in carnation (Dianthus caryophyllus L).

PubMed

Ardila, Harold Duban; Fernández, Raquel González; Higuera, Blanca Ligia; Redondo, Inmaculada; Martínez, Sixta Tulia

2014-01-01

We are currently using a 2-DE-based proteomics approach to study plant responses to pathogenic fungi by using the carnation (Dianthus caryophyllus L)-Fusarium oxysporum f. sp. dianthi pathosystem. It is clear that the protocols for the first stages of a standard proteomics workflow must be optimized to each biological system and objectives of the research. The optimization procedure for the extraction and separation of proteins by 1-DE and 2-DE in the indicated system is reported. This strategy can be extrapolated to other plant-pathogen interaction systems in order to perform an evaluation of the changes in the host protein profile caused by the pathogen and to identify proteins which, at early stages, are involved or implicated in the plant defense response.

Study on the extraction and purification of glycoprotein from the yellow seahorse, Hippocampus kuda Bleeker

PubMed Central

Su, Yuting; Xu, Yongjian

2015-01-01

The optimum parameters of extraction for glycoprotein from seahorse were examined and determined by Box-Behnken combined with ultrasonic extraction technology. Column chromatography of glycoprotein was used for further purification. The optimal extraction conditions of seahorse glycoprotein were extracting time 4.3 h, salt concentration 0.08 mol/L, extracting temperature 73°C, raw material, and water ratio 1:6. At the optimal conditions, the yield of saccharide reached to 1.123%, and the yield of protein reached to 5.898%. For purifying the crude glycoprotein, the stage renounces of DEAE-52 column chromatography were done, respectively, with 0.05, 0.1, 0.5 mol/L NaHCO3 solution, and further purification was done with Sephadex G-100 column chromatography. Finally, two pieces of seahorse glycoprotein were obtained by the column chromatography, that is, HG-11 and HG-21. The saccharide content was 56.7975% and 39.479%, the protein content was 30.5475% and 51.747%, respectively. PMID:26288722

A Novel Feature Extraction Method with Feature Selection to Identify Golgi-Resident Protein Types from Imbalanced Data

PubMed Central

Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

2016-01-01

The Golgi Apparatus (GA) is a major collection and dispatch station for numerous proteins destined for secretion, plasma membranes and lysosomes. The dysfunction of GA proteins can result in neurodegenerative diseases. Therefore, accurate identification of protein subGolgi localizations may assist in drug development and understanding the mechanisms of the GA involved in various cellular processes. In this paper, a new computational method is proposed for identifying cis-Golgi proteins from trans-Golgi proteins. Based on the concept of Common Spatial Patterns (CSP), a novel feature extraction technique is developed to extract evolutionary information from protein sequences. To deal with the imbalanced benchmark dataset, the Synthetic Minority Over-sampling Technique (SMOTE) is adopted. A feature selection method called Random Forest-Recursive Feature Elimination (RF-RFE) is employed to search the optimal features from the CSP based features and g-gap dipeptide composition. Based on the optimal features, a Random Forest (RF) module is used to distinguish cis-Golgi proteins from trans-Golgi proteins. Through the jackknife cross-validation, the proposed method achieves a promising performance with a sensitivity of 0.889, a specificity of 0.880, an accuracy of 0.885, and a Matthew’s Correlation Coefficient (MCC) of 0.765, which remarkably outperforms previous methods. Moreover, when tested on a common independent dataset, our method also achieves a significantly improved performance. These results highlight the promising performance of the proposed method to identify Golgi-resident protein types. Furthermore, the CSP based feature extraction method may provide guidelines for protein function predictions. PMID:26861308

Evaluation of different methods of protein extraction and identification of differentially expressed proteins upon ethylene-induced early-ripening in banana peels.

PubMed

Zhang, Li-Li; Feng, Ren-Jun; Zhang, Yin-Dong

2012-08-15

Banana peels (Musa spp.) are a good example of a plant tissue where protein extraction is challenging due to the abundance of interfering metabolites. Sample preparation is a critical step in proteomic research and is critical for good results. We sought to evaluate three methods of protein extraction: trichloroacetic acid (TCA)-acetone precipitation, phenol extraction, and TCA precipitation. We found that a modified phenol extraction protocol was the most optimal method. SDS-PAGE and two-dimensional gel electrophoresis (2-DE) demonstrated good protein separation and distinct spots of high quality protein. Approximately 300 and 550 protein spots were detected on 2-DE gels at pH values of 3-10 and 4-7, respectively. Several spots were excised from the 2-DE gels and identified by mass spectrometry. The protein spots identified were found to be involved in glycolysis, the tricarboxylic acid cycle, and the biosynthesis of ethylene. Several of the identified proteins may play important roles in banana ripening. Copyright © 2012 Society of Chemical Industry.

Extraction optimization and characterization of gelatine from fish dry skin of Spanish mackerel (Scomberromorus commersoni)

NASA Astrophysics Data System (ADS)

Kusumaningrum, I.; Pranoto, Y.; Hadiwiyoto, S.

2018-04-01

This work was to optimized gelatin extraction from dry skin of Spanish mackerel (Scomberromorus commersoni) using Response Surface Methodology (RSM). The aim of this study was to determine the optimal condition of temperature and time for extraction process and properties of the gelatin extracted from dry mackerel skin. The optimal condition for extraction was 59.71°C for 4.25 hours. Results showed that predicted yield by RSM was 13.69% and predicted gel strength was 291.93 Bloom, whereas the actual experiment for yield and gel strength were 13.03% and 291.33 Bloom, respectively. The gelatin extracted from dried skin were analyzed for their proximate composition, yield, gel strength, viscosity, color, and amino acid composition. The results of dried skin gelatin properties compared to the commercial gelatin. Gelatin extracted from the dried skin gave content lower moisture, ash and protein content but higher fat compared to commercial gelatin. This study also shows that the gelatin extracted from the dried skin gave higher gel strength and pH but the lower amino acid composition compared to commercial gelatin.

Optimization of extraction parameters of PTP1β (protein tyrosine phosphatase 1β), inhibitory polyphenols, and anthocyanins from Zea mays L. using response surface methodology (RSM).

PubMed

Hwang, Seung Hwan; Kwon, Shin Hwa; Wang, Zhiqiang; Kim, Tae Hyun; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

2016-08-26

Protein tyrosine phosphatase expressed in insulin-sensitive tissues (such as liver, muscle, and adipose tissue) has a key role in the regulation of insulin signaling and pathway activation, making protein tyrosine phosphatase a promising target for the treatment of type 2 diabetes mellitus and obesity and response surface methodology (RSM) is an effective statistical technique for optimizing complex processes using a multi-variant approach. In this study, Zea mays L. (Purple corn kernel, PCK) and its constituents were investigated for protein tyrosine phosphatase 1β (PTP1β) inhibitory activity including enzyme kinetic study and to improve total yields of anthocyanins and polyphenols, four extraction parameters, including temperature, time, solid-liquid ratio, and solvent volume, were optimized by RSM. Isolation of seven polyphenols and five anthocyanins was achieved by PTP1β assay. Among them, cyanidin-3-(6"malonylglucoside) and 3'-methoxyhirsutrin showed the highest PTP1β inhibition with IC50 values of 54.06 and 64.04 μM, respectively and 4.52 mg gallic acid equivalent/g (GAE/g) of total polyphenol content (TPC) and 43.02 mg cyanidin-3-glucoside equivalent/100 g (C3GE/100g) of total anthocyanin content (TAC) were extracted at 40 °C for 8 h with a 33 % solid-liquid ratio and a 1:15 solvent volume. Yields were similar to predictions of 4.58 mg GAE/g of TPC and 42.28 mg C3GE/100 g of TAC. These results indicated that PCK and 3'-methoxyhirsutrin and cyanidin-3-(6"malonylglucoside) might be active natural compounds and could be apply by optimizing of extraction process using response surface methodology.

Evaluation of extraction methods for the identification of proteins from date palm (Phoenix dactylifera L.) seed and flesh.

PubMed

Lee, Hooi Xian; Ahmad, Fisal; Saad, Bahruddin; Ismail, Mohd Nazri

2017-11-26

Date fruits are well known to be very nutritious. Nevertheless, the protein contents of the fruit, particularly the seed and flesh, are still understudied, largely due to their difficult physical characteristics. This study was conducted to compare three different protein extraction methods which were the trichloroacetic acid (TCA)-acetone (TCA-A), phenol (Phe), and TCA-acetone-phenol (TCA-A-Phe), and to perform proteomic analysis on date palm seed and flesh. Phe extraction method showed the highest protein yields for both seed (8.26 mg/g) and flesh (1.57 mg/g). Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Phe, and TCA-A-Phe extraction methods were shown to be efficient in removing interfering compounds and gave well-resolved bands over a wide range of molecular weights. Following liquid chromatography-tandem mass spectrometry analysis, about 50-64% of extracted proteins were identified with known functions including those involved in glycolysis, Krebs cycle, defense, and storage. Phe protein extraction method was proven to be the optimal method for date flesh and seed.

Extraction of Polysaccharide from Spirulina and Evaluation of Its Activities.

PubMed

Wang, Bingyue; Liu, Qian; Huang, Yinghong; Yuan, Yueling; Ma, Qianqian; Du, Manling; Cai, Tiange; Cai, Yu

2018-01-01

Polysaccharide of Spirulina platensis (PSP) is a kind of water-soluble polysaccharide extracted from Spirulina platensis . It has been proved to have antitumor, antioxidation, antiaging, and antivirus properties. And it has a promising prospect for wide application. This study aims to identify an extraction process for high-purity polysaccharide in Spirulina (PSP) through a series of optimization methods and then evaluates its initial antiaging activities. Four kinds of extraction methods-hot-water extraction, alkali extraction, ultrasonic-assisted extraction, and freeze-thaw extraction-were compared to find the optimal one, which was further optimized by response surface methodology. PSP was obtained after the crude PSP was deproteinized and depigmented. The antiaging effects of PSP were preliminarily evaluated through in vitro cell experiments. The alkali extraction method was determined as the optimal method, with the optimized extraction process consisting of a solid-liquid ratio of 1 : 50, a pH value of 10.25, a temperature of 89.24°C, and a time of 9.99 h. The final PSP contained 71.65% of polysaccharide and 8.54% of protein. At a concentration of 50  μ g/mL, PSP exerted a significant promoting effect on the proliferation and traumatic fusion of human immortalized epidermal cells HaCaT. An extraction method for high-purity PSP with a high extraction rate was established, and in vitro results suggest antioxidation and antiaging activities.

Defatting and Sonication Enhances Protein Extraction from Edible Insects

PubMed Central

2017-01-01

Edible insects are attracting growing interest as a sustainable source of protein for addition to processed meat and dairy products. The current study investigated the optimal method for protein extraction from mealworm larvae (Tenebrio molitor), cricket adults (Gryllus bimaculatus), and silkworm pupae (Bombyx mori), for use in further applications. After defatting with n-hexane for up to 48 h, sonication was applied for 1-20 min and the protein yield was measured. All samples showed a total residual fat percentage below 1.36%, and a 35% to 94% improvement in protein yield (%). In conclusion, defatting with n-hexane combined with sonication improves the protein yield from insect samples. PMID:29725219

Using Chlorella vulgaris to treat toxic excess sludge extract, and identification of its response mechanism by proteomics approach.

PubMed

Wang, Lu; Wang, Hualin; Chen, Xiurong; Xu, Yan; Zhou, Tianjun; Wang, Xiaoxiao; Lu, Qian; Ruan, Roger

2018-04-01

Chlorella vulgaris was cultivated in varying proportions of toxic sludge extracts obtained from a sequencing batch reactor for treating synthetic wastewater containing chlorophenols. C. vulgaris could reduce the ecotoxicity from sludge extracts, and a positive correlation was noted between ecotoxicity removal and total organic carbon removal. In terms of cell density, the optimal proportion of sludge extracts required for the cultivation of C. vulgaris was lower than 50%. The correlation between protein content in per 10 6 algae and inhibition extent of ecotoxicity of the 5 groups on the day of inoculation (0.9182, p < .05) indicated a positive relationship between algal protein secretion and ecotoxicity. According to the protein expression and differential protein expression analysis, we concluded that C. vulgaris produced proteins that involved in the stress response/redox system and energy metabolism/biosynthesis to respond to the toxic environment and some other proteins related to mixotrophic metabolism. Copyright © 2018 Elsevier Ltd. All rights reserved.

Impact of new ingredients obtained from brewer's spent yeast on bread characteristics.

PubMed

Martins, Z E; Pinho, O; Ferreira, I M P L V O

2018-05-01

The impact of bread fortification with β-glucans and with proteins/proteolytic enzymes from brewers' spent yeast on physical characteristics was evaluated. β-Glucans extraction from spent yeast cell wall was optimized and the extract was incorporated on bread to obtain 2.02 g β-glucans/100 g flour, in order to comply with the European Food Safety Authority guidelines. Protein/proteolytic enzymes extract from spent yeast was added to bread at 60 U proteolytic activity/100 g flour. Both β-glucans rich and proteins/proteolytic enzymes extracts favoured browning of bread crust. However, breads with proteins/proteolytic enzymes addition presented lower specific volume, whereas the incorporation of β-glucans in bread lead to uniform pores that was also noticeble in terms of higher specific volume. Overall, the improvement of nutritional/health promoting properties is highlighted with β-glucan rich extract, not only due to bread β-glucan content but also for total dietary fibre content (39% increase). The improvement was less noticeable for proteins/proteolytic enzymes extract. Only a 6% increase in bread protein content was noted with the addition of this extract and higher protein content would most likely accentuate the negative impact on bread specific volume that in turn could impair consumer acceptance. Therefore, only β-glucan rich extract is a promising bread ingredient.

Extracting physicochemical features to predict protein secondary structure.

PubMed

Huang, Yin-Fu; Chen, Shu-Ying

2013-01-01

We propose a protein secondary structure prediction method based on position-specific scoring matrix (PSSM) profiles and four physicochemical features including conformation parameters, net charges, hydrophobic, and side chain mass. First, the SVM with the optimal window size and the optimal parameters of the kernel function is found. Then, we train the SVM using the PSSM profiles generated from PSI-BLAST and the physicochemical features extracted from the CB513 data set. Finally, we use the filter to refine the predicted results from the trained SVM. For all the performance measures of our method, Q 3 reaches 79.52, SOV94 reaches 86.10, and SOV99 reaches 74.60; all the measures are higher than those of the SVMpsi method and the SVMfreq method. This validates that considering these physicochemical features in predicting protein secondary structure would exhibit better performances.

Extracting Physicochemical Features to Predict Protein Secondary Structure

PubMed Central

Chen, Shu-Ying

2013-01-01

We propose a protein secondary structure prediction method based on position-specific scoring matrix (PSSM) profiles and four physicochemical features including conformation parameters, net charges, hydrophobic, and side chain mass. First, the SVM with the optimal window size and the optimal parameters of the kernel function is found. Then, we train the SVM using the PSSM profiles generated from PSI-BLAST and the physicochemical features extracted from the CB513 data set. Finally, we use the filter to refine the predicted results from the trained SVM. For all the performance measures of our method, Q 3 reaches 79.52, SOV94 reaches 86.10, and SOV99 reaches 74.60; all the measures are higher than those of the SVMpsi method and the SVMfreq method. This validates that considering these physicochemical features in predicting protein secondary structure would exhibit better performances. PMID:23766688

Isolation and Characterization of Adhesive Secretion from Cuvierian Tubules of Sea Cucumber Holothuria forskåli (Echinodermata: Holothuroidea)

PubMed Central

Baranowska, Malgorzata; Schloßmacher, Ute; McKenzie, J. Douglas; Müller, Werner E. G.; Schröder, Heinz C.

2011-01-01

The sea cucumber Holothuria forskåli possesses a specialized system called Cuvierian tubules. During mechanical stimulation white filaments (tubules) are expelled and become sticky upon contact with any object. We isolated a protein with adhesive properties from protein extracts of Cuvierian tubules from H. forskåli. This protein was identified by antibodies against recombinant precollagen D which is located in the byssal threads of the mussel Mytilus galloprovincialis. To find out the optimal procedure for extraction and purification, the identified protein was isolated by several methods, including electroelution, binding to glass beads, immunoprecipitation, and gel filtration. Antibodies raised against the isolated protein were used for localization of the adhesive protein in Cuvierian tubules. Immunostaining and immunogold electron microscopical studies revealed the strongest immunoreactivity in the mesothelium; this tissue layer is involved in adhesion. Adhesion of Cuvierian tubule extracts was measured on the surface of various materials. The extracted protein showed the strongest adhesion to Teflon surface. Increased adhesion was observed in the presence of potassium and EDTA, while cadmium caused a decrease in adhesion. Addition of antibodies and trypsin abolished the adhesive properties of the extract. PMID:22013488

Comparison of Different Protein Extraction Methods for Gel-Based Proteomic Analysis of Ganoderma spp.

PubMed

Al-Obaidi, Jameel R; Saidi, Noor Baity; Usuldin, Siti Rokhiyah Ahmad; Hussin, Siti Nahdatul Isnaini Said; Yusoff, Noornabeela Md; Idris, Abu Seman

2016-04-01

Ganoderma species are a group of fungi that have the ability to degrade lignin polymers and cause severe diseases such as stem and root rot and can infect economically important plants and perennial crops such as oil palm, especially in tropical countries such as Malaysia. Unfortunately, very little is known about the complex interplay between oil palm and Ganoderma in the pathogenesis of the diseases. Proteomic technologies are simple yet powerful tools in comparing protein profile and have been widely used to study plant-fungus interaction. A critical step to perform a good proteome research is to establish a method that gives the best quality and a wide coverage of total proteins. Despite the availability of various protein extraction protocols from pathogenic fungi in the literature, no single extraction method was found suitable for all types of pathogenic fungi. To develop an optimized protein extraction protocol for 2-DE gel analysis of Ganoderma spp., three previously reported protein extraction protocols were compared: trichloroacetic acid, sucrose and phenol/ammonium acetate in methanol. The third method was found to give the most reproducible gels and highest protein concentration. Using the later method, a total of 10 protein spots (5 from each species) were successfully identified. Hence, the results from this study propose phenol/ammonium acetate in methanol as the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp.

Assay Development Process | Office of Cancer Clinical Proteomics Research

Cancer.gov

Typical steps involved in the development of a  mass spectrometry-based targeted assay include: (1) selection of surrogate or signature peptides corresponding to the targeted protein or modification of interest; (2) iterative optimization of instrument and method parameters for optimal detection of the selected peptide; (3) method development for protein extraction from biological matrices such as tissue, whole cell lysates, or blood plasma/serum and proteolytic digestion of proteins (usually with trypsin); (4) evaluation of the assay in the intended biological matrix to determine if e

Establishment of the optimum two-dimensional electrophoresis system of ovine ovarian tissue.

PubMed

Jia, J L; Zhang, L P; Wu, J P; Wang, J; Ding, Q

2014-08-26

Lambing performance of sheep is the most important economic trait and is regarded as a critic factoring affecting the productivity in sheep industry. Ovary plays the most roles in lambing trait. To establish the optimum two-dimensional electrophoresis system (2-DE) of ovine ovarian tissue, the common protein extraction methods of animal tissue (trichloroacetic acid/acetone precipitation and direct schizolysis methods) were used to extract ovine ovarian protein, and 17-cm nonlinear immobilized PH 3-10 gradient strips were used for 2-DE. The sample handling, loading quantity of the protein sample, and isoelectric focusing (IEF) steps were manipulated and optimized in this study. The results indicate that the direct schizolysis III method, a 200-μg loading quantity of the protein sample, and IEF steps II (20°C active hydration, 14 h→500 V, 1 h→1000 V 1 h→1000-9000 V, 6 h→80,000 VH→500 V 24 h) are optimal for 2-DE analysis of ovine ovarian tissue. Therefore, ovine ovarian tissue proteomics 2-DE was preliminarily established by the optimized conditions in this study; meanwhile, the conditions identified herein could provide a reference for ovarian sample preparation and 2-DE using tissues from other animals.

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.

PubMed

Katsura, Kazushige; Matsuda, Takayoshi; Tomabechi, Yuri; Yonemochi, Mayumi; Hanada, Kazuharu; Ohsawa, Noboru; Sakamoto, Kensaku; Takemoto, Chie; Shirouzu, Mikako

2017-11-01

Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers.

PubMed

Li, Ao; Wang, Ruyi; Li, Xianliang; Liu, Mingyong; Fan, Jian; Guo, Kai; Luo, Bing; Chen, Tingting; Feng, Shengqiu; Wang, Yanting; Wang, Bingrui; Peng, Liangcai; Xia, Tao

2016-05-19

Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in β-1,4-glucan and β-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in β-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction.

A multiplexed microfluidic toolbox for the rapid optimization of affinity-driven partition in aqueous two phase systems.

PubMed

Bras, Eduardo J S; Soares, Ruben R G; Azevedo, Ana M; Fernandes, Pedro; Arévalo-Rodríguez, Miguel; Chu, Virginia; Conde, João P; Aires-Barros, M Raquel

2017-09-15

Antibodies and other protein products such as interferons and cytokines are biopharmaceuticals of critical importance which, in order to be safely administered, have to be thoroughly purified in a cost effective and efficient manner. The use of aqueous two-phase extraction (ATPE) is a viable option for this purification, but these systems are difficult to model and optimization procedures require lengthy and expensive screening processes. Here, a methodology for the rapid screening of antibody extraction conditions using a microfluidic channel-based toolbox is presented. A first microfluidic structure allows a simple negative-pressure driven rapid screening of up to 8 extraction conditions simultaneously, using less than 20μL of each phase-forming solution per experiment, while a second microfluidic structure allows the integration of multi-step extraction protocols based on the results obtained with the first device. In this paper, this microfluidic toolbox was used to demonstrate the potential of LYTAG fusion proteins used as affinity tags to optimize the partitioning of antibodies in ATPE processes, where a maximum partition coefficient (K) of 9.2 in a PEG 3350/phosphate system was obtained for the antibody extraction in the presence of the LYTAG-Z dual ligand. This represents an increase of approx. 3.7 fold when compared with the same conditions without the affinity molecule (K=2.5). Overall, this miniaturized and versatile approach allowed the rapid optimization of molecule partition followed by a proof-of-concept demonstration of an integrated back extraction procedure, both of which are critical procedures towards obtaining high purity biopharmaceuticals using ATPE. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice.

PubMed

Laguía-Becher, Melina; Martín, Valentina; Kraemer, Mauricio; Corigliano, Mariana; Yacono, María L; Goldman, Alejandra; Clemente, Marina

2010-07-15

Codon optimization and subcellular targeting were studied with the aim to increase the expression levels of the SAG178-322 antigen of Toxoplasma gondii in tobacco leaves. The expression of the tobacco-optimized and native versions of the SAG1 gene was explored by transient expression from the Agrobacterium tumefaciens binary expression vector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER) and the apoplast. Finally, mice were subcutaneously and orally immunized with leaf extracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coli was used to optimize the oral immunization with leaf extracts-SAG1. Leaves agroinfiltrated with an unmodified SAG1 gene accumulated 5- to 10-fold more than leaves agroinfiltrated with a codon-optimized SAG1 gene. ER localization allowed the accumulation of higher levels of native SAG1. However, no significant differences were observed between the mRNA accumulations of the different versions of SAG1. Subcutaneous immunization with leaf extracts-SAG1 (SAG1) protected mice against an oral challenge with a non-lethal cyst dose, and this effect could be associated with the secretion of significant levels of IFN-gamma. The protection was increased when mice were ID boosted with rSAG1 (SAG1+boost). This group elicited a significant Th1 humoral and cellular immune response characterized by high levels of IFN-gamma. In an oral immunization assay, the SAG1+boost group showed a significantly lower brain cyst burden compared to the rest of the groups. Transient agroinfiltration was useful for the expression of all of the recombinant proteins tested. Our results support the usefulness of endoplasmic reticulum signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The results showed that this plant-produced protein has potential for use as vaccine and provides a potential means for protecting humans and animals against toxoplasmosis.

Isolation and Analysis of Keratins and Keratin-Associated Proteins from Hair and Wool.

PubMed

Deb-Choudhury, Santanu; Plowman, Jeffrey E; Harland, Duane P

2016-01-01

The presence of highly cross-linked protein networks in hair and wool makes them very difficult substrates for protein extraction, a prerequisite for further protein analysis and characterization. It is therefore imperative that these cross-links formed by disulfide bridges are first disrupted for the efficient extraction of proteins. Chaotropes such as urea are commonly used as efficient extractants. However, a combination of urea and thiourea not only improves recovery of proteins but also results in improved resolution of the keratins in 2DE gels. Reductants also play an important role in protein dissolution. Dithiothreitol effectively removes keratinous material from the cortex, whereas phosphines, like Tris(2-carboxyethyl)phosphine, remove material from the exocuticle. The relative extractability of the keratins and keratin-associated proteins is also dependent on the concentration of chaotropes, reductants, and pH, thus providing a means to preferentially extract these proteins. Ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIM(+)[Cl](-)) are known to solubilize wool by disrupting noncovalent interactions, specifically intermolecular hydrogen bonds. BMIM(+)[Cl](-) proved to be an effective extractant of wool proteins and complementary in nature to chaotropes such as urea and thiourea for identifying unique peptides of wool proteins using mass spectrometry (MS). Successful identification of proteins resolved by one- or two-dimensional electrophoresis and MS is highly dependent on the optimal recovery of its protease-digested peptides with an efficient removal of interfering substances. The detergent sodium deoxycholate used in conjunction with Empore™ disks improved identification of proteins by mass spectrometry leading to higher percentage sequence coverage, identification of unique peptides and higher score. Copyright © 2016 Elsevier Inc. All rights reserved.

An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins

PubMed Central

Kumar, Manoj; Singh, Rajendra; Meena, Anil; Patidar, Bhagwan S; Prasad, Rajendra; Chhabra, Sunil K; Bansal, Surendra K

2017-01-01

The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for different diseases, which may help to identify the proteins that may serve as markers for diagnostics as well as targets for development of new therapeutic potential. PMID:28469466

Efficient extraction strategies of tea (Camellia sinensis) biomolecules.

PubMed

Banerjee, Satarupa; Chatterjee, Jyotirmoy

2015-06-01

Tea is a popular daily beverage worldwide. Modulation and modifications of its basic components like catechins, alkaloids, proteins and carbohydrate during fermentation or extraction process changes organoleptic, gustatory and medicinal properties of tea. Through these processes increase or decrease in yield of desired components are evident. Considering the varied impacts of parameters in tea production, storage and processes that affect the yield, extraction of tea biomolecules at optimized condition is thought to be challenging. Implementation of technological advancements in green chemistry approaches can minimize the deviation retaining maximum qualitative properties in environment friendly way. Existed extraction processes with optimization parameters of tea have been discussed in this paper including its prospects and limitations. This exhaustive review of various extraction parameters, decaffeination process of tea and large scale cost effective isolation of tea components with aid of modern technology can assist people to choose extraction condition of tea according to necessity.

Direct analysis of in-gel proteins by carbon nanotubes-modified paper spray ambient mass spectrometry.

PubMed

Han, Feifei; Yang, Yuhan; Ouyang, Jin; Na, Na

2015-02-07

The in situ and direct extraction, desorption and ionization of in-gel intact proteins after electrophoresis has been achieved by carbon nanotubes (CNTs)-modified paper spray mass spectrometry at ambient conditions. Characteristics of CNTs (including larger surface area, smaller pore diameter and enhanced conductivity) were endowed to the porous filter paper substrate by uniformly dispersing the CNTs on the filter paper. Upon applying electric potential to the CNTs-modified paper, the in-gel proteins were extracted from the gel and subsequently migrated to the tip of the filter paper by electrophoresis-like behavior for paper spray ionization, which was monitored by extracted ion chronograms. The characterizations of modified filter papers and CNTs nanoparticles further confirmed the role of CNTs in in-gel protein extraction, protein migration as well as spray ionization at the paper tip. Under optimized conditions, a mixture of cytochrome c, lysozyme and myoglobin was successfully separated by native electrophoresis and subsequently analysed by the present method, showing a limit of detection of 10 ng per gel band. The present strategy offers a new pathway for the direct detection of in-gel intact proteins at ambient conditions without any pre-treatment (e.g. digestion, chemical extraction and desalting), which exhibits potential applications in top-down proteomics.

Comparison of the Anti-Inflammatory Activities of Supercritical Carbon Dioxide versus Ethanol Extracts from Leaves of Perilla frutescens Britt. Radiation Mutant.

PubMed

Jin, Chang Hyun; Park, Han Chul; So, Yangkang; Nam, Bomi; Han, Sung Nim; Kim, Jin-Baek

2017-02-17

In this study, we aimed to compare supercritical carbon dioxide extraction and ethanol extraction for isoegomaketone (IK) content in perilla leaf extracts and to identify the optimal method. We measured the IK concentration using HPLC and inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells from the extracts. The IK concentration was 10-fold higher in perilla leaf extracts by supercritical carbon dioxide extraction (SFE) compared with that in perilla leaf extracts by ethanol extraction (EE). When the extracts were treated in LPS-induced RAW 264.7 cells at 25 μg/mL, the SFE inhibited the expression of inflammatory mediators such as nitric oxide (NO), monocyte chemoattractant protein-1 (MCP-1), interleutkin-6 (IL-6), interferon-β (IFN-β), and inducible nitric oxide synthase (iNOS) to a much greater extent compared with EE. Taken together, supercritical carbon dioxide extraction is considered the optimal process for obtaining high IK content and anti-inflammatory activities in leaf extracts from the P. frutescens Britt. radiation mutant.

Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids.

PubMed

Martin, Rey W; Des Soye, Benjamin J; Kwon, Yong-Chan; Kay, Jennifer; Davis, Roderick G; Thomas, Paul M; Majewska, Natalia I; Chen, Cindy X; Marcum, Ryan D; Weiss, Mary Grace; Stoddart, Ashleigh E; Amiram, Miriam; Ranji Charna, Arnaz K; Patel, Jaymin R; Isaacs, Farren J; Kelleher, Neil L; Hong, Seok Hoon; Jewett, Michael C

2018-03-23

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

A green deep eutectic solvent-based aqueous two-phase system for protein extracting.

PubMed

Xu, Kaijia; Wang, Yuzhi; Huang, Yanhua; Li, Na; Wen, Qian

2015-03-15

As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n=3), 1.6057% (n=3) and 1.6132% (n=3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV-vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES-protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

Optimization of a wet microalgal lipid extraction procedure for improved lipid recovery for biofuel and bioproduct production.

PubMed

Sathish, Ashik; Marlar, Tyler; Sims, Ronald C

2015-10-01

Methods to convert microalgal biomass to bio based fuels and chemicals are limited by several processing and economic hurdles. Research conducted in this study modified/optimized a previously published procedure capable of extracting transesterifiable lipids from wet algal biomass. This optimization resulted in the extraction of 77% of the total transesterifiable lipids, while reducing the amount of materials and temperature required in the procedure. In addition, characterization of side streams generated demonstrated that: (1) the C/N ratio of the residual biomass or lipid extracted (LE) biomass increased to 54.6 versus 10.1 for the original biomass, (2) the aqueous phase generated contains nitrogen, phosphorous, and carbon, and (3) the solid precipitate phase was composed of up to 11.2 wt% nitrogen (70% protein). The ability to isolate algal lipids and the possibility of utilizing generated side streams as products and/or feedstock material for downstream processes helps promote the algal biorefinery concept. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bioactive characteristics and optimization of tamarind seed protein hydrolysate for antioxidant-rich food formulations.

PubMed

Bagul, Mayuri B; Sonawane, Sachin K; Arya, Shalini S

2018-04-01

Tamarind seed has been a source of valuable nutrients such as protein (contains high amount of many essential amino acids), essential fatty acids, and minerals which are recognized as additive to develop perfect balanced functional foods. The objective of present work was to optimize the process parameters for extraction and hydrolysis of protein from tamarind seeds. Papain-derived hydrolysates showed a maximum degree of hydrolysis (39.49%) and radical scavenging activity (42.92 ± 2.83%) at optimized conditions such as enzyme-to-substrate ratio (1:5), hydrolysis time (3 h), hydrolysis temperature (65 °C), and pH 6. From this study, papain hydrolysate can be considered as good source of natural antioxidants in developing food formulations.

Method optimization for proteomic analysis of soybean leaf: Improvements in identification of new and low-abundance proteins

PubMed Central

Mesquita, Rosilene Oliveira; de Almeida Soares, Eduardo; de Barros, Everaldo Gonçalves; Loureiro, Marcelo Ehlers

2012-01-01

The most critical step in any proteomic study is protein extraction and sample preparation. Better solubilization increases the separation and resolution of gels, allowing identification of a higher number of proteins and more accurate quantitation of differences in gene expression. Despite the existence of published results for the optimization of proteomic analyses of soybean seeds, no comparable data are available for proteomic studies of soybean leaf tissue. In this work we have tested the effects of modification of a TCA-acetone method on the resolution of 2-DE gels of leaves and roots of soybean. Better focusing was obtained when both mercaptoethanol and dithiothreitol were used in the extraction buffer simultaneously. Increasing the number of washes of TCA precipitated protein with acetone, using a final wash with 80% ethanol and using sonication to ressuspend the pellet increased the number of detected proteins as well the resolution of the 2-DE gels. Using this approach we have constructed a soybean protein map. The major group of identified proteins corresponded to genes of unknown function. The second and third most abundant groups of proteins were composed of photosynthesis and metabolism related genes. The resulting protocol improved protein solubility and gel resolution allowing the identification of 122 soybean leaf proteins, 72 of which were not detected in other published soybean leaf 2-DE gel datasets, including a transcription factor and several signaling proteins. PMID:22802721

A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTP.

PubMed

Koga, Hirohisa; Misawa, Satoru; Shibui, Tatsuro

2009-01-01

Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous-flow wheat embryo cell-free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC-based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 microM of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C-terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B(1-320)) gave almost the same result. The wheat embryo cell-free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48-h period at 26 degrees C. 2009 American Institute of Chemical Engineers Biotechnol.

Response surface methodology based extraction of Tribulus terrestris leads to an upsurge of antilithiatic potential by inhibition of calcium oxalate crystallization processes.

PubMed

Kaushik, Jyoti; Tandon, Simran; Gupta, Varun; Nayyar, Jasamrit; Singla, Surinder Kumar; Tandon, Chanderdeep

2017-01-01

Tribulus terrestris has significant antilithiatic efficacy established via both in vitro as well as in vivo studies and is used in numerous anti-urolithiatic herbal formulations viz. Cystone, Uriflow, Uritone and Neeri. However, to fully utilize its antilithiatic potential, the influence of different extraction parameters on antilithiatic ability of T. terrestris aqueous extract needs elucidation. Thus, the current study was undertaken using statistically optimized extraction conditions for aqueous extract preparation. Response surface methodology was employed to observe the influence of three variables i.e. temperature (°C), time (h) and solid: liquid ratio (S: L) on the extraction yield (%) and protein content (mg/g) of T. terrestris aqueous extract. RSM results revealed that the high S:L ratio, low temperature and reduced incubation time were optimal conditions for aqueous extraction. Under such extraction conditions the protein content reached the value of 26.6±1.22 mg/g and the obtained extraction yield was 27.32±1.62%. The assessment of antilithiatic activity of 4 selected extracts (AE1-4), revealed enhanced nucleation and aggregation inhibition of calcium oxalate crystals with AE1 and AE2, which in addition significantly altered the size and morphology of calcium oxalate monohydrate (COM) crystals compared to AE3 and AE4. In vitro cell culture based studies on renal epithelial cells (MDCK, NRK-52E and PK 15) proved that the AE1 showed higher cytoprotective potency by increasing cell viability as compared to the oxalate treated group. The free radical scavenging activity of aqueous extract lowered the reactive oxygen specie's induced damage and potentially reduced the signals of programmed cell death due to oxalate injury. In addition, modulation of the COM crystal morphology was enhanced by AE1 as compared to AE2. The FTIR and GC-MS analysis of AE1, showed the presence of biomolecules which could aid in the attenuation of lithiatic process. In the light of these results the utility of the RSM approach to fully optimize the antilithiatic potential of T. terrestris cannot be undermined.

Response surface methodology based extraction of Tribulus terrestris leads to an upsurge of antilithiatic potential by inhibition of calcium oxalate crystallization processes

PubMed Central

Kaushik, Jyoti; Tandon, Simran; Gupta, Varun; Nayyar, Jasamrit; Singla, Surinder Kumar; Tandon, Chanderdeep

2017-01-01

Tribulus terrestris has significant antilithiatic efficacy established via both in vitro as well as in vivo studies and is used in numerous anti-urolithiatic herbal formulations viz. Cystone, Uriflow, Uritone and Neeri. However, to fully utilize its antilithiatic potential, the influence of different extraction parameters on antilithiatic ability of T. terrestris aqueous extract needs elucidation. Thus, the current study was undertaken using statistically optimized extraction conditions for aqueous extract preparation. Response surface methodology was employed to observe the influence of three variables i.e. temperature (°C), time (h) and solid: liquid ratio (S: L) on the extraction yield (%) and protein content (mg/g) of T. terrestris aqueous extract. RSM results revealed that the high S:L ratio, low temperature and reduced incubation time were optimal conditions for aqueous extraction. Under such extraction conditions the protein content reached the value of 26.6±1.22 mg/g and the obtained extraction yield was 27.32±1.62%. The assessment of antilithiatic activity of 4 selected extracts (AE1-4), revealed enhanced nucleation and aggregation inhibition of calcium oxalate crystals with AE1 and AE2, which in addition significantly altered the size and morphology of calcium oxalate monohydrate (COM) crystals compared to AE3 and AE4. In vitro cell culture based studies on renal epithelial cells (MDCK, NRK-52E and PK 15) proved that the AE1 showed higher cytoprotective potency by increasing cell viability as compared to the oxalate treated group. The free radical scavenging activity of aqueous extract lowered the reactive oxygen specie’s induced damage and potentially reduced the signals of programmed cell death due to oxalate injury. In addition, modulation of the COM crystal morphology was enhanced by AE1 as compared to AE2. The FTIR and GC-MS analysis of AE1, showed the presence of biomolecules which could aid in the attenuation of lithiatic process. In the light of these results the utility of the RSM approach to fully optimize the antilithiatic potential of T. terrestris cannot be undermined. PMID:28846699

Identification and characterization of hydrocolloid from Cordia myxa leaf.

PubMed

Samavati, Vahid; Lorestani, Mohammad; Joolazadeh, Sajjad

2014-04-01

Hot water extraction technique was employed to extract the hydrocolloid from Cordia myxa leaf (PCM). The optimal conditions for extraction of PCM were determined using response surface methodology. A Box-Behnken design (BBD) was applied to evaluate the effects of three independent variables (extraction time (X1: 1-4 h), extraction temperature (X2: 55-95 °C), and water to raw material ratio (X3: 5-30 ml/g) on the extraction yield of PCM. The content of moisture, water-soluble and water-insoluble ash, crude protein and total phenol were determined in the extracted hydrocolloid by standard methods. The maximum hydrocolloid extraction yield (9.501±0.15%) was achieved by using extraction time of 4.94 h, extraction temperature of 94.91 °C and water to raw material ratio of 21.74 ml/g. The contents of moisture, crude protein, water-soluble and water-insoluble ash and total phenol were 21.63±0.94%, 14.27±0.55%, 3.07±0.16% and 2.61±0.19 mg galic acid/g, respectively. Copyright © 2014 Elsevier B.V. All rights reserved.

Immunochemical-based method for detection of hazelnut proteins in processed foods.

PubMed

Ben Rejeb, Samuel; Abbott, Michael; Davies, David; Querry, Jessica; Cléroux, Chantal; Streng, Christine; Delahaut, Philippe; Yeung, Jupiter M

2003-01-01

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect hazelnut by using polyclonal antibodies generated against a protein extract of roasted hazelnut. No cross-reactivity was observed in tests against 39 commodities, including many common allergens, tree nuts, and legumes. Hazelnut protein standard solutions at 0.45 ng/mL [inhibition concentration (IC80) of the competitive test] were clearly identified by the ELISA. An extraction and quantification method was developed and optimized for chocolate, cookies, breakfast cereals, and ice cream, major food commodities likely to be cross-contaminated with undeclared hazelnut during food processing. No sample cleanup was required when extracts were diluted 10-fold. Recovery results were generated with blank matrixes spiked at 4 levels from 1 to 10 microg/g hazelnut protein. With the developed extraction and sample handling procedure, hazelnut proteins were recovered at 64-83% from chocolate and at 78-97% from other matrixes. A confirmatory technique was developed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer. The developed methods were applied to a small market survey of chocolate products and allowed the identification of undeclared hazelnut in these products.

Establishing a high yielding streptomyces-based cell-free protein synthesis system.

PubMed

Li, Jian; Wang, He; Kwon, Yong-Chan; Jewett, Michael C

2017-06-01

Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Evaluation of Method-Specific Extraction Variability for the Measurement of Fatty Acids in a Candidate Infant/Adult Nutritional Formula Reference Material.

PubMed

Place, Benjamin J

2017-05-01

To address community needs, the National Institute of Standards and Technology has developed a candidate Standard Reference Material (SRM) for infant/adult nutritional formula based on milk and whey protein concentrates with isolated soy protein called SRM 1869 Infant/Adult Nutritional Formula. One major component of this candidate SRM is the fatty acid content. In this study, multiple extraction techniques were evaluated to quantify the fatty acids in this new material. Extraction methods that were based on lipid extraction followed by transesterification resulted in lower mass fraction values for all fatty acids than the values measured by methods utilizing in situ transesterification followed by fatty acid methyl ester extraction (ISTE). An ISTE method, based on the identified optimal parameters, was used to determine the fatty acid content of the new infant/adult nutritional formula reference material.

Optimization of Ultrasonic-Assisted Aqueous Two-Phase Extraction of Phloridzin from Malus Micromalus Makino with Ethanol/Ammonia Sulfate System.

PubMed

Zhang, Zhen; Liu, Fang; He, Caian; Yu, Yueli; Wang, Min

2017-12-01

Application of an aqueous two-phase system (ATPS) coupled with ultrasonic technology for the extraction of phloridzin from Malus micromalus Makino was evaluated and optimized by response surface methodology (RSM). The ethanol/ammonium sulfate ATPS was selected for detailed investigation, including the phase diagram, effect of phase composition and extract conditions on the partition of phloridzin, and the recycling of ammonium sulfate. In addition, the evaluation of extraction efficiency and the identification of phloridzin were investigated. The optimal partition coefficient (6.55) and recovery (92.86%) of phloridzin were obtained in a system composed of 35% ethanol (w/w) and 16% (NH 4 ) 2 SO 4 (w/w), 51:1 liquid-to-solid ratio, and extraction temperature of 36 °C. Comparing with the traditional solvent extraction with respective 35% and 80% ethanol, ultrasonic-assisted aqueous two-phase extraction (UAATPE) strategy had significant advantages with lower ethanol consumption, less impurity of sugar and protein, and higher extracting efficiency of phloridzin. Our result indicated that UAATPE was a valuable method for the extraction and preliminary purification of phloridzin from the fruit of Malus micromalus Makino, which has great potential in the deep processing of Malus micromalus Makino industry to increase these fruits' additional value and drive the local economic development. © 2017 Institute of Food Technologists®.

Membrane protein extraction and purification using styrene-maleic acid (SMA) copolymer: effect of variations in polymer structure.

PubMed

Morrison, Kerrie A; Akram, Aneel; Mathews, Ashlyn; Khan, Zoeya A; Patel, Jaimin H; Zhou, Chumin; Hardy, David J; Moore-Kelly, Charles; Patel, Roshani; Odiba, Victor; Knowles, Tim J; Javed, Masood-Ul-Hassan; Chmel, Nikola P; Dafforn, Timothy R; Rothnie, Alice J

2016-12-01

The use of styrene-maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5-10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Exploring the potential of 3D Zernike descriptors and SVM for protein-protein interface prediction.

PubMed

Daberdaku, Sebastian; Ferrari, Carlo

2018-02-06

The correct determination of protein-protein interaction interfaces is important for understanding disease mechanisms and for rational drug design. To date, several computational methods for the prediction of protein interfaces have been developed, but the interface prediction problem is still not fully understood. Experimental evidence suggests that the location of binding sites is imprinted in the protein structure, but there are major differences among the interfaces of the various protein types: the characterising properties can vary a lot depending on the interaction type and function. The selection of an optimal set of features characterising the protein interface and the development of an effective method to represent and capture the complex protein recognition patterns are of paramount importance for this task. In this work we investigate the potential of a novel local surface descriptor based on 3D Zernike moments for the interface prediction task. Descriptors invariant to roto-translations are extracted from circular patches of the protein surface enriched with physico-chemical properties from the HQI8 amino acid index set, and are used as samples for a binary classification problem. Support Vector Machines are used as a classifier to distinguish interface local surface patches from non-interface ones. The proposed method was validated on 16 classes of proteins extracted from the Protein-Protein Docking Benchmark 5.0 and compared to other state-of-the-art protein interface predictors (SPPIDER, PrISE and NPS-HomPPI). The 3D Zernike descriptors are able to capture the similarity among patterns of physico-chemical and biochemical properties mapped on the protein surface arising from the various spatial arrangements of the underlying residues, and their usage can be easily extended to other sets of amino acid properties. The results suggest that the choice of a proper set of features characterising the protein interface is crucial for the interface prediction task, and that optimality strongly depends on the class of proteins whose interface we want to characterise. We postulate that different protein classes should be treated separately and that it is necessary to identify an optimal set of features for each protein class.

Chemopreventive effects of free and bound phenolics associated to steep waters (nejayote) obtained after nixtamalization of different maize types.

PubMed

Rojas-García, Carlos; García-Lara, Silverio; Serna-Saldivar, Sergio O; Gutiérrez-Uribe, Janet A

2012-03-01

Free and bound phenolics extracts from nejayote solids were obtained after optimally lime-cooking blue, normal white, red, normal yellow, high-carotenoid and quality protein maize types. The extraction yield ranged from 4.47 to 10.05%. Bound phenolics extracts had higher content of total phenolics, antioxidant activity and ferulic acid compared to the free phenolics extracts. In general, free phenolics extracts were less cytotoxic than the bound phenolics counterparts. Bound phenolics extracts had higher induction of quinone reductase (QR) and particularly the normal yellow nejayote exerted the highest chemopreventive index tested in Hepa1c1c7 cells. When tested for monofunctional phase 2 induction capacity in BPrc1 cells, the bound phenolics extracts of blue, normal white and quality protein nejayotes were better inducers than the normal yellow counterpart. Particularly, the free phenolics extract of the white maize nejayote induced BPrc1 cells QR and exerted a higher chemopreventive index compared to the bound phenolics extract. Therefore, the nejayote of the normal white maize was the best source of monofunctional phase 2 enzyme inducers.

Playing biology's name game: identifying protein names in scientific text.

PubMed

Hanisch, Daniel; Fluck, Juliane; Mevissen, Heinz-Theodor; Zimmer, Ralf

2003-01-01

A growing body of work is devoted to the extraction of protein or gene interaction information from the scientific literature. Yet, the basis for most extraction algorithms, i.e. the specific and sensitive recognition of protein and gene names and their numerous synonyms, has not been adequately addressed. Here we describe the construction of a comprehensive general purpose name dictionary and an accompanying automatic curation procedure based on a simple token model of protein names. We designed an efficient search algorithm to analyze all abstracts in MEDLINE in a reasonable amount of time on standard computers. The parameters of our method are optimized using machine learning techniques. Used in conjunction, these ingredients lead to good search performance. A supplementary web page is available at http://cartan.gmd.de/ProMiner/.

Optimization of the cydex blue assay: A one-step colorimetric protein assay using cyclodextrins and compatible with detergents and reducers

PubMed Central

2018-01-01

Sodium dodecyl sulfate electrophoresis (SDS) is a protein separation technique widely used, for example, prior to immunoblotting. Samples are usually prepared in a buffer containing both high concentrations of reducers and high concentrations of SDS. This conjunction renders the samples incompatible with common protein assays. By chelating the SDS, cyclodextrins make the use of simple, dye-based colorimetric assays possible. In this paper, we describe the optimization of the assay, focussing on the cyclodextrin/SDS ratio and the use of commercial assay reagents. The adaptation of the assay to a microplate format and using other detergent-containing conventional extraction buffers is also described. PMID:29641569

A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments.

PubMed

Norman, Anders; Hansen, Lars Hestbjerg; Sørensen, Søren J

2006-02-28

Whole-cell biosensors have become popular tools for detection of ecotoxic compounds in environmental samples. We have developed an assay optimized for flow cytometry with detection of genotoxic compounds in mind. The assay features extended pre-incubation and a cell density of only 10(6)-10(7) cells/mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS-induction in whole soil samples. Soil microcosms were spiked with a dilution-series of crude broth extract from the mitomycin C-producing streptomycete Streptomyces caespitosus. Biosensors extracted from these microcosms after 1 day of incubation at 30 degrees C were easily distinguished from extracts of non-contaminated soil particles when using flow cytometry, and induction of the biosensor by mitomycin C was detectable at concentrations as low as 2.5 ng/g of soil.

Quantitative and qualitative optimization of allergen extraction from peanut and selected tree nuts. Part 2. Optimization of buffer and ionic strength using a full factorial experimental design.

PubMed

L'Hocine, Lamia; Pitre, Mélanie

2016-03-01

A full factorial design was used to assess the single and interactive effects of three non-denaturing aqueous (phosphate, borate, and carbonate) buffers at various ionic strengths (I) on allergen extractability from and immunoglobulin E (IgE) immunoreactivity of peanut, almond, hazelnut, and pistachio. The results indicated that the type and ionic strength of the buffer had different effects on protein recovery from the nuts under study. Substantial differences in protein profiles, abundance, and IgE-binding intensity with different combinations of pH and ionic strength were found. A significant interaction between pH and ionic strength was observed for pistachio and almond. The optimal buffer system conditions, which maximized the IgE-binding efficiency of allergens and provided satisfactory to superior protein recovery yield and profiles, were carbonate buffer at an ionic strength of I=0.075 for peanut, carbonate buffer at I=0.15 for almond, phosphate buffer at I=0.5 for hazelnut, and borate at I=0.15 for pistachio. The buffer type and its ionic strength could be manipulated to achieve the selective solubility of desired allergens. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Identification of compound-protein interactions through the analysis of gene ontology, KEGG enrichment for proteins and molecular fragments of compounds.

PubMed

Chen, Lei; Zhang, Yu-Hang; Zheng, Mingyue; Huang, Tao; Cai, Yu-Dong

2016-12-01

Compound-protein interactions play important roles in every cell via the recognition and regulation of specific functional proteins. The correct identification of compound-protein interactions can lead to a good comprehension of this complicated system and provide useful input for the investigation of various attributes of compounds and proteins. In this study, we attempted to understand this system by extracting properties from both proteins and compounds, in which proteins were represented by gene ontology and KEGG pathway enrichment scores and compounds were represented by molecular fragments. Advanced feature selection methods, including minimum redundancy maximum relevance, incremental feature selection, and the basic machine learning algorithm random forest, were used to analyze these properties and extract core factors for the determination of actual compound-protein interactions. Compound-protein interactions reported in The Binding Databases were used as positive samples. To improve the reliability of the results, the analytic procedure was executed five times using different negative samples. Simultaneously, five optimal prediction methods based on a random forest and yielding maximum MCCs of approximately 77.55 % were constructed and may be useful tools for the prediction of compound-protein interactions. This work provides new clues to understanding the system of compound-protein interactions by analyzing extracted core features. Our results indicate that compound-protein interactions are related to biological processes involving immune, developmental and hormone-associated pathways.

NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes.

PubMed

Maslennikov, Innokentiy; Kefala, Georgia; Johnson, Casey; Riek, Roland; Choe, Senyon; Kwiatkowski, Witek

2007-11-08

Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required.

NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes

PubMed Central

Maslennikov, Innokentiy; Kefala, Georgia; Johnson, Casey; Riek, Roland; Choe, Senyon; Kwiatkowski, Witek

2007-01-01

Background Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Results Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. Conclusion The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required. PMID:17988403

Development of an Efficient Protein Extraction Method Compatible with LC-MS/MS for Proteome Mapping in Two Australian Seagrasses Zostera muelleri and Posidonia australis

PubMed Central

Jiang, Zhijian; Kumar, Manoj; Padula, Matthew P.; Pernice, Mathieu; Kahlke, Tim; Kim, Mikael; Ralph, Peter J.

2017-01-01

The availability of the first complete genome sequence of the marine flowering plant Zostera marina (commonly known as seagrass) in early 2016, is expected to significantly raise the impact of seagrass proteomics. Seagrasses are marine ecosystem engineers that are currently declining worldwide at an alarming rate due to both natural and anthropogenic disturbances. Seagrasses (especially species of the genus Zostera) are compromised for proteomic studies primarily due to the lack of efficient protein extraction methods because of their recalcitrant cell wall which is rich in complex polysaccharides and a high abundance of secondary metabolites in their cells. In the present study, three protein extraction methods that are commonly used in plant proteomics i.e., phenol (P); trichloroacetic acid/acetone/SDS/phenol (TASP); and borax/polyvinyl-polypyrrolidone/phenol (BPP) extraction, were evaluated quantitatively and qualitatively based on two dimensional isoelectric focusing (2D-IEF) maps and LC-MS/MS analysis using the two most abundant Australian seagrass species, namely Zostera muelleri and Posidonia australis. All three tested methods produced high quality protein extracts with excellent 2D-IEF maps in P. australis. However, the BPP method produces better results in Z. muelleri compared to TASP and P. Therefore, we further modified the BPP method (M-BPP) by homogenizing the tissue in a modified protein extraction buffer containing both ionic and non-ionic detergents (0.5% SDS; 1.5% Triton X-100), 2% PVPP and protease inhibitors. Further, the extracted proteins were solubilized in 0.5% of zwitterionic detergent (C7BzO) instead of 4% CHAPS. This slight modification to the BPP method resulted in a higher protein yield, and good quality 2-DE maps with a higher number of protein spots in both the tested seagrasses. Further, the M-BPP method was successfully utilized in western-blot analysis of phosphoenolpyruvate carboxylase (PEPC—a key enzyme for carbon metabolism). This optimized protein extraction method will be a significant stride toward seagrass proteome mining and identifying the protein biomarkers to stress response of seagrasses under the scenario of global climate change and anthropogenic perturbations. PMID:28861098

Development of an Efficient Protein Extraction Method Compatible with LC-MS/MS for Proteome Mapping in Two Australian Seagrasses Zostera muelleri and Posidonia australis.

PubMed

Jiang, Zhijian; Kumar, Manoj; Padula, Matthew P; Pernice, Mathieu; Kahlke, Tim; Kim, Mikael; Ralph, Peter J

2017-01-01

The availability of the first complete genome sequence of the marine flowering plant Zostera marina (commonly known as seagrass) in early 2016, is expected to significantly raise the impact of seagrass proteomics. Seagrasses are marine ecosystem engineers that are currently declining worldwide at an alarming rate due to both natural and anthropogenic disturbances. Seagrasses (especially species of the genus Zostera ) are compromised for proteomic studies primarily due to the lack of efficient protein extraction methods because of their recalcitrant cell wall which is rich in complex polysaccharides and a high abundance of secondary metabolites in their cells. In the present study, three protein extraction methods that are commonly used in plant proteomics i.e., phenol (P); trichloroacetic acid/acetone/SDS/phenol (TASP); and borax/polyvinyl-polypyrrolidone/phenol (BPP) extraction, were evaluated quantitatively and qualitatively based on two dimensional isoelectric focusing (2D-IEF) maps and LC-MS/MS analysis using the two most abundant Australian seagrass species, namely Zostera muelleri and Posidonia australis . All three tested methods produced high quality protein extracts with excellent 2D-IEF maps in P. australis . However, the BPP method produces better results in Z. muelleri compared to TASP and P. Therefore, we further modified the BPP method (M-BPP) by homogenizing the tissue in a modified protein extraction buffer containing both ionic and non-ionic detergents (0.5% SDS; 1.5% Triton X-100), 2% PVPP and protease inhibitors. Further, the extracted proteins were solubilized in 0.5% of zwitterionic detergent (C7BzO) instead of 4% CHAPS. This slight modification to the BPP method resulted in a higher protein yield, and good quality 2-DE maps with a higher number of protein spots in both the tested seagrasses. Further, the M-BPP method was successfully utilized in western-blot analysis of phosphoenolpyruvate carboxylase (PEPC-a key enzyme for carbon metabolism). This optimized protein extraction method will be a significant stride toward seagrass proteome mining and identifying the protein biomarkers to stress response of seagrasses under the scenario of global climate change and anthropogenic perturbations.

Optimization of extraction and antioxidant activity of polysaccharides from Salvia miltiorrhiza Bunge residue.

PubMed

Jiang, Yuanyuan; Wang, Long; Zhang, Li; Wang, Tao; Zhou, Yonghong; Ding, Chunbang; Yang, Ruiwu; Wang, Xiaoli; Yu, Lin

2015-08-01

In this study, the process of extracting polysaccharides from Salvia miltiorrhiza Bunge residue was optimized by using a Box-Behnken design. Statistical analysis of the results showed that the linear and quadratic terms of the three variables of the extraction process had significant effects. The optimal conditions are as follows: extracting time of 2.6 h, extraction temperature of 89 °C, and ratio of water to raw material of 32 mL/g. Moreover, a new polysaccharide with antioxidant activity [i.e., SMWP-1 (∼5.27×10(5) Da)] was isolated from S. miltiorrhiza residue. The carbohydrate, uronic acid, and protein contents of SMWP-1 were 90.11%, 0.13%, and 0.53%, respectively. The SMWP-1 is composed of glucose, xylose, mannose, and galactose. The preliminary structural characterization of SMWP-1 was determined via Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM) analyses. This polysaccharide exhibited strong reducing power and free-radical scavenging activities in vitro against 2,2-diphenyl-1-picrylhydrazyl, superoxide anion, and hydroxyl. Therefore, SMWP-1 can be investigated further as a novel natural antioxidant. Copyright © 2015 Elsevier B.V. All rights reserved.

Optimization of the trienzyme extraction for the microbiological assay of folate in vegetables.

PubMed

Chen, Liwen; Eitenmiller, Ronald R

2007-05-16

Response surface methodology was applied to optimize the trienzyme digestion for the extraction of folate from vegetables. Trienzyme extraction is a combined enzymatic digestion by protease, alpha-amylase, and conjugase (gamma-glutamyl hydrolase) to liberate the carbohydrate and protein-bound folates from food matrices for total folate analysis. It is the extraction method used in AOAC Official Method 2004.05 for assay of total folate in cereal grain products. Certified reference material (CRM) 485 mixed vegetables was used to represent the matrix of vegetables. Regression and ridge analysis were performed by statistical analysis software. The predicted second-order polynomial model was adequate (R2 = 0.947), without significant lack of fit (p > 0.1). Both protease and alpha-amylase have significant effects on the extraction. Ridge analysis gave an optimum trienzyme digestion time: Pronase, 1.5 h; alpha-amylase, 1.5 h; and conjugase, 3 h. The experimental value for CRM 485 under this optimum digestion was close to the predicted value from the model, confirming the validity and adequacy of the model. The optimized trienzyme digestion condition was applied to five vegetables and yielded higher folate levels than the trienzyme digestion parameters employed in AOAC Official Method 2004.05.

Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

PubMed

Agardh, Elisabet; Gustavsson, Carin; Hagert, Per; Nilsson, Marie; Agardh, Carl-David

2006-02-01

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

Profilin Is Required for Optimal Actin-Dependent Transcription of Respiratory Syncytial Virus Genome RNA

PubMed Central

Burke, Emily; Mahoney, Nicole M.; Almo, Steven C.; Barik, Sailen

2000-01-01

Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-l-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-l-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-l-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome. PMID:10623728

Box-Behnken design for extraction optimization of crude polysaccharides from Tunisian Phormidium versicolor cyanobacteria (NCC 466): Partial characterization, in vitro antioxidant and antimicrobial activities.

PubMed

Belhaj, Dalel; Frikha, Donyez; Athmouni, Khaled; Jerbi, Bouthaina; Ahmed, Mohammad Boshir; Bouallagui, Zouhaier; Kallel, Monem; Maalej, Sami; Zhou, John; Ayadi, Habib

2017-12-01

In this study, response surface methodology (RSM) based on Box-Behnken design (BBD) was employed to optimize the aqueous extraction of crude polysaccharides from Tunisian cyanobacteria Phormidium versicolor (NCC 466). The optimal extraction conditions with an extraction yield of 21.56±0.92% were as follows: extraction temperature at 81.05°C, extraction time of 3.99h, and water to raw material ratio of 21.52mLg -1 . Crude Phormidium versicolor polysaccharides (CPv-PS) are found to be a hetero-sulfated-anionic polysaccharides that contained carbohydrate (79.37±1.58%), protein (0.45±0.11%), uronic acids (4.37±0.19%) and sulfate (6.83±0.28%). The carbohydrate fraction was composed of arabinose, xylose, ribose, rhamnose, N-acetyl glucosamine, galactose, glucose, mannose, glucuronic acid and saccharose with corresponding mole percentages of 2.41, 14.58, 2.18, 6.23, 7.04, 28.21, 26.04, 3.02, 0.86 and 5.07, respectively. Evaluation of the antioxidant activity in vitro suggested that CPv-PS strongly scavenged radicals, prevented bleaching of β-carotene and reduced activity. Furthermore, the CPv-PS exhibited effective antimicrobial properties. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Diagnosis and Prevention of Infection by Phlebotomus Fever Group Viruses

DTIC Science & Technology

1991-05-03

proficient, but non- optimal expression vectors [e.g., pAcRP6, see below]. The two types of recombinant viruses were used to infect Spodoptera ... frugiperda cells and the expressed PT viral proteins characterised (Overton et al., 1987). Recombinant AcNPV having the S DNA in one orientation expressed...proteins virus were raised in mice using the corresponding S. frugiperda infected cell extracts and were employed to identify N and NSs proteins in PT virus

Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qian, Chen; Hettich, Robert L.

The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient cycling, perhaps most specifically with respect to impact on plant growth but also more broadly to global impact on carbon and nitrogen-cycling. Liquid chromatography coupled to high performance mass spectrometry provides a powerful approach to characterize soil microbiomes; however, the limited microbial biomass and the presence of abundant interferences in soil samples present major challenges to soil proteome extraction and subsequent MS measurement. To address some of the major issues, we have designed and optimized an experimental method to enhance microbialmore » proteome extraction concomitant with minimizing the soil-borne humic substances co-extraction from soils. Among the range of interferences, humic substances are often the worst in terms of adversely impacting proteome extraction and mass spectrometry measurement. Our approach employs an in-situ detergent-based microbial lysis / TCA precipitation coupled with an additional acidification precipitation step at the peptide level which efficiently removes humic acids. By combing filtration and pH adjustment of the final peptide solution, the remaining humic acids can be differentially precipitated and removed with a membrane filter, thereby leaving much cleaner proteolytic peptide samples for MS measurement. As a result, this modified method is a reliable and straight-forward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or reducing or biasing protein identification in mass spectrometry.« less

Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements

DOE PAGES

Qian, Chen; Hettich, Robert L.

2017-05-24

The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient cycling, perhaps most specifically with respect to impact on plant growth but also more broadly to global impact on carbon and nitrogen-cycling. Liquid chromatography coupled to high performance mass spectrometry provides a powerful approach to characterize soil microbiomes; however, the limited microbial biomass and the presence of abundant interferences in soil samples present major challenges to soil proteome extraction and subsequent MS measurement. To address some of the major issues, we have designed and optimized an experimental method to enhance microbialmore » proteome extraction concomitant with minimizing the soil-borne humic substances co-extraction from soils. Among the range of interferences, humic substances are often the worst in terms of adversely impacting proteome extraction and mass spectrometry measurement. Our approach employs an in-situ detergent-based microbial lysis / TCA precipitation coupled with an additional acidification precipitation step at the peptide level which efficiently removes humic acids. By combing filtration and pH adjustment of the final peptide solution, the remaining humic acids can be differentially precipitated and removed with a membrane filter, thereby leaving much cleaner proteolytic peptide samples for MS measurement. As a result, this modified method is a reliable and straight-forward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or reducing or biasing protein identification in mass spectrometry.« less

Effects of heat treatment on the antioxidative and anti-inflammatory properties of orange by-products.

PubMed

Li, Chien-Chun; Hsu, Hui-Jin; Wang, Yu-Shiang; Cassidy, Jennifer; Sheen, Shiowshuh; Liu, Shih-Chuan

2017-07-19

This study investigated the changes in the functional components, antioxidative activities, antibacterial activities, anti-inflammatory activities of orange (Citrus sinensis (L.) Osbeck) by-products (OBP) on heat treatment at 50 and 100 °C (hereafter denoted 50D and 100D extracts, respectively). Optimal heating conditions were also investigated. The total phenolic content, flavonoid content and antioxidative activities of OBP extracts significantly increased on heat treatment. The lag time of Cu 2+ -induced oxidation of human LDL was increased by 2.61, 8.61 and 8.76-fold with the addition of 0.6, 0.8 and 1.0 mg ml -1 100D extracts, respectively. The 100D extracts may significantly inhibit the growth of E. coli O157, Salmonella typhimurium and Listeria monocytogenes. 1 μg mL -1 of 100D extract may suppress the TNF-α-induced ICAM-1 protein expression. The optimal heating time for OBP was 26 h at 100 °C, which resulted in the highest antioxidant activities.

Oleosins (24 and 18 kDa) are hydrolyzed not only in extracted soybean oil bodies but also in soybean germination.

PubMed

Chen, Yeming; Zhao, Luping; Cao, Yanyun; Kong, Xiangzhen; Hua, Yufei

2014-01-29

After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, β-conglycinin, γ-conglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35-50 °C and pH 6.0-6.5, while 60 °C or pH 11.0 could denature them.

Novel Displacement Agents for Aqueous 2-Phase Extraction Can Be Estimated Based on Hybrid Shortcut Calculations.

PubMed

Kress, Christian; Sadowski, Gabriele; Brandenbusch, Christoph

2016-10-01

The purification of therapeutic proteins is a challenging task with immediate need for optimization. Besides other techniques, aqueous 2-phase extraction (ATPE) of proteins has been shown to be a promising alternative to cost-intensive state-of-the-art chromatographic protein purification. Most likely, to enable a selective extraction, protein partitioning has to be influenced using a displacement agent to isolate the target protein from the impurities. In this work, a new displacement agent (lithium bromide [LiBr]) allowing for the selective separation of the target protein IgG from human serum albumin (represents the impurity) within a citrate-polyethylene glycol (PEG) ATPS is presented. In order to characterize the displacement suitability of LiBr on IgG, the mutual influence of LiBr and the phase formers on the aqueous 2-phase system (ATPS) and partitioning is investigated. Using osmotic virial coefficients (B22 and B23) accessible by composition gradient multiangle light-scattering measurements, the precipitating effect of LiBr on both proteins and an estimation of both protein partition coefficients is estimated. The stabilizing effect of LiBr on both proteins was estimated based on B22 and experimentally validated within the citrate-PEG ATPS. Our approach contributes to an efficient implementation of ATPE within the downstream processing development of therapeutic proteins. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Automated, high-throughput platform for protein solubility screening using a split-GFP system

PubMed Central

Listwan, Pawel; Terwilliger, Thomas C.

2010-01-01

Overproduction of soluble and stable proteins for functional and structural studies is a major bottleneck for structural genomics programs and traditional biochemistry laboratories. Many high-payoff proteins that are important in various biological processes are “difficult to handle” as protein reagents in their native form. We have recently made several advances in enabling biochemical technologies for improving protein stability (http://www.lanl.gov/projects/gfp/), allowing stratagems for efficient protein domain trapping, solubility-improving mutations, and finding protein folding partners. In particular split-GFP protein tags are a very powerful tool for detection of stable protein domains. Soluble, stable proteins tagged with the 15 amino acid GFP fragment (amino acids 216–228) can be detected in vivo and in vitro using the engineered GFP 1–10 “detector” fragment (amino acids 1–215). If the small tag is accessible, the detector fragment spontaneously binds resulting in fluorescence. Here, we describe our current and on-going efforts to move this process from the bench (manual sample manipulation) to an automated, high-throughput, liquid-handling platform. We discuss optimization and validation of bacterial culture growth, lysis protocols, protein extraction, and assays of soluble and insoluble protein in multiple 96 well plate format. The optimized liquid-handling protocol can be used for rapid determination of the optimal, compact domains from single ORFS, collections of ORFS, or cDNA libraries. PMID:19039681

Ranking support vector machine for multiple kernels output combination in protein-protein interaction extraction from biomedical literature.

PubMed

Yang, Zhihao; Lin, Yuan; Wu, Jiajin; Tang, Nan; Lin, Hongfei; Li, Yanpeng

2011-10-01

Knowledge about protein-protein interactions (PPIs) unveils the molecular mechanisms of biological processes. However, the volume and content of published biomedical literature on protein interactions is expanding rapidly, making it increasingly difficult for interaction database curators to detect and curate protein interaction information manually. We present a multiple kernel learning-based approach for automatic PPI extraction from biomedical literature. The approach combines the following kernels: feature-based, tree, and graph and combines their output with Ranking support vector machine (SVM). Experimental evaluations show that the features in individual kernels are complementary and the kernel combined with Ranking SVM achieves better performance than those of the individual kernels, equal weight combination and optimal weight combination. Our approach can achieve state-of-the-art performance with respect to the comparable evaluations, with 64.88% F-score and 88.02% AUC on the AImed corpus. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Subcritical Water Technology for Enhanced Extraction of Biochemical Compounds from Chlorella vulgaris

PubMed Central

Awaluddin, S. A.; Thiruvenkadam, Selvakumar; Izhar, Shamsul; Hiroyuki, Yoshida; Danquah, Michael K.; Harun, Razif

2016-01-01

Subcritical water extraction (SWE) technology has been used for the extraction of active compounds from different biomass materials with low process cost, mild operating conditions, short process times, and environmental sustainability. With the limited application of the technology to microalgal biomass, this work investigates parametrically the potential of subcritical water for high-yield extraction of biochemicals such as carbohydrates and proteins from microalgal biomass. The SWE process was optimized using central composite design (CCD) under varying process conditions of temperature (180–374°C), extraction time (1–20 min), biomass particulate size (38–250 μm), and microalgal biomass loading (5–40 wt.%). Chlorella vulgaris used in this study shows high volatile matter (83.5 wt.%) and carbon content (47.11 wt.%), giving advantage as a feedstock for biofuel production. The results showed maximum total carbohydrate content and protein yields of 14.2 g/100 g and 31.2 g/100 g, respectively, achieved under the process conditions of 277°C, 5% of microalgal biomass loading, and 5 min extraction time. Statistical analysis revealed that, of all the parameters investigated, temperature is the most critical during SWE of microalgal biomass for protein and carbohydrate production. PMID:27366748

Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis

PubMed Central

Field, Christine M.; Pelletier, James F.; Mitchison, Timothy J.

2017-01-01

We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen, and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant ~11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization. It can be spread as thin layers, and naturally depletes oxygen though mitochondrial metabolism, which makes it ideal for fluorescence imaging. When combined with artificial lipid bilayers, it allows reconstitution and analysis of the spatially controlled signaling that positions the cleavage furrow during early cytokinesis. Actin-intact extract is generally useful for probing the biochemistry and biophysics of the large Xenopus egg. Protocols are provided for preparation of actin-intact egg extract, control of the cell cycle, fluorescent probes for cytoskeleton and cytoskeleton-dependent signaling, preparation of glass surfaces for imaging experiments, and immunodepletion to probe the role of specific proteins and protein complexes. We also describe methods for adding supported lipid bilayers to mimic the plasma membrane and for confining in microfluidic droplets to explore size scaling issues. PMID:28065319

Maca polysaccharides: Extraction optimization, structural features and anti-fatigue activities.

PubMed

Li, Yujuan; Xin, Yizhou; Xu, Fangxue; Zheng, Mengmeng; Xi, Xiaozhi; Cui, Xiaowei; Cao, Hui; Guo, Hong; Han, Chunchao

2018-08-01

The maca polysaccharides optimal extraction conditions were obtained by using response surface methodology (RSM) method and the anti-fatigue activity of maca polysaccharides (MCP) was explored. The maca polysaccharides extract yield of RSM could reach 9.97 mg/g by using the model predicts, and the total sugar and protein purity were 61.00% and 4.46% with the further isolation process, respectively. And the monosaccharide compositions obtained by gas chromatograph (GC) were composed of rhamnose (rha), glucose (glc), galactose (gal) with the ratio of 2.34:10.21:1.00. Furthermore, the anti-fatigue activity was evaluated by the swimming parameter, biochemistry parameters (liver glycogen (LG), blood urea nitrogen (BUN), and lactic acid (LD)), the result indicated that the low-dose maca polysaccharides group had the significant anti-fatigue activity. Copyright © 2018 Elsevier B.V. All rights reserved.

Mass spectrometry compatible surfactant for optimized in-gel protein digestion.

PubMed

Saveliev, Sergei V; Woodroofe, Carolyn C; Sabat, Grzegorz; Adams, Christopher M; Klaubert, Dieter; Wood, Keith; Urh, Marjeta

2013-01-15

Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5-2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20-30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment.

Affinity monolith-integrated poly(methyl methacrylate) microchips for on-line protein extraction and capillary electrophoresis.

PubMed

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T

2008-07-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with eight reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in a porogenic solvent consisting of 70% 1-dodecanol and 30% cyclohexanol. Antifluorescein isothiocyanate was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of fluorescein isothiocyanate (FITC)-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis.

Affinity Monolith-Integrated Poly(methyl Methacrylate) Microchips for On-Line Protein Extraction and Capillary Electrophoresis

PubMed Central

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T.

2008-01-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with 8 reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene dimethacrylate in a porogenic solvent consisting of 70% dodecanol and 30% hexanol. Anti-fluorescein isothiocyanate (FITC) was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of FITC-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis. PMID:18479142

Optimisation of low temperature extraction of banana juice using commercial pectinase.

PubMed

Sagu, Sorel Tchewonpi; Nso, Emmanuel Jong; Karmakar, Sankha; De, Sirshendu

2014-05-15

The objective of this work was to develop a process with optimum conditions for banana juice. The procedure involves hydrolyzing the banana pulp by commercial pectinase followed by cloth filtration. Response surface methodology with Doehlert design was utilised to optimize the process parameters. The temperature of incubation (30-60 °C), time of reaction (20-120 min) and concentration of pectinase (0.01-0.05% v/w) were the independent variables and viscosity, clarity, alcohol insoluble solids (AIS), total polyphenol and protein concentration were the responses. Total soluble sugar, pH, conductivity, calcium, sodium and potassium concentration in the juice were also evaluated. The results showed reduction of AIS and viscosity with reaction time and pectinase concentration and reduction of polyphenol and protein concentration with temperature. Using numerical optimization, the optimum conditions for the enzymatic extraction of banana juice were estimated. Depectinization kinetics was also studied at optimum temperature and variation of kinetic constants with enzyme dose was evaluated. Copyright © 2013 Elsevier Ltd. All rights reserved.

Microwave-assisted extraction of cyclotides from Viola ignobilis.

PubMed

Farhadpour, Mohsen; Hashempour, Hossein; Talebpour, Zahra; A-Bagheri, Nazanin; Shushtarian, Mozhgan Sadat; Gruber, Christian W; Ghassempour, Alireza

2016-03-15

Cyclotides are an interesting family of circular plant peptides. Their unique three-dimensional structure, comprising a head-to-tail circular backbone chain and three disulfide bonds, confers them stability against thermal, chemical, and enzymatic degradation. Their unique stability under extreme conditions creates an idea about the possibility of using harsh extraction methods such as microwave-assisted extraction (MAE) without affecting their structures. MAE has been introduced as a potent extraction method for extraction of natural compounds, but it is seldom used for peptide and protein extraction. In this work, microwave irradiation was applied to the extraction of cyclotides. The procedure was performed in various steps using a microwave instrument under different conditions. High-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) results show stability of cyclotide structures on microwave radiation. The influential parameters, including time, temperature, and the ratio of solvents that are affecting the MAE potency, were optimized. Optimal conditions were obtained at 20 min of irradiation time, 1200 W of system power in 60 °C, and methanol/water at the ratio of 90:10 (v/v) as solvent. The comparison of MAE results with maceration extraction shows that there are similarities between cyclotide sequences and extraction yields. Copyright © 2015 Elsevier Inc. All rights reserved.

[Establishment of optimal conditions at laboratory and pilot plant levels for the preparation of a protein isolated from Lupinus mutabilis].

PubMed

Rodríguez Pacheco, T; Aliaga, T; Schoeneberger, H; Gross, R

1981-12-01

Laboratory conditions were first investigated to determine are optimum processing parameters for the preparation of a protein isolate from the ground, defatted, commercial flakes of Lupinus mutabilis. The extraction variables were: pH (2-10); solvent to lupine ratio (5:1 to 40:1); temperature (28 degrees C - 60 degrees C) and time (10-50 min). The isoelectric point of the lupine protein was found to be pH 4.5 with a protein solubility higher than 80% above pH 8.0. Using 70-100 mesh, ground defatted flakes, and extracting at pH 8.7 for 60 min, a protein isolate was obtained on acidification to pH 4.5 which was 99.8 protein (dry basis), compared to 55.25% protein for the original material. This protein isolate represented 32% of the initial material and 57.6% of the initial nitrogen. When making pilot plant assays we found that the yield of protein isolate decreased to 20.4% of the original material and 36.4% of the initial nitrogen. The protein efficiency ratio for the protein isolate was 2.15 when supplemented with methionine, and had a digestibility of 89.33

[Biotechnological optimization of nutrient composition of fermented dairy drink].

PubMed

Donskaya, G A

2014-01-01

The receipt based on the results of carried out studies is substantiated and technology of the new fermented dairy drink containing whole milk and whey with inulin (Jerusalem artichoke extract) and optimizing initial mineral composition of raw material has been developed. The starters ascertaining optimal organoleptic properties of the drink have been selected. It has been established that Jerusalem artichoke and its derivatives in the form of syrups and extracts stimulate fermentative processes of technological microflora, with maximum activity observed with Jerusalem artichoke extract. Physical-chemical and microbiological characteristics of the drink have been defined during storage. The possibility to optimize the nutrient composition of fermented dairy product by means of introducing of Jerusalem artichoke extract into milk-protein base has been demonstrated. It has been calculated that consumption of 100 g of fermented dairy drink enriched with Jerusalem artichoke extract makes it possible to satisfy the physiological needs (recommended daily allowance--RDA) for babies from 0 to 3 months in vitamins B1, B2 and B6 by 25-35% and in minerals P, K, and Ca by 20, 68, 34, 26%. For adults receiving 250 g of fermented beverage meets RDA for vitamins B1, B2 and B6 by 10-19% and in the macronutrients P, K, Ca-by 25-35%. Designed fermented dairy drink supplemented with natural plant ingredient possesses increased antioxidant activity and may be recommended for mass consumption without any limitations.

Optimization of protein electroextraction from microalgae by a flow process.

PubMed

Coustets, Mathilde; Joubert-Durigneux, Vanessa; Hérault, Josiane; Schoefs, Benoît; Blanckaert, Vincent; Garnier, Jean-Pierre; Teissié, Justin

2015-06-01

Classical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2 ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size. Copyright © 2014 Elsevier B.V. All rights reserved.

Spider Silk Processing for Spidroin Recovery from Crossopriza Lyoni Web

NASA Astrophysics Data System (ADS)

Mohtar, J. A.; Ooi, W. L.; Yusuf, F.

2018-03-01

Spider silk is a potential biomaterial that can be used in various applications for its outstanding physicomechanical properties attributed by the spidroin composition. Efforts for commercializing spider silks have been mainly focused on the characterization of spidroins from the Entelegyne spiders for exceptional fibre construction. Hence, studies on silk proteins from the Haplogyne species remain neglected. The aim of this study is to isolate spidroin from Crossopriza lyoni web. Silk processing involved the pretreatment of fibres for the shell layer removal from the surface. A screening study was conducted to analyze the effect of temperature, incubation time and agitation speed on spidroin extraction using Ajisawa’s reagent by OFAT analysis followed by statistical optimization of the extraction process via RSM for maximal protein recovery. All parameters exerted significant effect on spidroin recovery (p<0.05) in which the maximum protein concentration (451.78 ± 0.110 µg/ml) was obtained at optimal condition of 70°C, 350 rpm and 1.25 hours. The discovery of spidroin from this study provides a basic platform for engineering spider silk to meet the demand for a variety of silk-based products in the near future.

Expression and Characterization of Acidothermus celluloyticus E1 Endoglucanase in Transgenic Duckweed Lemna minor 8627

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Y.; Cheng, J. J.; Himmel, M. E.

2007-01-01

Endoglucanase E1 from Acidothermus cellulolyticus was expressed cytosolically under control of the cauliflower mosaic virus 35S promoter in transgenic duckweed, Lemna minor 8627 without any obvious observable phenotypic effects on morphology or rate of growth. The recombinant enzyme co-migrated with the purified catalytic domain fraction of the native E1 protein on western blot analysis, revealing that the cellulose-binding domain was cleaved near or in the linker region. The duckweed-expressed enzyme was biologically active and the expression level was up to 0.24% of total soluble protein. The endoglucanase activity with carboxymethylcellulose averaged 0.2 units mg protein{sup -1} extracted from fresh duckweed.more » The optimal temperature and pH for E1 enzyme activity were about 80 C and pH 5, respectively. While extraction with HEPES (N-[2-hydroxyethyl]piperazine-N{prime}-[2-ethanesulfonic acid]) buffer (pH 8) resulted in the highest recovery of total soluble proteins and E1 enzyme, extraction with citrate buffer (pH 4.8) at 65 C enriched relative amounts of E1 enzyme in the extract. This study demonstrates that duckweed may offer new options for the expression of cellulolytic enzymes in transgenic plants.« less

Reproducible Tissue Homogenization and Protein Extraction for Quantitative Proteomics Using MicroPestle-Assisted Pressure-Cycling Technology.

PubMed

Shao, Shiying; Guo, Tiannan; Gross, Vera; Lazarev, Alexander; Koh, Ching Chiek; Gillessen, Silke; Joerger, Markus; Jochum, Wolfram; Aebersold, Ruedi

2016-06-03

The reproducible and efficient extraction of proteins from biopsy samples for quantitative analysis is a critical step in biomarker and translational research. Recently, we described a method consisting of pressure-cycling technology (PCT) and sequential windowed acquisition of all theoretical fragment ions-mass spectrometry (SWATH-MS) for the rapid quantification of thousands of proteins from biopsy-size tissue samples. As an improvement of the method, we have incorporated the PCT-MicroPestle into the PCT-SWATH workflow. The PCT-MicroPestle is a novel, miniaturized, disposable mechanical tissue homogenizer that fits directly into the microTube sample container. We optimized the pressure-cycling conditions for tissue lysis with the PCT-MicroPestle and benchmarked the performance of the system against the conventional PCT-MicroCap method using mouse liver, heart, brain, and human kidney tissues as test samples. The data indicate that the digestion of the PCT-MicroPestle-extracted proteins yielded 20-40% more MS-ready peptide mass from all tissues tested with a comparable reproducibility when compared to the conventional PCT method. Subsequent SWATH-MS analysis identified a higher number of biologically informative proteins from a given sample. In conclusion, we have developed a new device that can be seamlessly integrated into the PCT-SWATH workflow, leading to increased sample throughput and improved reproducibility at both the protein extraction and proteomic analysis levels when applied to the quantitative proteomic analysis of biopsy-level samples.

Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts

PubMed Central

Ahn, Jin-Ho; Hwang, Mi-Yeon; Lee, Kyung-Ho; Choi, Cha-Yong; Kim, Dong-Myung

2007-01-01

This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesized protein was enhanced by as much as 10 times in the presence of an optimal second codon. The signal peptides were in situ cleaved and the target proteins were produced in their native sizes by carrying out the cell-free synthesis reactions in the presence of Triton X-100, most likely through the activation of signal peptidase in the S30 extract. The amplification of the template DNA and the addition of the signal sequences were accomplished by PCR. Hence, elevated levels of recombinant proteins were generated within several hours. PMID:17185295

Pectin from Opuntia ficus indica: Optimization of microwave-assisted extraction and preliminary characterization.

PubMed

Lefsih, Khalef; Giacomazza, Daniela; Dahmoune, Farid; Mangione, Maria Rosalia; Bulone, Donatella; San Biagio, Pier Luigi; Passantino, Rosa; Costa, Maria Assunta; Guarrasi, Valeria; Madani, Khodir

2017-04-15

Optimization of microwave-assisted extraction (MAE) of water-soluble pectin (WSP) from Opuntia ficus indica cladodes was performed using Response Surface Methodology. The effect of extraction time (X 1 ), microwave power (X 2 ), pH (X 3 ) and solid-to-liquid ratio (X 4 ) on the extraction yield was examined. The optimum conditions of MAE were as follows: X 1 =2.15min; X 2 =517W; X 3 =2.26 and X 4 =2g/30.6mL. The maximum obtained yield of pectin extraction was 12.57%. Total carbohydrate content of WSP is about 95.5% including 34.4% of Galacturonic acid. Pectin-related proteins represent only the 0.66% of WSP mass. HPSEC and light scattering analyses reveal that WSP is mostly constituted of high molecular pectin and FTIR measurements show that the microwave treatment does not alter the chemical structure of WSP, in which Galacturonic acid content and yield are 34.4% and 4.33%, respectively. Overall, application of MAE can give rise to high quality pectin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Blending protein separation and peptide analysis through real-time proteolytic digestion.

PubMed

Slysz, Gordon W; Schriemer, David C

2005-03-15

Typical liquid- or gel-based protein separations require enzymatic digestion as an important first step in generating protein identifications. Traditional protocols involve long-term proteolytic digestion of the separated protein, often leading to sample loss and reduced sensitivity. Previously, we presented a rapid method of proteolytic digestion that showed excellent digestion of resistant and low concentrations of protein without requiring reduction and alkylation. Here, we demonstrate on-line, real-time tryptic digestion in conjunction with reversed-phase protein separation. The studies were aimed at optimizing pH and ionic strength and the size of the digestion element, to produce maximal protein digestion with minimal effects on chromatographic integrity. Upon establishing optimal conditions, the digestion element was attached downstream from a capillary C4 reversed-phase column. A four-protein mixture was processed through the combined system, and the resulting peptides were analyzed on-line by electrospray mass spectrometry. Extracted ion chromatograms for protein chromatography based on peptide elution were generated. These were shown to emulate ion chromatograms produced in a subsequent run without the digestion element, based on protein elution. The methodology will enable rapid and sensitive analysis of liquid-based protein separations using the power of bottom-up proteomics methodologies.

Optimization of ELISA Conditions to Quantify Colorectal Cancer Antigen-Antibody Complex Protein (GA733-FcK) Expressed in Transgenic Plant

PubMed Central

Ahn, Junsik; Lee, Kyung Jin

2014-01-01

The purpose of this study is to optimize ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment fused to KDEL, an ER retention motif (GA733-FcK) expressed in transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from transgenic plant expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG recognizing the GA733 did not detect any GA733-FcK whereas anti-human Fc IgG recognizing the human Fc existed in plant leaf extracts. For blocking buffer conditions, 3% BSA buffer clearly blocked the plate, compared to the 5% skim-milk buffer. For capture antibody, monoclonal antibody (MAb) CO17-1A was applied to coat the plate with different amounts (1, 0.5, and 0.25 μg/well). Among the amounts of the capture antibody, 1 and 0.5 μg/well (capture antibody) showed similar absorbance, whereas 0.25 μg/well of the capture antibody showed significantly less absorbance. Taken together, the optimized conditions to quantify plant-derived GA733-FcK were 0.5 μg/well of MAb CO17-1A per well for the capture antibody, 3% BSA for blocking buffer, and anti-human Fc conjugated HRP. To confirm the optimized ELISA conditions, correlation analysis was conducted between the quantified amount of GA733-FcK in ELISA and its protein density values of different leaf samples in Western blot. The co-efficient value R2 between the ELISA quantified value and protein density was 0.85 (p<0.01), which indicates that the optimized ELISA conditions feasibly provides quantitative information of GA733-FcK expression in transgenic plant. PMID:24555929

Proteomic profiling of an undefined microbial consortium cultured in fermented dairy manure: Methods development.

PubMed

Hanson, Andrea J; Paszczynski, Andrzej J; Coats, Erik R

2016-03-01

The production of polyhydroxyalkanoates (PHA; bioplastics) from waste or surplus feedstocks using mixed microbial consortia (MMC) and aerobic dynamic feeding (ADF) is a growing field within mixed culture biotechnology. This study aimed to optimize a 2DE workflow to investigate the proteome dynamics of an MMC synthesizing PHA from fermented dairy manure. To mitigate the challenges posed to effective 2DE by this complex sample matrix, the bacterial biomass was purified using Accudenz gradient centrifugation (AGC) before protein extraction. The optimized 2DE method yielded high-quality gels suitable for quantitative comparative analysis and subsequent protein identification by LC-MS/MS. The optimized 2DE method could be adapted to other proteomic investigations involving MMC in complex organic or environmental matrices. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of microextraction by packed sorbent, liquid-liquid microextraction and derivatization pretreatment of diet-derived phenolic acids in plasma by gas chromatography with triple quadrupole mass spectrometry.

PubMed

Bustamante, Luis; Cárdenas, Diana; von Baer, Dietrich; Pastene, Edgar; Duran-Sandoval, Daniel; Vergara, Carola; Mardones, Claudia

2017-09-01

Miniaturized sample pretreatments for the analysis of phenolic metabolites in plasma, involving protein precipitation, enzymatic deconjugation, extraction procedures, and different derivatization reactions were systematically evaluated. The analyses were conducted by gas chromatography with mass spectrometry for the evaluation of 40 diet-derived phenolic compounds. Enzyme purification was necessary for the phenolic deconjugation before extraction. Trimethylsilanization reagent and two different tetrabutylammonium salts for derivatization reactions were compared. The optimum reaction conditions were 50 μL of trimethylsilanization reagent at 90°C for 30 min, while tetrabutylammonium salts were associated with loss of sensitivity due to rapid activation of the inert gas chromatograph liner. Phenolic acids extractions from plasma were optimized. Optimal microextraction by packed sorbent performance was achieved using an octadecylsilyl packed bed and better recoveries for less polar compounds, such as methoxylated derivatives, were observed. Despite the low recovery for many analytes, repeatability using an automated extraction procedure in the gas chromatograph inlet was 2.5%. Instead, using liquid-liquid microextraction, better recoveries (80-110%) for all analytes were observed at the expense of repeatability (3.8-18.4%). The phenolic compounds in gerbil plasma samples, collected before and 4 h after the administration of a calafate extract, were analyzed with the optimized methodology. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Accurate prediction of subcellular location of apoptosis proteins combining Chou's PseAAC and PsePSSM based on wavelet denoising.

PubMed

Yu, Bin; Li, Shan; Qiu, Wen-Ying; Chen, Cheng; Chen, Rui-Xin; Wang, Lei; Wang, Ming-Hui; Zhang, Yan

2017-12-08

Apoptosis proteins subcellular localization information are very important for understanding the mechanism of programmed cell death and the development of drugs. The prediction of subcellular localization of an apoptosis protein is still a challenging task because the prediction of apoptosis proteins subcellular localization can help to understand their function and the role of metabolic processes. In this paper, we propose a novel method for protein subcellular localization prediction. Firstly, the features of the protein sequence are extracted by combining Chou's pseudo amino acid composition (PseAAC) and pseudo-position specific scoring matrix (PsePSSM), then the feature information of the extracted is denoised by two-dimensional (2-D) wavelet denoising. Finally, the optimal feature vectors are input to the SVM classifier to predict subcellular location of apoptosis proteins. Quite promising predictions are obtained using the jackknife test on three widely used datasets and compared with other state-of-the-art methods. The results indicate that the method proposed in this paper can remarkably improve the prediction accuracy of apoptosis protein subcellular localization, which will be a supplementary tool for future proteomics research.

Accurate prediction of subcellular location of apoptosis proteins combining Chou’s PseAAC and PsePSSM based on wavelet denoising

PubMed Central

Chen, Cheng; Chen, Rui-Xin; Wang, Lei; Wang, Ming-Hui; Zhang, Yan

2017-01-01

Apoptosis proteins subcellular localization information are very important for understanding the mechanism of programmed cell death and the development of drugs. The prediction of subcellular localization of an apoptosis protein is still a challenging task because the prediction of apoptosis proteins subcellular localization can help to understand their function and the role of metabolic processes. In this paper, we propose a novel method for protein subcellular localization prediction. Firstly, the features of the protein sequence are extracted by combining Chou's pseudo amino acid composition (PseAAC) and pseudo-position specific scoring matrix (PsePSSM), then the feature information of the extracted is denoised by two-dimensional (2-D) wavelet denoising. Finally, the optimal feature vectors are input to the SVM classifier to predict subcellular location of apoptosis proteins. Quite promising predictions are obtained using the jackknife test on three widely used datasets and compared with other state-of-the-art methods. The results indicate that the method proposed in this paper can remarkably improve the prediction accuracy of apoptosis protein subcellular localization, which will be a supplementary tool for future proteomics research. PMID:29296195

Bioactivities and extraction optimization of crude polysaccharides from the fruits and leaves of Rubus chingii Hu.

PubMed

Zhang, Tian-Tian; Lu, Chuan-Li; Jiang, Jian-Guo; Wang, Min; Wang, Dong-Mei; Zhu, Wei

2015-10-05

Polysaccharides of Rubus chingii Hu fruit and leaf were extracted to compare their antioxidant, anti-inflammatory, and anticancer activities against breast cancer cells MCF-7 and liver cancer cells Bel-7402. Results showed that all the tested bioactivities of polysaccharides from leaf (L-Ps) were better than those of polysaccharides from fruit (F-Ps). Response surface methodology was then used to optimize the extraction conditions of polysaccharides from leaf. Additionally, polysaccharides from fruit and leaf were characterized and their contents of total sugars, proteins and uronic acid were compared. It was found that polysaccharides from fruit and leaf were similar in IR and UV absorption, but significantly different in contents of total sugars, protein and uronic acid. Their elution profiles of DEAE-Sepharose fast flow column were different too. The main peak of polysaccharides from fruit was eluted with 0.3 mol/l NaCl solution and the main peak of polysaccharides from leaf was eluted with deionized water. The differences between the two polysaccharides may be responsible for their differences in bioactivities. Further studies are required to explore their complete structural characteristics, structure-activity relationship and the mechanism of their activities. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of temperature, pH and NaCl on protease activity in digestive tract of young turbot, Scophthalmus maximus

NASA Astrophysics Data System (ADS)

Chen, Muyan; Zhang, Xiumei; Gao, Tianxiang; Chen, Chao

2006-09-01

The protease activity in digestive tract of young turbot Scophthalmus maximum was studied, and the optimal pH, temperature and NaCl concentration were determined for different portions of the fish's internal organs. The optimal activity in the fish's stomach was at pH of 2.2, while that in the intestinal extracts was within the alkaline range from 9.5 to 10.0. In hepatopancreas, the optimal pH was in low alkalinity at 8.5. The optimal reaction temperature was above 40°C in stomach, intestine and hepatopancreas. With increasing temperature, the pH value increased in stomach, while in the intestine, an opposite tendency was observed due to combined effect of pH and temperature. NaCl concentration showed inhibitory impact on protein digestion in hepatopancreas. The main protease for protein digestion in turbot seemed to be pepsin. Moreover, the maximum protease activity in different segments of intestine existed in the hindgut.

Longevity extension of worker honey bees (Apis mellifera) by royal jelly: optimal dose and active ingredient

PubMed Central

Han, Mingfeng

2017-01-01

In the Western honey bee, Apis mellifera, queens and workers have different longevity although they share the same genome. Queens consume royal jelly (RJ) as the main food throughout their life, including as adults, but workers only eat worker jelly when they are larvae less than 3 days old. In order to explore the effect of RJ and the components affecting longevity of worker honey bees, we first determined the optimal dose for prolonging longevity of workers as 4% RJ in 50% sucrose solution, and developed a method of obtaining long lived workers. We then compared the effects of longevity extension by RJ 4% with bee-collected pollen from rapeseed (Brassica napus). Lastly, we determined that a water soluble RJ protein obtained by precipitation with 60% ammonium sulfate (RJP60) contained the main component for longevity extension after comparing the effects of RJ crude protein extract (RJCP), RJP30 (obtained by precipitation with 30% ammonium sulfate), and RJ ethanol extract (RJEE). Understanding what regulates worker longevity has potential to help increase colony productivity and improve crop pollination efficiency. PMID:28367370

Longevity extension of worker honey bees (Apis mellifera) by royal jelly: optimal dose and active ingredient.

PubMed

Yang, Wenchao; Tian, Yuanyuan; Han, Mingfeng; Miao, Xiaoqing

2017-01-01

In the Western honey bee, Apis mellifera , queens and workers have different longevity although they share the same genome. Queens consume royal jelly (RJ) as the main food throughout their life, including as adults, but workers only eat worker jelly when they are larvae less than 3 days old. In order to explore the effect of RJ and the components affecting longevity of worker honey bees, we first determined the optimal dose for prolonging longevity of workers as 4% RJ in 50% sucrose solution, and developed a method of obtaining long lived workers. We then compared the effects of longevity extension by RJ 4% with bee-collected pollen from rapeseed ( Brassica napus ). Lastly, we determined that a water soluble RJ protein obtained by precipitation with 60% ammonium sulfate (RJP 60 ) contained the main component for longevity extension after comparing the effects of RJ crude protein extract (RJCP), RJP 30 (obtained by precipitation with 30% ammonium sulfate), and RJ ethanol extract (RJEE). Understanding what regulates worker longevity has potential to help increase colony productivity and improve crop pollination efficiency.

A proteomics performance standard to support measurement quality in proteomics.

PubMed

Beasley-Green, Ashley; Bunk, David; Rudnick, Paul; Kilpatrick, Lisa; Phinney, Karen

2012-04-01

The emergence of MS-based proteomic platforms as a prominent technology utilized in biochemical and biomedical research has increased the need for high-quality MS measurements. To address this need, National Institute of Standards and Technology (NIST) reference material (RM) 8323 yeast protein extract is introduced as a proteomics quality control material for benchmarking the preanalytical and analytical performance of proteomics-based experimental workflows. RM 8323 yeast protein extract is based upon the well-characterized eukaryote Saccharomyces cerevisiae and can be utilized in the design and optimization of proteomics-based methodologies from sample preparation to data analysis. To demonstrate its utility as a proteomics quality control material, we coupled LC-MS/MS measurements of RM 8323 with the NIST MS Quality Control (MSQC) performance metrics to quantitatively assess the LC-MS/MS instrumentation parameters that influence measurement accuracy, repeatability, and reproducibility. Due to the complexity of the yeast proteome, we also demonstrate how NIST RM 8323, along with the NIST MSQC performance metrics, can be used in the evaluation and optimization of proteomics-based sample preparation methods. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives

PubMed Central

Thoring, Lena; Wüstenhagen, Doreen A.; Borowiak, Maria; Stech, Marlitt; Sonnabend, Andrei; Kubick, Stefan

2016-01-01

Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called “difficult-to-express” proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of “difficult-to-express” proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called “cell-free” protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various “difficult-to-express” proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA. PMID:27684475

The synthesis of recombinant membrane proteins in yeast for structural studies.

PubMed

Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

2016-02-15

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions.

PubMed

Tian, Ruijun; Jin, Jing; Taylor, Lorne; Larsen, Brett; Quaggin, Susan E; Pawson, Tony

2013-04-01

Gangliosides are ubiquitous components of cell membranes. Their interactions with bacterial toxins and membrane-associated proteins (e.g. receptor tyrosine kinases) have important roles in the regulation of multiple cellular functions. Currently, an effective approach for measuring ganglioside-protein interactions especially in a large-scale fashion is largely missing. To this end, we report a facile MS-based approach to explore gangliosides extracted from cells and measure their interactions with protein of interest globally. We optimized a two-step protocol for extracting total gangliosides from cells within 2 h. Easy-to-use magnetic beads conjugated with a protein of interest were used to capture interacting gangliosides. To measure ganglioside-protein interaction on a global scale, we applied a high-sensitive LC-MS system, containing hydrophilic interaction LC separation and multiple reaction monitoring-based MS for ganglioside detection. Sensitivity for ganglioside GM1 is below 100 pg, and the whole analysis can be done in 20 min with isocratic elution. To measure ganglioside interactions with soluble vascular endothelial growth factor receptor 1 (sFlt1), we extracted and readily detected 36 species of gangliosides from perivascular retinal pigment epithelium cells across eight different classes. Twenty-three ganglioside species have significant interactions with sFlt1 as compared with IgG control based on p value cutoff <0.05. These results show that the described method provides a rapid and high-sensitive approach for systematically measuring ganglioside-protein interactions. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preheated milk proteins improve the stability of grape skin anthocyanins extracts.

PubMed

He, Zhiyong; Xu, Mingzhu; Zeng, Maomao; Qin, Fang; Chen, Jie

2016-11-01

The effects of casein and whey proteins, preheated at 40-100°C and 45-60°C for 15min, respectively, on color loss and anthocyanins degradation in grape skin anthocyanins extracts (GSAE) at pH 3.2 and 6.3 were evaluated. Preheating milk proteins effectively improved their protective effects against color loss and anthocyanins degradation in GSAE solutions during thermal treatment (at 80°C for 2h), H2O2 oxidation (0.005% H2O2 for 1h) and illumination (at 5000lx for 5 d). Whey proteins and casein, preheated at 50°C and 60°C for 15min, respectively, demonstrated the optimal protective effects. However, preheated whey proteins had a better protective effect on the thermal, oxidation and photo stability of GSAE, decreasing the thermal, oxidative and photo degradation of anthocyanins in GSAE 71.59%, 32.22% and 56.92% at pH 3.2 and 54.91%, 22.89% and 46.68% at pH 6.3, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

Process optimization for reverse micellar extraction of stem bromelain with a focus on back extraction.

PubMed

Dhaneshwar, Amrut D; Chaurasiya, Ram Saran; Hebbar, H Umesh

2014-01-01

In the current study, reverse micellar extraction (RME) for the purification of stem bromelain was successfully achieved using the sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane system. A maximum forward extraction efficiency of 58.0% was obtained at 100 mM AOT concentration, aqueous phase pH of 8.0 and 0.2 M NaCl. Back extraction studies on altering stripping phase pH and KCl concentration, addition of counter-ion and iso-propyl alcohol (IPA) and mechanical agitation with glass beads indicated that IPA addition and agitation with glass beads have significant effects on extraction efficiency. The protein extraction was higher (51.9%) in case of the IPA (10% v/v) added system during back extraction as compared to a cetyltrimethylammonium bromide (100 mM) added system (9.42%). The central composite design technique was used to optimize the back extraction conditions further. Concentration of IPA, amount of glass beads, mixing time, and agitation speed (in rpm) were the variables selected. IPA concentration of 8.5% (v/v), glass bead concentration of 0.6 (w/v), and mixing time of 45 min at 400 rpm resulted in higher back extraction efficiency of 45.6% and activity recovery of 88.8% with purification of 3.04-fold. The study indicated that mechanical agitation using glass beads could be used for destabilizing the reverse micelles and release of bromelain back into the fresh aqueous phase. © 2014 American Institute of Chemical Engineers.

Biomarkers of Aspergillus spores

NASA Astrophysics Data System (ADS)

Sulc, Miroslav; Peslova, Katerina; Zabka, Martin; Hajduch, Marian; Havlicek, Vladimir

2009-02-01

We applied both matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometric and 1D sodium dodecylsulfate polyacrylamide gel electrophoretic (1D-PAGE) approaches for direct analysis of intact fungal spores of twenty four Aspergillus species. In parallel, we optimized various protocols for protein extraction from Aspergillus spores using acidic conditions, step organic gradient and variable sonication treatment. The MALDI-TOF mass spectra obtained from optimally prepared samples provided a reproducible fingerprint demonstrating the capability of the MALDI-TOF approach to type and characterize different fungal strains within the Aspergillus genus. Mass spectra of intact fungal spores provided signals mostly below 20 kDa. The minimum material amount represented 0.3 [mu]g (10,000 spores). Proteins with higher molecular weight were detected by 1D-PAGEE Eleven proteins were identified from three selected strains in the range 5-25 kDa by the proteomic approach. Hemolysin and hydrophobin have the highest relevance in host-pathogen interactions.

GH10 XynA is the main xylanase identified in the crude enzymatic extract of Paenibacillus sp. A59 when grown on xylan or lignocellulosic biomass.

PubMed

Ghio, Silvina; Insani, Ester M; Piccinni, Florencia E; Talia, Paola M; Grasso, Daniel H; Campos, Eleonora

2016-01-01

A novel bacterial isolate with polysaccharides degrading activity was identified as Paenibacillus sp., and named Paenibacillus sp. A59. Even though it is a strict mesophile, optimal xylanase activity of the crude enzymatic extract was achieved between 50°C and 70°C and more than 60% of the activity was retained after incubation for 48h at 50°C, indicating thermotolerance of the enzymes involved. The extract was also active on pre-treated sugarcane residue (SCR) and wheat straw, releasing xylobiose and xylose as the main products, therefore confirming its predominantly xylanolytic activity. By zymograms and mass spectrometry of crude enzymatic extracts of xylan or SCR cultures, a 32kDa GH10 beta- 1,4- endoxylanase with xylanase and no CMCase activity was identified. We named this enzyme XynA and it was the only xylanase identified under both conditions assayed, suggesting that it is a good candidate for recombinant expression and evaluation in hemicelluloses deconstruction applications. Also, a protein with two S-layer homology domains (SLH) and a large uncharacterized C-terminal domain as well as an ABC substrate binding protein were identified in crude extracts of SCR cultures. We propose that Paenibacillus sp. A59 uses a system similar to anaerobic and other Gram positive bacteria, with SLH-domain proteins anchoring polysaccharide-degrading enzymes close to the membrane and the substrate binding protein assisting translocation of simple sugars to the cell interior. Copyright © 2016 Elsevier GmbH. All rights reserved.

Deproteinization of water-soluble ß-glucan during acid extraction from fruiting bodies of Pleurotus ostreatus mushrooms.

PubMed

Szwengiel, Artur; Stachowiak, Barbara

2016-08-01

Some ß-glucans can be easily extracted from Basidiomycete mushrooms but commonly used extraction procedures are not satisfactory. A simultaneous method for acid extraction and deproteinization in the case of Pleurotus ostreatus was developed using response surface methodology. The optimized extraction conditions proposed here (30°C, 3.8% HCl, 300min, stirring) allow for the simultaneous extraction and deproteinization of polysaccharides. Additionally, the acid extraction yield was 7 times greater than that of hot water extraction. The combined enzymatic digestion with lyticase, ß-glucanase, exo-1,3-ß-d-glucanase, and ß-glucosidase results elucidated that an extract containing ß-1,3-ß-1,6-ß-1,4-glucan. The gel permeation chromatography (GPC) results showed that the two glucan fractions obtained do not contain linked proteins. The weight average molecular weight of the first fraction (Mw=1137kDa) was 60 times higher than that of the second fraction (Mw=19kDa). Copyright © 2016 Elsevier Ltd. All rights reserved.

Purification and some properties of the protein component of tissue thromboplastin from human brain.

PubMed Central

Bjorklid, E; Storm, E

1977-01-01

The protein component of tissue thromboplastib (Factor III) from human brain was purified by extraction of a microsomal fraction with sodium deoxycholate, gel filtration of the extract on Sephadex G-100 and preparative polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The product, apoprotein III, was homogeneous by anayltical polyacrylamide-gel electrophoresis, and it induced monospecific antibodies in rabbits and goat as shown by immunodiffusion and immunoelectrophoresis. Amino acid- and carbohydrate-analysis data for apoprotein III are presented. The carbohydrate moiety of the protein consists of fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminate, amounting to a total content of 6.3g/100g. The apoprotein alone had no procoagulant activity. When Factor III was reconstituted by combining the pure apoprotein with a purified lipid fraction from the deoxycholate extract of crude Factor III, a high and optimal procoagulant activity was obtained at a phospholipid/protein ratio of 1.1g/g. Phosphatidylethanolamine alone had a weak but significant ability to restore activity, whereas phosphatidylcholine and phosphatidylserine separately had almost none. Two-component mixtures were on average more effective, and three-component mixtures far more effective, than the single phospholipids. The inclusion of a small amount of phosphatidylserine was very important for high activity. Images Fig. 2. PLATE 1 PMID:889578

Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on a HAB causing Alexandrium tamarense.

PubMed

Li, Yi; Zhu, Hong; Zhang, Huajun; Chen, Zhangran; Tian, Yun; Xu, Hong; Zheng, Tianling; Zheng, Wei

2014-08-15

Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on Alexandrium tamarense were measured through studying the algicidal procedure, nuclear damage and transcription of related genes. Medium components were optimized to improve algicidal activity, and characteristics of algicidal extracts were determined. Transmission electron microscope analysis revealed that the cell structure was broken. Cell membrane integrity destruction and nuclear structure degradation were monitored using confocal laser scanning microscope, and the rbcS, hsp and proliferating cell nuclear antigen (PCNA) gene expressions were studied. Results showed that 1.0% tryptone, 0.4% glucose and 0.8% MgCl2 were the optimal nutrient sources. The algicidal extracts were heat and pH stable, non-protein and less than 1kD. Cell membrane and nuclear structure integrity were lost, and the transcription of the rbcS and PCNA genes were significantly inhibited and there was up-regulation of hsp gene expression during the exposure procedure. The algicidal extracts destroyed the cell membrane and nuclear structure integrity, inhibited related gene expression and, eventually, lead to the inhibition of algal growth. All the results may elaborate firstly the cell death process and nuclear damage in A. tamarense which was induced by algicidal extracts, and the algicidal extracts could be potentially used as bacterial control of HABs in future. Copyright © 2014 Elsevier B.V. All rights reserved.

Investigating the importance of Delaunay-based definition of atomic interactions in scoring of protein-protein docking results.

PubMed

Jafari, Rahim; Sadeghi, Mehdi; Mirzaie, Mehdi

2016-05-01

The approaches taken to represent and describe structural features of the macromolecules are of major importance when developing computational methods for studying and predicting their structures and interactions. This study attempts to explore the significance of Delaunay tessellation for the definition of atomic interactions by evaluating its impact on the performance of scoring protein-protein docking prediction. Two sets of knowledge-based scoring potentials are extracted from a training dataset of native protein-protein complexes. The potential of the first set is derived using atomic interactions extracted from Delaunay tessellated structures. The potential of the second set is calculated conventionally, that is, using atom pairs whose interactions were determined by their separation distances. The scoring potentials were tested against two different docking decoy sets and their performances were compared. The results show that, if properly optimized, the Delaunay-based scoring potentials can achieve higher success rate than the usual scoring potentials. These results and the results of a previous study on the use of Delaunay-based potentials in protein fold recognition, all point to the fact that Delaunay tessellation of protein structure can provide a more realistic definition of atomic interaction, and therefore, if appropriately utilized, may be able to improve the accuracy of pair potentials. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of matrix effects in developing rugged high-throughput LC-MS/MS methods for bioanalysis.

PubMed

Li, Fumin; Wang, Jun; Jenkins, Rand

2016-05-01

There is an ever-increasing demand for high-throughput LC-MS/MS bioanalytical assays to support drug discovery and development. Matrix effects of sofosbuvir (protonated) and paclitaxel (sodiated) were thoroughly evaluated using high-throughput chromatography (defined as having a run time ≤1 min) under 14 elution conditions with extracts from protein precipitation, liquid-liquid extraction and solid-phase extraction. A slight separation, in terms of retention time, between underlying matrix components and sofosbuvir/paclitaxel can greatly alleviate matrix effects. High-throughput chromatography, with proper optimization, can provide rapid and effective chromatographic separation under 1 min to alleviate matrix effects and enhance assay ruggedness for regulated bioanalysis.

Blood derived products in pediatrics: New laboratory tools for optimizing potency assignment and reducing side effects.

PubMed

Amiral, Jean; Seghatchian, Jerard

2017-04-01

Neonates and children can develop rare bleeding disorders due to congenital/acquired coagulation Factor deficiencies, or allo-immune/autoimmune complications, or can undergo surgeries at high haemorrhagic risk. They then need specialized transfusion of blood components/products, or purified blood extracted products or recombinant proteins. Blood-derived therapies conventionally used for management of affected infants with genetic/acquired deficiencies, bleeding problems (coagulation Factor reduced or missing) or thrombotic disorders (reduced or missing anticoagulant proteins) pose some additional risks. These remedial therapies can cause tolerance when used very early in life and, sometimes needed, repeatedly. The introduction of recombinant proteins has allowed manufacturers to produce large amounts of the proteins usually present at very low concentration in blood. This has also changed the risk pattern of plasma-extracted products, especially in terms of continual reduction of viral transmission. Many efforts have been made over these past decades to reduce the risks associated with the use of all these products in terms of viral and bacterial safety, as well as immune disorders but they are not the objective of this article. Other associated side effects are the presence of undesired activities in blood products, which can produce thrombotic events or adverse reactions. The progressive introduction of blood derived products has greatly improved the prognosis and quality of life of affected patients. This concerns whole blood, but also blood cell concentrates, mainly platelets and red blood cells, plasma, while the blood extracted products are increasingly replaced by recombinant proteins. All these therapeutic products, i.e. blood extracted drugs, improve health and quality of life for hemophiliac's A or B, or patients with auto/allo-immune thrombocytopenias or with rare bleeding disorders, and those with thrombotic events occurring in childhood, which are mainly due to Protein C or Protein S deficiencies (congenital or acquired). Progress in analytical methods and biotechnology allow better control of the manufacturing processes for all blood derived or plasma extracted products and recombinant proteins, and contribute to improved manufacturing processes to minimize the occurrence of side effects. These adverse events can be due to the aging of the blood cell concentrate with release of their granule content, and generation of EVs, which can produce anaphylactic reactions and risk of thrombosis, but also to the presence of activated coagulation Factors in purified products, such as Factor Xia as recently identified in immunoglobulin concentrates. Characterization and measurement of contaminant products is of special usefulness during product preparation and for optimization of manufacturing processes for purified extracted products, but also for recombinant proteins. The pharmaceutical industry introduces these new methods for validating manufacturing processes, or for quality control assessments. The objective is first to warrant the full quality and safety of the lots produced, and assure the highest efficacy with the lowest risks when used in patients. For cell concentrates and fresh blood, storage conditions are critical and measurement of analytes such as EVs or Annexin V allows evaluation of quality of each individual transfused pouch. In addition to all the rules around viral and bacterial transmission risk, and immune tolerance, our available laboratory methods contribute to reducing the side effects of blood cell concentrates and derived plasma products, as well as those of the therapeutic recombinant proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Purification of Plant Receptor Kinases from Plant Plasma Membranes.

PubMed

Lee, Jin Suk

2017-01-01

Receptor kinases play a central role in various biological processes, but due to their low abundance and highly hydrophobic and dynamic nature, only a few of them have been functionally characterized, and their partners and ligands remain unidentified. Receptor protein extraction and purification from plant tissues is one of the most challenging steps for the success of various biochemical analyses to characterize their function. Immunoprecipitation is a widely used and selective method for enriching or purifying a specific protein. Here we describe two different optimized protein purification protocols, batch and on-chip immunoprecipitation, which efficiently isolate plant membrane receptor kinases for functional analysis.

A Combination of Soybean and Haematococcus Extract Alleviates Ultraviolet B-Induced Photoaging

PubMed Central

Shin, Jieun; Kim, Jong-Eun; Pak, Kum-Ju; Kang, Jung Il; Kim, Tae-Seok; Lee, Sang-Yoon; Yeo, Ik-Hyun; Park, Jung Han Yoon; Kim, Jong Hun; Kang, Nam Joo; Lee, Ki Won

2017-01-01

Soybean-derived isoflavones have been investigated for their preventative effects against UV-induced symptoms of skin damage including wrinkle formation and inflammation. Haematococcus pluvialis is a freshwater species of Chlorophyta that contains high concentrations of the natural carotenoid pigment astaxanthin. Astaxanthin is known to be involved in retinoic acid receptor (RAR) signaling and previously been associated with the inhibition of activator protein (AP)-1 dependent transcription. Based on previous studies, we hypothesized that a combination of soy extract (SE) and Haematococcus extract (HE) may prevent UVB-induced photoaging through specific signaling pathways, as measured by UVB-induced wrinkling on hairless mice skin and expression changes in human dermal fibroblasts (HDFs). The 1:2 ratio of SE and HE mixture (SHM) showed the optimal benefit in vivo. SHM was found to inhibit wrinkle formation via the downregulation of matrix metalloproteinase (MMP)-1 mRNA and protein expression. SHM also inhibited mitogen-activated protein kinase (MAPK) phosphorylation and the transactivation of AP-1 which plays an important role in regulating MMP expression. These results highlight the potential for SHM to be developed as a therapeutic agent to prevent UVB-induced skin wrinkling. PMID:28327532

A Combination of Soybean and Haematococcus Extract Alleviates Ultraviolet B-Induced Photoaging.

PubMed

Shin, Jieun; Kim, Jong-Eun; Pak, Kum-Ju; Kang, Jung Il; Kim, Tae-Seok; Lee, Sang-Yoon; Yeo, Ik-Hyun; Park, Jung Han Yoon; Kim, Jong Hun; Kang, Nam Joo; Lee, Ki Won

2017-03-22

Soybean-derived isoflavones have been investigated for their preventative effects against UV-induced symptoms of skin damage including wrinkle formation and inflammation. Haematococcus pluvialis is a freshwater species of Chlorophyta that contains high concentrations of the natural carotenoid pigment astaxanthin. Astaxanthin is known to be involved in retinoic acid receptor (RAR) signaling and previously been associated with the inhibition of activator protein (AP)-1 dependent transcription. Based on previous studies, we hypothesized that a combination of soy extract (SE) and Haematococcus extract (HE) may prevent UVB-induced photoaging through specific signaling pathways, as measured by UVB-induced wrinkling on hairless mice skin and expression changes in human dermal fibroblasts (HDFs). The 1:2 ratio of SE and HE mixture (SHM) showed the optimal benefit in vivo. SHM was found to inhibit wrinkle formation via the downregulation of matrix metalloproteinase (MMP)-1 mRNA and protein expression. SHM also inhibited mitogen-activated protein kinase (MAPK) phosphorylation and the transactivation of AP-1 which plays an important role in regulating MMP expression. These results highlight the potential for SHM to be developed as a therapeutic agent to prevent UVB-induced skin wrinkling.

Subcritical Fluid Extraction of Chinese Quince Seed: Optimization and Product Characterization.

PubMed

Wang, Li; Wu, Min; Liu, Hua-Min; Ma, Yu-Xiang; Wang, Xue-De; Qin, Guang-Yong

2017-03-25

Chinese quince seed (CQS) is an underutilized oil source and a potential source of unsaturated fatty acids and α-tocopherol-rich oil. Subcritical fluid (SCF) extraction is executed at lower pressures and temperatures than the pressures and temperatures used in supercritical fluid extraction. However, no studies on the SCF extraction of CQS oil are reported. Therefore, the objective of this study was to evaluate the use of SCF for the extraction of CQS oil and to compare the use of SCF with the classical Soxhlet (CS) and supercritical CO₂ (SC-CO₂) extraction methods. Response surface methodology (RSM) was used to investigate the extraction conditions: temperature (45-65 °C), time (30-50 min), and solvent/solid ratio (5-15 mL/g). The optimization results showed that the highest yield (27.78%) was obtained at 56.18 °C, 40.20 min, and 12.57 mL/g. The oil extracted by SCF had a higher unsaturated fatty acid content (86.37%-86.75%), higher α-tocopherol content (576.0-847.6 mg/kg), lower acid value (3.97 mg/g), and lower peroxide value (0.02 meq O₂/kg) than extractions using CS and SC-CO 2 methods. The SCF-defatted meal of oilseed exhibited the highest nitrogen solubility index (49.64%) and protein dispersibility index (50.80%), demonstrating that SCF extraction was a promising and efficient technique as an alternative to CS and SC-CO 2 methods, as very mild operating conditions and an eco-friendly solvent can be used in the process with maximum preservation of the quality of the meal.

Sea buckthorn (Hippophae rhamnoides) proanthocyanidins inhibit in vitro enzymatic hydrolysis of protein.

PubMed

Arimboor, Ranjith; Arumughan, C

2011-08-01

Interactions of phenolics with other food constituents and digestive enzymes are likely to have interference with the digestion and bioavailability of food and phenolics. In this study the effect of sea buckthorn proanthocyanidins on in vitro digestion of protein was evaluated. Optimization of the extraction conditions showed that maximum recovery of sea buckthorn proanthocyanidins was obtained with acidified acetone; water mixture (60% to 70%, v/v). Crude proanthocyanidin extracts thus prepared were purified using sephadex gel column chromatography and their average degree of polymerization and the effects on enzymatic hydrolysis of bovine serum albumin as influenced by their protein precipitation capacities were studied. Average degree of polymerization of proanthocyanidins in berry pulp, kernel, seed coat, and leaves was 7.4, 5.6, 8.2, and 10.6, respectively. The EC50 values for the protein precipitation by the PA of berry pulp, kernel seed coat, and leaves were 44.2, 44.1, 65.8, and 39.8 μg, respectively. Relative enzymatic hydrolysis of the protein-proanthocyanidin complexes was 44.1% to 60.3% for pepsin and 57.5% to 67.7% for trypsin. Interactions of sea buckthorn proanthocyanidins with food proteins and digestive enzymes might alter the protein digestibility and phenolic bioavailabilty. © 2011 Institute of Food Technologists®

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

PubMed Central

Li, Liang; Mustafi, Debarshi; Fu, Qiang; Tereshko, Valentina; Chen, Delai L.; Tice, Joshua D.; Ismagilov, Rustem F.

2006-01-01

High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a “hybrid” droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as ≈140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in ≈10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 μl of protein solution, ≈1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization. PMID:17159147

Development and characterization of an allergoid of cat dander for immunotherapy.

PubMed

Sola, J P; Pedreño, Y; Cerezo, A; Peñalver-Mellado, M

2018-01-13

Allergy to cats is a frequent cause of sensitization to indoor allergens and currently there are few alternatives to specific immunotherapy with cat native extracts. The objective is to develop and characterize a new allergoid to increase the tools available for use in clinical practice. The allergoid cat dander extract (ACD) was developed from a native cat dander extract (NCD) by modification with glutaraldehyde, and the optimal process control was determined by SDS-PAGE, DOT BLOT and determination of free amine groups. The ACD was characterized in protein profile by SDS-PAGE, size exclusion chromatography (SEC) and peptide footprint. The allergenic profile of ACD was determined by immunoblot, IgE CAP inhibition and IgG competition ELISA. The major allergen content in NCD was obtained by the ELISA sandwich protocol and was extrapolated to ACD. The control process determined the optimal development of the allergoid. The ACD obtained contains 182.28μg/mg of protein and 11.90μg/mg of Fel d 1. SDS-PAGE and SEC confirmed the presence of high molecular weight proteins in ACD, and the peptide footprint showed the presence of Fel d 1 and Fel d 7. The high degree of polymerization was evidenced with the determination of the reduction of lysine residues in the allergoid, resulting 91.96%. The ACD showed a significant loss of allergenicity respect to NCD, while the IgG-binding capacity was maintained. The ACD obtained presents a good safety profile, so would be a good alternative for treatment of cat allergy. Copyright © 2017 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.

ComplexQuant: high-throughput computational pipeline for the global quantitative analysis of endogenous soluble protein complexes using high resolution protein HPLC and precision label-free LC/MS/MS.

PubMed

Wan, Cuihong; Liu, Jian; Fong, Vincent; Lugowski, Andrew; Stoilova, Snejana; Bethune-Waddell, Dylan; Borgeson, Blake; Havugimana, Pierre C; Marcotte, Edward M; Emili, Andrew

2013-04-09

The experimental isolation and characterization of stable multi-protein complexes are essential to understanding the molecular systems biology of a cell. To this end, we have developed a high-throughput proteomic platform for the systematic identification of native protein complexes based on extensive fractionation of soluble protein extracts by multi-bed ion exchange high performance liquid chromatography (IEX-HPLC) combined with exhaustive label-free LC/MS/MS shotgun profiling. To support these studies, we have built a companion data analysis software pipeline, termed ComplexQuant. Proteins present in the hundreds of fractions typically collected per experiment are first identified by exhaustively interrogating MS/MS spectra using multiple database search engines within an integrative probabilistic framework, while accounting for possible post-translation modifications. Protein abundance is then measured across the fractions based on normalized total spectral counts and precursor ion intensities using a dedicated tool, PepQuant. This analysis allows co-complex membership to be inferred based on the similarity of extracted protein co-elution profiles. Each computational step has been optimized for processing large-scale biochemical fractionation datasets, and the reliability of the integrated pipeline has been benchmarked extensively. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Residues with similar hexagon neighborhoods share similar side-chain conformations.

PubMed

Li, Shuai Cheng; Bu, Dongbo; Li, Ming

2012-01-01

We present in this study a new approach to code protein side-chain conformations into hexagon substructures. Classical side-chain packing methods consist of two steps: first, side-chain conformations, known as rotamers, are extracted from known protein structures as candidates for each residue; second, a searching method along with an energy function is used to resolve conflicts among residues and to optimize the combinations of side chain conformations for all residues. These methods benefit from the fact that the number of possible side-chain conformations is limited, and the rotamer candidates are readily extracted; however, these methods also suffer from the inaccuracy of energy functions. Inspired by threading and Ab Initio approaches to protein structure prediction, we propose to use hexagon substructures to implicitly capture subtle issues of energy functions. Our initial results indicate that even without guidance from an energy function, hexagon structures alone can capture side-chain conformations at an accuracy of 83.8 percent, higher than 82.6 percent by the state-of-art side-chain packing methods.

Optimized manual and automated recovery of amplifiable DNA from tissues preserved in buffered formalin and alcohol-based fixative.

PubMed

Duval, Kristin; Aubin, Rémy A; Elliott, James; Gorn-Hondermann, Ivan; Birnboim, H Chaim; Jonker, Derek; Fourney, Ron M; Frégeau, Chantal J

2010-02-01

Archival tissue preserved in fixative constitutes an invaluable resource for histological examination, molecular diagnostic procedures and for DNA typing analysis in forensic investigations. However, available material is often limited in size and quantity. Moreover, recovery of DNA is often severely compromised by the presence of covalent DNA-protein cross-links generated by formalin, the most prevalent fixative. We describe the evaluation of buffer formulations, sample lysis regimens and DNA recovery strategies and define optimized manual and automated procedures for the extraction of high quality DNA suitable for molecular diagnostics and genotyping. Using a 3-step enzymatic digestion protocol carried out in the absence of dithiothreitol, we demonstrate that DNA can be efficiently released from cells or tissues preserved in buffered formalin or the alcohol-based fixative GenoFix. This preparatory procedure can then be integrated to traditional phenol/chloroform extraction, a modified manual DNA IQ or automated DNA IQ/Te-Shake-based extraction in order to recover DNA for downstream applications. Quantitative recovery of high quality DNA was best achieved from specimens archived in GenoFix and extracted using magnetic bead capture.

Optimization for ultrasound extraction of polysaccharides from mulberry fruits with antioxidant and hyperglycemic activity in vitro.

PubMed

Chen, Chun; You, Li-Jun; Abbasi, Arshad Mehmood; Fu, Xiong; Liu, Rui Hai

2015-10-05

Single-factor experiment and Box-Behnken design (BBD) were applied to optimize the ultrasound-assisted extraction of mulberry fruits polysaccharides (MFP). Under optimum conditions: ratio of water to raw material 40.25, extraction temperature 69°C, ultrasonic power 190W and extraction time 75 min, the MFP yield was 3.13% (±0.07%), in accordance to the predicted value of 3.04%. The mulberry fruits polysaccharides fractions was obtained by deproteinization (MFP-1), followed by decolorization and deionization (MFP-2). Carbohydrate content in MFP, MFP-1 and MFP-2 was 58.61% (±1.47%), 69.98% (±0.91%), 81.18% (±1.29%), as well as proteins was estimated 16.50% (±0.86%), 1.57% (±0.63%), 1.02% (±0.18%), respectively. The FT-IR indicated that MFP, MFP-1 and MFP-2 were acidic polysaccharides. The MFP-1 exhibited the strongest antioxidant activity, while MFP-2 showed the strongest hyperglycemic activity in vitro. This may be caused by their different compositions and physical properties in the different mulberry fruit polysaccharides fractions. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Establishment of optimun conditions in order to obtain a protein isolate from Chilean Hazelnut].

PubMed

Villarroel, Mario; Zapata, Constanza; Pino, Leonardo; Rubilar, Mónica

2012-03-01

An alternative to solve the problem of the overall deficit of proteins has been the use ofdefatted cakes generated by the extraction of oil from vegetable sources such as rapeseed, soybean, lupin, etc. This process at the same time increases the protein content, making this feasible to be used to enrich some types of food. This is the case of the chilean hazelnut (Gevuina avellana, Mol), monotypic species characterized by their high percentage of oil (50%) and whose defatted cake isolated protein could be used to obtain an isolated protein. For this purpose optimized conditions of extraction of protein were carried out using the surface response methodology (SRM) and a central composite design with three independent variables: time of contact of the cake with the solvent, sample/solvent ratio and pH was used. All variables were controlled at five different levels. The data were subjected to an analysis of regression and ANOVA, the first to determine the polynomial equation and the second to select the control factors with significant effect on the extraction of the protein. The best combination of factors turned out to be: time between 30 and 40 minutes, pH between 9 and 9.5 and a relationship sample/solvent between 1/15 to 1/16 with a final yield of 76%. The physical characteristics were: density 0,504 g/cm3, compaction 43, 34% apparent and pale yellow. Proximal analysis showed a concentration of protein of 76%, 13%, raw fiber carbohydrate 0.68% and oil 1.29%. With regard to the functional properties emphasized water absorption (320 g/100 g), absorption of oil (410 g/100 g) and foaming capacity (221%).

An Optimized Trichloroacetic Acid/Acetone Precipitation Method for Two-Dimensional Gel Electrophoresis Analysis of Qinchuan Cattle Longissimus Dorsi Muscle Containing High Proportion of Marbling.

PubMed

Hao, Ruijie; Adoligbe, Camus; Jiang, Bijie; Zhao, Xianlin; Gui, Linsheng; Qu, Kaixing; Wu, Sen; Zan, Linsen

2015-01-01

Longissimus dorsi muscle (LD) proteomics provides a novel opportunity to reveal the molecular mechanism behind intramuscular fat deposition. Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis. Trichloroacetic acid (TCA)/acetone precipitation is a widely used method to remove contaminants from protein samples. However, the high speed centrifugation employed in this method produces hard precipitates, which restrict contaminant elimination and protein re-dissolution. To address the problem, the centrifugation precipitates were first grinded with a glass tissue grinder and then washed with 90% acetone (TCA/acetone-G-W) in the present study. According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis. Additionally, we also evaluated the effect of sample drying on 2-DE profile as well as protein yield. It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min. In summary, we developed an optimized TCA/acetone precipitation method for protein extraction of LD, in which the modifications improved the effectiveness of TCA/acetone method.

An Optimized Trichloroacetic Acid/Acetone Precipitation Method for Two-Dimensional Gel Electrophoresis Analysis of Qinchuan Cattle Longissimus Dorsi Muscle Containing High Proportion of Marbling

PubMed Central

Hao, Ruijie; Adoligbe, Camus; Jiang, Bijie; Zhao, Xianlin; Gui, Linsheng; Qu, Kaixing; Wu, Sen; Zan, Linsen

2015-01-01

Longissimus dorsi muscle (LD) proteomics provides a novel opportunity to reveal the molecular mechanism behind intramuscular fat deposition. Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis. Trichloroacetic acid (TCA)/acetone precipitation is a widely used method to remove contaminants from protein samples. However, the high speed centrifugation employed in this method produces hard precipitates, which restrict contaminant elimination and protein re-dissolution. To address the problem, the centrifugation precipitates were first grinded with a glass tissue grinder and then washed with 90% acetone (TCA/acetone-G-W) in the present study. According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis. Additionally, we also evaluated the effect of sample drying on 2-DE profile as well as protein yield. It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min. In summary, we developed an optimized TCA/acetone precipitation method for protein extraction of LD, in which the modifications improved the effectiveness of TCA/acetone method. PMID:25893432

Reduction of L-phenylalanine in protein hydrolysates using L-phenylalanine ammonia-lyase from Rhodosporidium toruloides.

PubMed

Castañeda, María Teresita; Adachi, Osao; Hours, Roque Alberto

2015-10-01

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.25) from Rhodosporidium toruloides was utilized to remove L-phenylalanine (L-Phe) from different commercial protein hydrolysates. A casein acid hydrolysate (CAH, L-Phe ~2.28 %) was employed as a model substrate. t-Cinnamic acid resulting from deamination of L-Phe was extracted, analyzed at λ = 290 nm, and used for PAL activity determination. Optimum reaction conditions, optimized using successive Doehlert design, were 35 mg mL(-1) of CAH and 800 mU mL(-1) of PAL, while temperature and pH were 42 °C and 8.7, respectively. Reaction kinetics of PAL with CAH was determined under optimized conditions. Then, removal of L-Phe from CAH was tested. Results showed that more than 92 % of initial L-Phe was eliminated. Similar results were obtained with other protein hydrolysates. These findings demonstrate that PAL is a useful biocatalyst for L-Phe removal from protein hydrolysates, which can be evaluated as potential ingredients in foodstuffs for PKU patients.

Chromatographic studies of drug interactions with alpha1-acid glycoprotein by ultrafast affinity extraction and peak profiling.

PubMed

Beeram, Sandya; Bi, Cong; Zheng, Xiwei; Hage, David S

2017-05-12

Interactions with serum proteins such as alpha 1 -acid glycoprotein (AGP) can have a significant effect on the behavior and pharmacokinetics of drugs. Ultrafast affinity extraction and peak profiling were used with AGP microcolumns to examine these processes for several model drugs (i.e., chlorpromazine, disopyramide, imipramine, lidocaine, propranolol and verapamil). The association equilibrium constants measured for these drugs with soluble AGP by ultrafast affinity extraction were in the general range of 10 4 -10 6 M -1 at pH 7.4 and 37°C and gave good agreement with literature values. Some of these values were dependent on the relative drug and protein concentrations that were present when using a single-site binding model; these results suggested a more complex mixed-mode interaction was actually present, which was also then used to analyze the data. The apparent dissociation rate constants that were obtained by ultrafast affinity extraction when using a single-site model varied from 0.14 to 7.0s -1 and were dependent on the relative drug and protein concentrations. Lower apparent dissociation rate constants were obtained by this approach as the relative amount of drug versus protein was decreased, with the results approaching those measured by peak profiling at low drug concentrations. This information should be useful in better understanding how these and other drugs interact with AGP in the circulation. In addition, the chromatographic approaches that were optimized and used in this report to examine these systems can be adapted for the analysis of other solute-protein interactions of biomedical interest. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of Moringa oleifera seed extract on rumen fermentation in vitro.

PubMed

Hoffmann, E M; Muetzel, S; Becker, K

2003-02-01

Moringa oleifera is a pantropical tree of the family Moringaceae. A previously undescribed property of an aqueous extract from the seeds of this plant is the modulation of ruminal fermentation patterns, especially protein degradation, as demonstrated in a short-term batch incubation system. Gas, short chain fatty acids (SCFA) and cellulolytic enzyme activities were determined as general fermentation parameters. A dot blot assay able to directly detect true protein in rumen fluid samples was used to quantify protein degradation. For complex substrates the interpretation of protein degradation profiles was amended by polyacrylamide gel electrophoresis (PAGE) of the samples. When incubated with pure carbohydrates at a concentration of 1 mg ml(-1), the extract reduced microbial degradation of the model protein, bovine serum albumin (BSA), such that its concentration was at least 40% above the control after 12 h of incubation. Total protein degradation was thus delayed by approximately 9 h. When fermented along with wheat straw, leaf protein (Rubisco) was almost entirely protected during 12 h of fermentation. The degradation of soy proteins was retarded by at least 4-6 h, depending on the protein band. There were strong side effects on the fermentation of pure cellulose (SCFA yield-60% after 12 h), whereas cellobiose and starch fermentation were less affected (-18 and -8%, respectively). When the complex substrates were fermented, SCFA yield was reduced by approximately 30% after 12 h. In our work we clearly demonstrate the efficacy of the new substance, which is neither a tannin nor a saponin, in an in vitro system, using pure as well as complex substrates. The properties shown in vitro for the crude extract suggest that it could have a positive effect on the protein metabolism of ruminants under intensive management and that negative side effects can be overcome by an optimized dosage. If the chemical nature of the active substance and its mechanism of action can be clarified, it may provide an alternative to replace critical synthetic feed additives (such as antibiotics) for high yielding dairy cows.

Optimization of Maillard reaction with ribose for enhancing anti-allergy effect of fish protein hydrolysates using response surface methodology.

PubMed

Yang, Sung-Yong; Kim, Se-Wook; Kim, Yoonsook; Lee, Sang-Hoon; Jeon, Hyeonjin; Lee, Kwang-Won

2015-06-01

Halibut is served on sushi and as sliced raw fish fillets. We investigated the optimal conditions of the Maillard reaction (MR) with ribose using response surface methodology to reduce the allergenicity of its protein. A 3-factored and 5-leveled central composite design was used, where the independent variables were substrate (ribose) concentration (X1, %), reaction time (X2, min), and pH (X3), while the dependent variables were browning index (Y1, absorbance at 420nm), DPPH scavenging (Y2, EC50 mg/mL), FRAP (Y3, mM FeSO4/mg extract) and β-hexosaminidase release (Y4, %). The optimal conditions were obtained as follows: X1, 28.36%; X2, 38.09min; X3, 8.26. Maillard reaction products of fish protein hydrolysate (MFPH) reduced the amount of nitric oxide synthesis compared to the untreated FPH, and had a significant anti-allergy effect on β-hexosaminidase and histamine release, compared with that of the FPH control. We concluded that MFPH, which had better antioxidant and anti-allergy activities than untreated FPH, can be used as an improved dietary source. Copyright © 2014 Elsevier Ltd. All rights reserved.

Optimization of Evans blue quantitation in limited rat tissue samples

PubMed Central

Wang, Hwai-Lee; Lai, Ted Weita

2014-01-01

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting. PMID:25300427

Optimization of Evans blue quantitation in limited rat tissue samples

NASA Astrophysics Data System (ADS)

Wang, Hwai-Lee; Lai, Ted Weita

2014-10-01

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting.

Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneity.

PubMed

Nakano, Keiichi; Tamura, Shogo; Otuka, Kohei; Niizeki, Noriyasu; Shigemura, Masahiko; Shimizu, Chikara; Matsuno, Kazuhiko; Kobayashi, Seiichi; Moriyama, Takanori

2013-07-15

Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

Optimization of microfabricated nanoliter-scale solid-phase extraction device for detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Chen, Wenzhang; Shen, Jing; Yin, Xuefeng; Yu, Yingnian

2007-01-01

A nano-scale solid-phase extraction (SPE) device was developed for the detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with a simplified microfabrication technology. By using SU-8 photoresist instead of epoxy glue to connect the microchannel and transfer capillary, polymeric contaminant signals in MS analysis were significantly reduced. Micro SPE columns with different capacities and geometric characteristics were investigated in order to increase the detection sensitivity and decrease spot size for MALDI-TOF-MS analysis. It is shown that enhancements in sensitivities for the detection of proteins in low abundance were correlated with the reduction in column capacity and increase in column aspect ratio. Fifty nanoliters of matrix solution were sufficient to elute the sample completely from the optimized micro SPE column with 3.5 nL capacity. The mass spectrum of a 5 fmol in-gel tryptic digest of bovine serum albumin (BSA), processed by the micro SPE column, demonstrated that 29 peptides matched the protein giving a sequence coverage of 51%, which was better than that obtained from analysis of 25 fmol of the same sample prepared by the dried-droplet method. With the micro SPE column treatment of 2 microL of digestion supernatant of a gel spot of the IQGAP1 protein, 15 peptides were detected from the mass spectrum with the highest individual score of 111, while, with a ZipTip procedure, only nine peaks were detected with the highest individual score of 71. Analytical results demonstrated that this approach greatly improved the sequence coverage and identification specificity for the tested protein. It can serve as a very useful tool in proteomics studies, especially for low abundance proteins. Copyright (c) 2006 John Wiley & Sons, Ltd.

Culturing and extraction of Coprococcus comes, absorption of serumagglutinins by soluble fractions and relation between agglutinins and antibodies in sera of patients with Crohn's disease.

PubMed

Hazenberg, M P; Pennock-Schröder, A M; van de Merwe, J P

1986-01-01

Agglutinating antibodies to Coprococcus comes and three other obligately anaerobic coccoid rods from the intestinal flora are used in the diagnosis of Crohn's disease. Further studies on the pathogenetic role as well as the development of more sensitive and specific methods for detecting antibodies require extraction of the antigen fractions. Culturing methods to obtain C. comes with optimal antigen presentation and isolation of soluble antigen fractions were therefore developed. Hot water extraction of whole cells and subsequent removal of proteins with trichloroacetic acid provided a fraction that absorbed serum agglutinins, was useful for an enzyme-linked immunosorbent assay and induced agglutinating antibodies in rats.

Direct extraction of lead (II) from untreated human blood serum using restricted access carbon nanotubes and its determination by atomic absorption spectrometry.

PubMed

Barbosa, Valéria Maria Pereira; Barbosa, Adriano Francisco; Bettini, Jefferson; Luccas, Pedro Orival; Figueiredo, Eduardo Costa

2016-01-15

Oxidized carbon nanotubes were covered with layers of bovine serum albumin to result in so-called restricted-access carbon nanotubes (RACNTs). This material can extract Pb(2+) ions directly from untreated human blood serum while excluding all the serum proteins. The RACNTs have a protein exclusion capacity of almost 100% and a maximum Pb(2+) adsorption capacity of 34.5mg g(-1). High resolution transmission electron microscopy, scanning transmission electron microscopy and energy dispersive spectroscopy were used to confirm the BSA layer and Pb(2+) adsorption sites. A mini-column filled with RACNTs was used in an on-line solid phase extraction system coupled to a thermospray flame furnace atomic absorption spectrometry. At optimized experimental conditions, the method has a detection limit as low as 2.1µg L(-1), an enrichment factor of 5.5, and inter- and intra-day precisions (expressed as relative standard deviation) of <8.1%. Recoveries of the Pb(2+) spiked samples ranged from 89.4% to 107.3% for the extraction from untreated human blood serum. Copyright © 2015 Elsevier B.V. All rights reserved.

[Extraction and antioxidant activity of collagen from elephant skin, pig skin and fish scales].

PubMed

Li, Chunnan; Sun, Jiaming; Zhang, Hui

2011-08-01

To study collagen structure of the traditional Chinese medicine elephant skin and the proposed alternatives such as pig skin, fish scale, and antioxidant activity. Orthogonal experimental design method was employed to determine the optimal extraction condition of collagen from the elephant skin, and the structure and content of collagen of proposed alternatives were compared, their scavenging ability were determined by salicylic acid. Collagen extracted from elephant skin with the optimal conditions was the structural integrity and good quality first time, and collagen structure of the elephant skin was similar to the proposed alternatives. Free radical scavenging capacity of collagen, values of IC50, were 0.51 g x L(-1) of elephant skin, 0.60 g x L(-1) of pig skin and 0.42 g x L(-1) of fish scale. By comparing and identification of proteins that the collagen of elephant skin is type I collagen, with a strong antioxidant capacity, is the active ingredients of elephant skin. It provides a further study of alternatives as an important reference.

Microbial production of nattokinase: current progress, challenge and prospect.

PubMed

Cai, Dongbo; Zhu, Chengjun; Chen, Shouwen

2017-05-01

Nattokinase (EC 3.4.21.62) is a profibrinolytic serine protease with a potent fibrin-degrading activity, and it has been produced by many host strains. Compared to other fibrinolytic enzymes (urokinase, t-PA and streprokinase), nattokinase shows the advantages of having no side effects, low cost and long life-time, and it has the potential to be used as a drug for treating cardiovascular disease and served as a functional food additive. In this review, we focused on screening of producing strains, genetic engineering, fermentation process optimization for microbial nattokinase production, and the extraction and purification of nattokinase were also discussed in this particular chapter. The selection of optimal nattokinase producing strain was the crucial starting element for improvement of nattokinase production. Genetic engineering, protein engineering, fermentation optimization and process control have been proved to be the effective strategies for enhancement of nattokinase production. Also, extraction and purification of nattokinase are critical for the quality evaluation of nattokinase. Finally, the prospect of microbial nattokinase production was also discussed regarding the recent progress, challenge, and trends in this field.

Nonparametric variational optimization of reaction coordinates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Banushkina, Polina V.; Krivov, Sergei V., E-mail: s.krivov@leeds.ac.uk

State of the art realistic simulations of complex atomic processes commonly produce trajectories of large size, making the development of automated analysis tools very important. A popular approach aimed at extracting dynamical information consists of projecting these trajectories into optimally selected reaction coordinates or collective variables. For equilibrium dynamics between any two boundary states, the committor function also known as the folding probability in protein folding studies is often considered as the optimal coordinate. To determine it, one selects a functional form with many parameters and trains it on the trajectories using various criteria. A major problem with such anmore » approach is that a poor initial choice of the functional form may lead to sub-optimal results. Here, we describe an approach which allows one to optimize the reaction coordinate without selecting its functional form and thus avoiding this source of error.« less

An effective placental cotyledons proteins extraction method for 2D gel electrophoresis.

PubMed

Tan, Niu J; Daim, Leona D J; Jamil, Amilia A M; Mohtarrudin, Norhafizah; Thilakavathy, Karuppiah

2017-03-01

Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction.

PubMed

Beeram, Sandya R; Zheng, Xiwei; Suh, Kyungah; Hage, David S

2018-03-03

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha 1 -acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered. Copyright © 2018 Elsevier Inc. All rights reserved.

The Network Organization of Cancer-associated Protein Complexes in Human Tissues

PubMed Central

Zhao, Jing; Lee, Sang Hoon; Huss, Mikael; Holme, Petter

2013-01-01

Differential gene expression profiles for detecting disease genes have been studied intensively in systems biology. However, it is known that various biological functions achieved by proteins follow from the ability of the protein to form complexes by physically binding to each other. In other words, the functional units are often protein complexes rather than individual proteins. Thus, we seek to replace the perspective of disease-related genes by disease-related complexes, exemplifying with data on 39 human solid tissue cancers and their original normal tissues. To obtain the differential abundance levels of protein complexes, we apply an optimization algorithm to genome-wide differential expression data. From the differential abundance of complexes, we extract tissue- and cancer-selective complexes, and investigate their relevance to cancer. The method is supported by a clustering tendency of bipartite cancer-complex relationships, as well as a more concrete and realistic approach to disease-related proteomics. PMID:23567845

Optimal subset selection of primary sequence features using the genetic algorithm for thermophilic proteins identification.

PubMed

Wang, LiQiang; Li, CuiFeng

2014-10-01

A genetic algorithm (GA) coupled with multiple linear regression (MLR) was used to extract useful features from amino acids and g-gap dipeptides for distinguishing between thermophilic and non-thermophilic proteins. The method was trained by a benchmark dataset of 915 thermophilic and 793 non-thermophilic proteins. The method reached an overall accuracy of 95.4 % in a Jackknife test using nine amino acids, 38 0-gap dipeptides and 29 1-gap dipeptides. The accuracy as a function of protein size ranged between 85.8 and 96.9 %. The overall accuracies of three independent tests were 93, 93.4 and 91.8 %. The observed results of detecting thermophilic proteins suggest that the GA-MLR approach described herein should be a powerful method for selecting features that describe thermostabile machines and be an aid in the design of more stable proteins.

[Effect of solution environments on ceramic membrane microfiltration of model system of Chinese medicines].

PubMed

Zhang, Lianjun; Lu, Jin; Le, Kang; Fu, Tingming; Guo, Liwei

2010-07-01

To investigate the effect of differents solution environments on the ceramic membrane microfiltration of model system of Chinese medicines. Taking binary system of soybean protein-berberine as the research object, flux, transmittance of berberine and traping rate of protein as indexes, different solution environment on membrane process were investigated. When the concentration of soybean protein was under 1 g x L(-1), the membrane flux was minimum with the traping of berberine decreased slightly as the concentration increased. When pH was 4, the flux was maximum with the traping rate of protein was 99%, and the transmittance of berberine reached above 60%. The efficiency of membrane separation can be improved by optimizing the solution environment of water-extraction of chinese medicines. The efficiency of membrane separation is the best when adjust the pH to the isoelectric point of proteins for the proteins as the main pollutant in aqueous solution.

Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

PubMed

Maggi, Maristella; Scotti, Claudia

2017-08-01

Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

Simultaneous determination of 9 heterocyclic aromatic amines in pork products by liquid chromatography coupled with triple quadrupole tandem mass spectrometry

NASA Astrophysics Data System (ADS)

Shen, X. C.; Zhang, Y. L.; Cui, Y. Q.; Xu, L. Y.; Li, X.; Qi, J. H.

2017-07-01

Heterocyclic aromatic amines (HAAs) are potent mutagens that formed at high temperature in cooked, protein-rich food. Owing to their frequent intake, an accurate method is essential to access human health risk of HAAs exposure through detecting these compounds in various heat-treated meat products. In this study, a liquid chromatography-electrospray tandem mass spectrometry (LC--ESI-MS/MS) method was developed to perform the determination of 9 mutagenic heterocyclic amines (HAAs) in meat samples with multiple reaction monitoring (MRM) mode. Ultrasound assisted extraction and diatomaceous earth was employed to extract HAAs from food samples, and the analytes were purified and enriched using tandem solid phase extraction, with propyl sulfonic acid coupled to a C18 cartridge. Two parameters, extraction time and eluent, were carefully optimized to improve the extraction and purification efficiency. The LC separation was carried out using a Zorbax SB-C18 (3.5 μm particle size, 2.1 × 150 mm i.d.) column and optimized some parameters, such as pH, concentration and volume. Under the optimal experimental conditions, recoveries ranged from 52.97% to 97.11% with good quality parameters: limit of detection values between 0.02 and 0.24 ng mL-1, linearity (R2>0.998), and run-to-run and day-to-day precisions lower than 9.81% achieved. To evaluate the performance of the method in high throughput analysis of complex meat samples, the LC-MS/MS method was applied to the analysis of HAAs in three food samples, and the results demonstrated that the method can be used for the trace determination of HAAs in pork samples.

Influence of extraction pH on the foaming, emulsification, oil-binding and visco-elastic properties of marama protein.

PubMed

Gulzar, Muhammad; Taylor, John Rn; Minnaar, Amanda

2017-11-01

Marama bean protein, as extracted previously at pH 8, forms a viscous, adhesive and extensible dough. To obtain a protein isolate with optimum functional properties, protein extraction under slightly acidic conditions (pH 6) was investigated. Two-dimensional electrophoresis showed that pH 6 extracted marama protein lacked some basic 11S legumin polypeptides, present in pH 8 extracted protein. However, it additionally contained acidic high molecular weight polypeptides (∼180 kDa), which were disulfide crosslinked into larger proteins. pH 6 extracted marama proteins had similar emulsification properties to soy protein isolate and several times higher foaming capacity than pH 8 extracted protein, egg white and soy protein isolate. pH 6 extracted protein dough was more elastic than pH 8 extracted protein, approaching the elasticity of wheat gluten. Marama protein extracted at pH 6 has excellent food-type functional properties, probably because it lacks some 11S polypeptides but has additional high molecular weight proteins. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Reverse micellar extraction of bromelain from pineapple peel--Effect of surfactant structure.

PubMed

Wan, Jing; Guo, Jingjing; Miao, Zhitong; Guo, Xia

2016-04-15

Pineapple peel is generally disposed or used as compost. This study was focused on extracting bromelain from pineapple peel by using reverse micelles. It was found that gemini surfactant C12-8-C12·2Br (octamethylene-α,ω-bis(dimethyldodecylammonium bromide)) showed distinctive advantage over its monomeric counterpart DTAB (dodecyl trimethyl ammonium bromide); under optimized condition, the bromelain extracted with C12-8-C12·2Br reverse micelle had an activity recovery of 163% and a purification fold of 3.3, while when using DTAB reverse micelle, the activity recovery was 95% and the purification fold was 1.7. Therefore, the spacer of gemini surfactant should play a positive role in bromelain extraction and may suggest the potential of gemini surfactant in protein separation since it has been so far rarely used in relative experiments or technologies. Copyright © 2015 Elsevier Ltd. All rights reserved.

How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves.

PubMed

Zhang, Chen; Sanders, Johan P M; Xiao, Ting T; Bruins, Marieke E

2015-01-01

Leaf protein can be obtained cost-efficiently by alkaline extraction, but overuse of chemicals and low quality of (denatured) protein limits its application. The research objective was to investigate how alkali aids protein extraction of green tea leaf residue, and use these results for further improvements in alkaline protein biorefinery. Protein extraction yield was studied for correlation to morphology of leaf tissue structure, protein solubility and hydrolysis degree, and yields of non-protein components obtained at various conditions. Alkaline protein extraction was not facilitated by increased solubility or hydrolysis of protein, but positively correlated to leaf tissue disruption. HG pectin, RGII pectin, and organic acids were extracted before protein extraction, which was followed by the extraction of cellulose and hemi-cellulose. RGI pectin and lignin were both linear to protein yield. The yields of these two components were 80% and 25% respectively when 95% protein was extracted, which indicated that RGI pectin is more likely to be the key limitation to leaf protein extraction. An integrated biorefinery was designed based on these results.

Extraction process optimization of polyphenols from Indian Citrus sinensis – as novel antiglycative agents in the management of diabetes mellitus

PubMed Central

2014-01-01

Background Diabetes mellitus is a chronic metabolic disorder characterized by increased blood glucose level. It has become an epidemic disease in the 21st century where, India leads the world with largest number of diabetic subjects. Non-enzymatic glycosylation (glycation) is severe form of diabetes, occurs between reducing sugar and proteins which results in the formation of advanced glycation end products (AGEs) that leads to the other complicated secondary disorders. In this context, Mangifera indica (Mango), Syzygium cumini (Jambul), Vitis vinifera (Grapes), Citrus sinensis (Orange), Artocarpus heterophyllus (Jackfruit), Manilkara zapota (Sapodilla) seeds were evaluated for their antiglyation activity. Attempts were made to isolate the polyphenols in the seeds that have recorded the maximum activity. Methods Different extraction methods (shake flask, centrifugation and pressurized hot water) using various extractants (organic solvents, hot water and pressurized hot water) were adopted to investigate the in vitro antiglycation activity. Central composite (CCD) design based Response Surface Methodology (RSM) was espoused to optimize the extraction process of polyphenols from the fruit seeds that have recorded poor antiglycation activity. The PTLC analysis was performed to isolate the polyphenols (Flavonoids and phenolic acids) and LC-PDA-MS analysis was done for structure prediction. Results Pressurized hot water extraction of Artocarpus heterophyllus (87.52%) and Citrus sinensis seeds (74.79%) was found to possess high and low antiglycation activity, respectively. The RSM mediated optimization process adopted for the Citrus sinensis seeds have revealed that 1:15 solvent ratio (hexane to heptane), 6 minutes and 1:20 solid to liquid ratio as the optimal conditions for the extraction of polyphenols with a maximum antiglycation activity (89.79%). The LC-PDA-MS analysis of preparative thin layer chromatography (PTLC) eluates of Artocarpus heterophyllus seed has showed the presence of compounds like quercetin (301.2), 4-hydroxy phenyl acetic acid (149.0), rhamnosyl-di-hexosyl quercetin sulphate (857.6), quercetin-3-O-xyloside (428.2), rutin (613.4), diosmetin (298.1) and luteolin (283.0). Conclusion The Artocarpus heterophyllus was observed to possess a significant antiglycation activity and the activity of Citrus sinensis was improved after the optimization process, which proved that both the seeds may be used as a traditional medicine in the management of chronic diabetes mellitus. PMID:24397983

Extraction process optimization of polyphenols from Indian Citrus sinensis - as novel antiglycative agents in the management of diabetes mellitus.

PubMed

Shakthi Deve, Asaithambi; Sathish Kumar, Thiyagarajan; Kumaresan, Kuppamuthu; Rapheal, Vinohar Stephen

2014-01-07

Diabetes mellitus is a chronic metabolic disorder characterized by increased blood glucose level. It has become an epidemic disease in the 21st century where, India leads the world with largest number of diabetic subjects. Non-enzymatic glycosylation (glycation) is severe form of diabetes, occurs between reducing sugar and proteins which results in the formation of advanced glycation end products (AGEs) that leads to the other complicated secondary disorders. In this context, Mangifera indica (Mango), Syzygium cumini (Jambul), Vitis vinifera (Grapes), Citrus sinensis (Orange), Artocarpus heterophyllus (Jackfruit), Manilkara zapota (Sapodilla) seeds were evaluated for their antiglyation activity. Attempts were made to isolate the polyphenols in the seeds that have recorded the maximum activity. Different extraction methods (shake flask, centrifugation and pressurized hot water) using various extractants (organic solvents, hot water and pressurized hot water) were adopted to investigate the in vitro antiglycation activity. Central composite (CCD) design based Response Surface Methodology (RSM) was espoused to optimize the extraction process of polyphenols from the fruit seeds that have recorded poor antiglycation activity. The PTLC analysis was performed to isolate the polyphenols (Flavonoids and phenolic acids) and LC-PDA-MS analysis was done for structure prediction. Pressurized hot water extraction of Artocarpus heterophyllus (87.52%) and Citrus sinensis seeds (74.79%) was found to possess high and low antiglycation activity, respectively. The RSM mediated optimization process adopted for the Citrus sinensis seeds have revealed that 1:15 solvent ratio (hexane to heptane), 6 minutes and 1:20 solid to liquid ratio as the optimal conditions for the extraction of polyphenols with a maximum antiglycation activity (89.79%). The LC-PDA-MS analysis of preparative thin layer chromatography (PTLC) eluates of Artocarpus heterophyllus seed has showed the presence of compounds like quercetin (301.2), 4-hydroxy phenyl acetic acid (149.0), rhamnosyl-di-hexosyl quercetin sulphate (857.6), quercetin-3-O-xyloside (428.2), rutin (613.4), diosmetin (298.1) and luteolin (283.0). The Artocarpus heterophyllus was observed to possess a significant antiglycation activity and the activity of Citrus sinensis was improved after the optimization process, which proved that both the seeds may be used as a traditional medicine in the management of chronic diabetes mellitus.

Enzyme-assisted extraction enhancing the umami taste amino acids recovery from several cultivated mushrooms.

PubMed

Poojary, Mahesha M; Orlien, Vibeke; Passamonti, Paolo; Olsen, Karsten

2017-11-01

In this study, enzyme-assisted extraction was performed to extract umami taste and total free amino acids (FAAs) from the six different mushrooms including shiitake (Lentinus edodes), oyster (Pleurotus ostreatus), tea tree (Agrocybe aegerita) and, white, brown and portobello champignons (Agaricus bisporus). β-Glucanase and Flavourzyme® were used as the enzymes for cell wall and proteins hydrolysis, respectively. It was found that β-glucanase treatment alone did not enhance the extraction efficiency, however in combination, β-glucanase and Flavourzyme® enhanced the extraction efficiency significantly up to 20-fold compared to conventional HCl mediated extraction, depending on the mushroom species. The optimal conditions for the enzyme treatment were: water as extraction solvent (initial pH = 7), enzyme concentration of 5% v/w each of β-glucanase and Flavourzyme®, temperature 50°C and an incubation time of 1h. White and brown champignons were found to be the richest source of umami taste FAAs (26.75±1.07 and 25.6±0.9mg/g DM, respectively). Copyright © 2017 Elsevier Ltd. All rights reserved.

Optimization of squalene produced from crude palm oil waste

NASA Astrophysics Data System (ADS)

Wandira, Irda; Legowo, Evita H.; Widiputri, Diah I.

2017-01-01

Squalene is a hydrocarbon originally and still mostly extracted from shark liver oil. Due to environmental issues over shark hunting, there have been efforts to extract squalene from alternative sources, such as Palm Fatty Acid Distillate (PFAD), one of crude palm oil (CPO) wastes. Previous researches have shown that squalene can be extracted from PFAD using saponification process followed with liquid-liquid extraction process although the method had yet to be optimized in order to optimize the amount of squalene extracted from PFAD. The optimization was done by optimizing both processes of squalene extraction method: saponification and liquid-liquid extraction. The factors utilized in the saponification process optimization were KOH concentration and saponification duration while during the liquid-liquid extraction (LLE) process optimization, the factors used were the volumes of distilled water and dichloromethane. The optimum percentage of squalene content in the extract (24.08%) was achieved by saponifying the PFAD with 50%w/v KOH for 60 minutes and subjecting the saponified PFAD to LLE, utilizing 100 ml of distilled water along with 3 times addition of fresh dichloromethane, 75 ml each; those factors would be utilized in the optimum squalene extraction method.

Characterization of the immune response of domestic fowl following immunization with proteins extracted from Dermanyssus gallinae.

PubMed

Harrington, David; Din, Hatem Mohi El; Guy, Jonathan; Robinson, Karen; Sparagano, Olivier

2009-03-23

Dermanyssus gallinae is the most significant ectoparasite of European poultry egg laying production systems due to high costs of control and associated production losses as well as adverse effects on bird welfare. In this study, soluble proteins were extracted from unfed D. gallinae (DGE) using a urea-based detergent and ultra-filtration, passed through a 0.22 microm filter and blended aseptically with adjuvant. One group of laying hens was immunized with DGE and adjuvant (Montanide ISA 50 V) whilst another group (Control) received physiological saline and adjuvant. All birds were immunized on two occasions, 21 days apart. Antibody response to immunization was determined by ELISA and western blotting using immunoglobulins (Igs) extracted from egg yolk. DGE immunization of hens resulted in a significant (P<0.05) IgY response compared to controls, although there was no significant difference in IgM response between treatments. A number of proteins were identified by western blotting using IgY antibodies from DGE immunized birds, most prominently at 40 and 230kDa. Analysis of proteins from approximately corresponding bands on SDS-PAGE confirmed the identity of tropomyosin, whilst other proteins showed high sequence homology with myosin and actin from other arachnid and insect species. Immunization of hens with DGE resulted in a 50.6% increase in mite mortality (P<0.001) 17h after feeding when tested by an in vitro mite feeding model. Data in this study demonstrate that somatic antigens from D. gallinae can be used to stimulate a protective immune response in laying hens. Further work is needed to identify other proteins of interest that could confer higher protection against D. gallinae, as well as optimization of the vaccination and in vitro testing protocol.

[Optimization of dissolution process for superfine grinding technology on total saponins of Panax ginseng fibrous root by response surface methodology].

PubMed

Zhao, Ya; Lai, Xiao-Pin; Yao, Hai-Yan; Zhao, Ran; Wu, Yi-Na; Li, Geng

2014-03-01

To investigate the effects of superfine comminution extraction technology of ginseng total saponins from Panax ginseng fibrous root, and to make sure the optimal extraction condition. Optimal condition of ginseng total saponins from Panax ginseng fibrous root was based on single factor experiment to study the effects of crushing degree, extraction time, alcohol concentration and extraction temperature on extraction rate. Response surface method was used to investigate three main factors such as superfine comminution time, extraction time and alcohol concentration. The relationship between content of ginseng total saponins in Panax ginseng fibrous root and three factors fitted second degree polynomial models. The optimal extraction condition was 9 min of superfine comminution time, 70% of alcohol, 50 degrees C of extraction temperature and 70 min of extraction time. Under the optimal condition, ginseng total saponins from Panax ginseng fibrous root was average 94. 81%, which was consistent with the predicted value. The optimization of technology is rapid, efficient, simple and stable.

Optimization of a Bioluminescence Resonance Energy Transfer-Based Assay for Screening of Trypanosoma cruzi Protein/Protein Interaction Inhibitors.

PubMed

Mild, Jesica G; Fernandez, Lucia R; Gayet, Odile; Iovanna, Juan; Dusetti, Nelson; Edreira, Martin M

2018-05-01

Chagas disease, a parasitic disease caused by Trypanosoma cruzi, is a major public health burden in poor rural populations of Central and South America and a serious emerging threat outside the endemic region, since the number of infections in non-endemic countries continues to rise. In order to develop more efficient anti-trypanosomal treatments to replace the outdated therapies, new molecular targets need to be explored and new drugs discovered. Trypanosoma cruzi has distinctive structural and functional characteristics with respect to the human host. These exclusive features could emerge as interesting drug targets. In this work, essential and differential protein-protein interactions for the parasite, including the ribosomal P proteins and proteins involved in mRNA processing, were evaluated in a bioluminescence resonance energy transfer-based assay as a starting point for drug screening. Suitable conditions to consider using this simple and robust methodology to screening compounds and natural extracts able to inhibit protein-protein interactions were set in living cells and lysates.

Depth Filters Containing Diatomite Achieve More Efficient Particle Retention than Filters Solely Containing Cellulose Fibers

PubMed Central

Buyel, Johannes F.; Gruchow, Hannah M.; Fischer, Rainer

2015-01-01

The clarification of biological feed stocks during the production of biopharmaceutical proteins is challenging when large quantities of particles must be removed, e.g., when processing crude plant extracts. Single-use depth filters are often preferred for clarification because they are simple to integrate and have a good safety profile. However, the combination of filter layers must be optimized in terms of nominal retention ratings to account for the unique particle size distribution in each feed stock. We have recently shown that predictive models can facilitate filter screening and the selection of appropriate filter layers. Here we expand our previous study by testing several filters with different retention ratings. The filters typically contain diatomite to facilitate the removal of fine particles. However, diatomite can interfere with the recovery of large biopharmaceutical molecules such as virus-like particles and aggregated proteins. Therefore, we also tested filtration devices composed solely of cellulose fibers and cohesive resin. The capacities of both filter types varied from 10 to 50 L m−2 when challenged with tobacco leaf extracts, but the filtrate turbidity was ~500-fold lower (~3.5 NTU) when diatomite filters were used. We also tested pre–coat filtration with dispersed diatomite, which achieved capacities of up to 120 L m−2 with turbidities of ~100 NTU using bulk plant extracts, and in contrast to the other depth filters did not require an upstream bag filter. Single pre-coat filtration devices can thus replace combinations of bag and depth filters to simplify the processing of plant extracts, potentially saving on time, labor and consumables. The protein concentrations of TSP, DsRed and antibody 2G12 were not affected by pre-coat filtration, indicating its general applicability during the manufacture of plant-derived biopharmaceutical proteins. PMID:26734037

Depth Filters Containing Diatomite Achieve More Efficient Particle Retention than Filters Solely Containing Cellulose Fibers.

PubMed

Buyel, Johannes F; Gruchow, Hannah M; Fischer, Rainer

2015-01-01

The clarification of biological feed stocks during the production of biopharmaceutical proteins is challenging when large quantities of particles must be removed, e.g., when processing crude plant extracts. Single-use depth filters are often preferred for clarification because they are simple to integrate and have a good safety profile. However, the combination of filter layers must be optimized in terms of nominal retention ratings to account for the unique particle size distribution in each feed stock. We have recently shown that predictive models can facilitate filter screening and the selection of appropriate filter layers. Here we expand our previous study by testing several filters with different retention ratings. The filters typically contain diatomite to facilitate the removal of fine particles. However, diatomite can interfere with the recovery of large biopharmaceutical molecules such as virus-like particles and aggregated proteins. Therefore, we also tested filtration devices composed solely of cellulose fibers and cohesive resin. The capacities of both filter types varied from 10 to 50 L m(-2) when challenged with tobacco leaf extracts, but the filtrate turbidity was ~500-fold lower (~3.5 NTU) when diatomite filters were used. We also tested pre-coat filtration with dispersed diatomite, which achieved capacities of up to 120 L m(-2) with turbidities of ~100 NTU using bulk plant extracts, and in contrast to the other depth filters did not require an upstream bag filter. Single pre-coat filtration devices can thus replace combinations of bag and depth filters to simplify the processing of plant extracts, potentially saving on time, labor and consumables. The protein concentrations of TSP, DsRed and antibody 2G12 were not affected by pre-coat filtration, indicating its general applicability during the manufacture of plant-derived biopharmaceutical proteins.

[Optimization of Extraction Technology for Sericin from Silkworm Cocoon with Orthogonal Design].

PubMed

Zhao, Chun-ying; Wang, Yan; Li, Yun-feng; Chen, Zhi-hong

2015-05-01

To optimize the appropriate extracting technology for sericin from Silkworm cocoon. Using sericin extraction rates and sericin content as the indices. The single and orthogonal experiments were used to determine the best conditions. The optimal extraction technology for sericin from Silkworm cocoon was as follows: 1: 30 for the ratio of solid to liquid, 3 h reflux for 2 times of extraction and water temperature at 100 degrees C. The extraction rate of sericin from Silkworm cocoon was 27.1%. The optimal extraction technology is stable, feasible, and can provide reference for further pharmacological study on cocoon sericin.

Characterization of pectic polysaccharides extracted from apple pomace by hot-compressed water.

PubMed

Wang, Xin; Lü, Xin

2014-02-15

Response surface methodology (RSM) was used to optimize the extraction of pectic polysaccharides from apple pomace by hot-compressed water, by which the optimum levels of the parameters were obtained as follows: extraction temperature 140 °C, extraction time 5 min, S:W ratio 1:14. Compared with commercial pectin, the Mw, galacturonic acid content, DM and protein of the extracted pectic polysaccharides were lower while ash content and neutral sugars were higher. The endothermic transition temperature and fusion heat of the extracted pectic polysaccharides was lower than commercial one according to DSC analysis. For its rheological properties, it was found that the viscosity of the extracted pectic polysaccharides solution was slightly lower than commercial pectin at lower shear rate region while it decreased sharply when the shear rate increased. Besides, both G' and G" moduli of the extracted pectic polysaccharides were lower than the commercial pectin's possibly because of weaker polymer chain interaction, which was also reflected in gel textural properties. However, the extracted pectic polysaccharides showed higher in vitro antioxidant capability and inhibitory effect on HT-29 colon adenocarcinoma cells than commercial pectin. Copyright © 2013 Elsevier Ltd. All rights reserved.

Seafood-like flavour obtained from the enzymatic hydrolysis of the protein by-products of seaweed (Gracilaria sp.).

PubMed

Laohakunjit, Natta; Selamassakul, Orrapun; Kerdchoechuen, Orapin

2014-09-01

An enzymatic bromelain seaweed protein hydrolysate (eb-SWPH) was characterised as the precursor for thermally processed seafood flavour. Seaweed (Gracilaria fisheri) protein after agar extraction was hydrolysed using bromelain (enzyme activity=119,325 U/g) at 0-20% (w/w) for 0.5-24 h. Optimal hydrolysis conditions were determined using response surface methodology. The proposed model took into account the interaction effect of the enzyme concentration and hydrolysis time on the physicochemical properties and volatile components of eb-SWPH. The optimal hydrolysis conditions for the production of eb-SWPH were 10% bromelain for 3h, which resulted in a 38.15% yield and a 62.91% degree of hydrolysis value. Three free amino acids, arginine, lysine, and leucine, were abundant in the best hydrolysate. Ten volatile flavours of the best eb-SWPH were identified using gas chromatography/mass spectrometry. The predominant odourants were hexanal, hexanoic acid, nonanoic acid, and dihydroactinidiolide. The thermally processed seafood flavour produced from eb-SWPH exhibited a roasted seafood-like flavouring. Copyright © 2014 Elsevier Ltd. All rights reserved.

pLoc-mPlant: predict subcellular localization of multi-location plant proteins by incorporating the optimal GO information into general PseAAC.

PubMed

Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

2017-08-22

One of the fundamental goals in cellular biochemistry is to identify the functions of proteins in the context of compartments that organize them in the cellular environment. To realize this, it is indispensable to develop an automated method for fast and accurate identification of the subcellular locations of uncharacterized proteins. The current study is focused on plant protein subcellular location prediction based on the sequence information alone. Although considerable efforts have been made in this regard, the problem is far from being solved yet. Most of the existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions. This kind of multiplex protein is particularly important for both basic research and drug design. Using the multi-label theory, we present a new predictor called "pLoc-mPlant" by extracting the optimal GO (Gene Ontology) information into the Chou's general PseAAC (Pseudo Amino Acid Composition). Rigorous cross-validation on the same stringent benchmark dataset indicated that the proposed pLoc-mPlant predictor is remarkably superior to iLoc-Plant, the state-of-the-art method for predicting plant protein subcellular localization. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at , by which users can easily get their desired results without the need to go through the complicated mathematics involved.

pLoc-mVirus: Predict subcellular localization of multi-location virus proteins via incorporating the optimal GO information into general PseAAC.

PubMed

Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

2017-09-10

Knowledge of subcellular locations of proteins is crucially important for in-depth understanding their functions in a cell. With the explosive growth of protein sequences generated in the postgenomic age, it is highly demanded to develop computational tools for timely annotating their subcellular locations based on the sequence information alone. The current study is focused on virus proteins. Although considerable efforts have been made in this regard, the problem is far from being solved yet. Most existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions. This kind of multiplex proteins is particularly important for both basic research and drug design. Using the multi-label theory, we present a new predictor called "pLoc-mVirus" by extracting the optimal GO (Gene Ontology) information into the general PseAAC (Pseudo Amino Acid Composition). Rigorous cross-validation on a same stringent benchmark dataset indicated that the proposed pLoc-mVirus predictor is remarkably superior to iLoc-Virus, the state-of-the-art method in predicting virus protein subcellular localization. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc-mVirus/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. Copyright © 2017 Elsevier B.V. All rights reserved.

A Straightforward and Highly Efficient Precipitation/On-pellet Digestion Procedure Coupled to a Long Gradient Nano-LC Separation and Orbitrap Mass Spectrometry for Label-free Expression Profiling of the Swine Heart Mitochondrial Proteome

PubMed Central

Duan, Xiaotao; Young, Rebecca; Straubinger, Robert M.; Page, Brian J.; Cao, Jin; Wang, Hao; Yu, Haoying; Canty, John M.; Qu, Jun

2009-01-01

For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/in-gel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals vs. those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n=10), and samples were analyzed in random order to minimize quantitative false-positives. Using this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were compared with the results of a parallel 2D-DIGE analysis. The sample preparation and analytical strategy developed here represents an advancement that can be adapted to analyze other tissue proteomes. PMID:19290621

[Optimization for supercritical CO2 extraction with response surface methodology of Prunus armeniaca oil].

PubMed

Chen, Fei-Fei; Wu, Yan; Ge, Fa-Huan

2012-03-01

To optimize the extraction conditions of Prunus armeniaca oil by Supercritical CO2 extraction and identify its components by GC-MS. Optimized of SFE-CO extraction by response surface methodology and used GC-MS to analysis Prunus armeniaca oil compounds. Established the model of an equation for the extraction rate of Prunus armeniaca oil by supercritical CO2 extraction, and the optimal parameters for the supercritical CO2 extraction determined by the equation were: the extraction pressure was 27 MPa, temperature was 39 degrees C, the extraction rate of Prunus armeniaca oil was 44.5%. 16 main compounds of Prunus armeniaca oil extracted by supercritical CO2 were identified by GC-MS, unsaturated fatty acids were 92.6%. This process is simple, and can be used for the extraction of Prunus armeniaca oil.

Automated structure determination of proteins with the SAIL-FLYA NMR method.

PubMed

Takeda, Mitsuhiro; Ikeya, Teppei; Güntert, Peter; Kainosho, Masatsune

2007-01-01

The labeling of proteins with stable isotopes enhances the NMR method for the determination of 3D protein structures in solution. Stereo-array isotope labeling (SAIL) provides an optimal stereospecific and regiospecific pattern of stable isotopes that yields sharpened lines, spectral simplification without loss of information, and the ability to collect rapidly and evaluate fully automatically the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as those that can be analyzed using conventional methods. Here, we describe a protocol for the preparation of SAIL proteins by cell-free methods, including the preparation of S30 extract and their automated structure analysis using the FLYA algorithm and the program CYANA. Once efficient cell-free expression of the unlabeled or uniformly labeled target protein has been achieved, the NMR sample preparation of a SAIL protein can be accomplished in 3 d. A fully automated FLYA structure calculation can be completed in 1 d on a powerful computer system.

Extraction optimization of mucilage from Basil (Ocimum basilicum L.) seeds using response surface methodology.

PubMed

Nazir, Sadaf; Wani, Idrees Ahmed; Masoodi, Farooq Ahmad

2017-05-01

Aqueous extraction of basil seed mucilage was optimized using response surface methodology. A Central Composite Rotatable Design (CCRD) for modeling of three independent variables: temperature (40-91 °C); extraction time (1.6-3.3 h) and water/seed ratio (18:1-77:1) was used to study the response for yield. Experimental values for extraction yield ranged from 7.86 to 20.5 g/100 g. Extraction yield was significantly ( P  < 0.05) affected by all the variables. Temperature and water/seed ratio were found to have pronounced effect while the extraction time was found to have minor possible effects. Graphical optimization determined the optimal conditions for the extraction of mucilage. The optimal condition predicted an extraction yield of 20.49 g/100 g at 56.7 °C, 1.6 h, and a water/seed ratio of 66.84:1. Optimal conditions were determined to obtain highest extraction yield. Results indicated that water/seed ratio was the most significant parameter, followed by temperature and time.

"One-pot" ethyl chloroformate derivatization and liquid-liquid extraction of reduced glutathione in erythrocyte and its quantitative GC-MS analysis.

PubMed

Manca, Alessandra; Alladio, Eugenio; Massarenti, Paola; Puccinelli, M Paola; De Francesco, Antonella; Del Grosso, Erika; Mengozzi, Giulio; Pazzi, Marco; Vincenti, Marco

2017-12-01

A simple "one-pot" derivatization and liquid-liquid extraction (LLE) procedure was developed for GC-MS analysis of reduced glutathione (GSH) analysis in erythrocytes. The metabolite was extracted by 5% (w/v) TCA, the supernatant treated with ECF and ethanol-pyridine media, the derivative separated and detected by gas chromatography-mass spectrometry using a short non-polar capillary GC column at a high column-head pressure. Total analysis time was 11min. The process was optimized by a Design of Experiment. The method was validated showing a good linearity over the 25.4-813.4μM concentration range, providing satisfactory results in terms of intra-day and inter-day precision as well as an optimal accuracy. The new method was evaluated in a pilot study involving patients with severe protein malnutrition. Comparison of this group with a group of healthy subjects revealed significantly lower GSH concentrations in erythrocytes in the former, thus proving that the described GC-MS method could be employed for fast and simple GSH analysis in clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimization of Pressurized Liquid Extraction of Three Major Acetophenones from Cynanchum bungei Using a Box-Behnken Design

PubMed Central

Li, Wei; Zhao, Li-Chun; Sun, Yin-Shi; Lei, Feng-Jie; Wang, Zi; Gui, Xiong-Bin; Wang, Hui

2012-01-01

In this work, pressurized liquid extraction (PLE) of three acetophenones (4-hydroxyacetophenone, baishouwubenzophenone, and 2,4-dihydroxyacetophenone) from Cynanchum bungei (ACB) were investigated. The optimal conditions for extraction of ACB were obtained using a Box-Behnken design, consisting of 17 experimental points, as follows: Ethanol (100%) as the extraction solvent at a temperature of 120 °C and an extraction pressure of 1500 psi, using one extraction cycle with a static extraction time of 17 min. The extracted samples were analyzed by high-performance liquid chromatography using an UV detector. Under this optimal condition, the experimental values agreed with the predicted values by analysis of variance. The ACB extraction yield with optimal PLE was higher than that obtained by soxhlet extraction and heat-reflux extraction methods. The results suggest that the PLE method provides a good alternative for acetophenone extraction. PMID:23203079

Selection of an Appropriate Protein Extraction Method to Study the Phosphoproteome of Maize Photosynthetic Tissue

PubMed Central

Luís, Inês M.; Alexandre, Bruno M.; Oliveira, M. Margarida

2016-01-01

Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK. PMID:27727304

Evidence-based recommendations for optimal dietary protein intake in older people: a position paper from the PROT-AGE Study Group.

PubMed

Bauer, Jürgen; Biolo, Gianni; Cederholm, Tommy; Cesari, Matteo; Cruz-Jentoft, Alfonso J; Morley, John E; Phillips, Stuart; Sieber, Cornel; Stehle, Peter; Teta, Daniel; Visvanathan, Renuka; Volpi, Elena; Boirie, Yves

2013-08-01

New evidence shows that older adults need more dietary protein than do younger adults to support good health, promote recovery from illness, and maintain functionality. Older people need to make up for age-related changes in protein metabolism, such as high splanchnic extraction and declining anabolic responses to ingested protein. They also need more protein to offset inflammatory and catabolic conditions associated with chronic and acute diseases that occur commonly with aging. With the goal of developing updated, evidence-based recommendations for optimal protein intake by older people, the European Union Geriatric Medicine Society (EUGMS), in cooperation with other scientific organizations, appointed an international study group to review dietary protein needs with aging (PROT-AGE Study Group). To help older people (>65 years) maintain and regain lean body mass and function, the PROT-AGE study group recommends average daily intake at least in the range of 1.0 to 1.2 g protein per kilogram of body weight per day. Both endurance- and resistance-type exercises are recommended at individualized levels that are safe and tolerated, and higher protein intake (ie, ≥ 1.2 g/kg body weight/d) is advised for those who are exercising and otherwise active. Most older adults who have acute or chronic diseases need even more dietary protein (ie, 1.2-1.5 g/kg body weight/d). Older people with severe kidney disease (ie, estimated GFR <30 mL/min/1.73 m(2)), but who are not on dialysis, are an exception to this rule; these individuals may need to limit protein intake. Protein quality, timing of ingestion, and intake of other nutritional supplements may be relevant, but evidence is not yet sufficient to support specific recommendations. Older people are vulnerable to losses in physical function capacity, and such losses predict loss of independence, falls, and even mortality. Thus, future studies aimed at pinpointing optimal protein intake in specific populations of older people need to include measures of physical function. Copyright © 2013 American Medical Directors Association, Inc. Published by Elsevier Inc. All rights reserved.

Box-Behnken design for investigation of microwave-assisted extraction of patchouli oil

NASA Astrophysics Data System (ADS)

Kusuma, Heri Septya; Mahfud, Mahfud

2015-12-01

Microwave-assisted extraction (MAE) technique was employed to extract the essential oil from patchouli (Pogostemon cablin). The optimal conditions for microwave-assisted extraction of patchouli oil were determined by response surface methodology. A Box-Behnken design (BBD) was applied to evaluate the effects of three independent variables (microwave power (A: 400-800 W), plant material to solvent ratio (B: 0.10-0.20 g mL-1) and extraction time (C: 20-60 min)) on the extraction yield of patchouli oil. The correlation analysis of the mathematical-regression model indicated that quadratic polynomial model could be employed to optimize the microwave extraction of patchouli oil. The optimal extraction conditions of patchouli oil was microwave power 634.024 W, plant material to solvent ratio 0.147648 g ml-1 and extraction time 51.6174 min. The maximum patchouli oil yield was 2.80516% under these optimal conditions. Under the extraction condition, the experimental values agreed with the predicted results by analysis of variance. It indicated high fitness of the model used and the success of response surface methodology for optimizing and reflect the expected extraction condition.

Intercellular Distribution of Glutathione Synthesis in Maize Leaves and Its Response to Short-Term Chilling1

PubMed Central

Gómez, Leonardo D.; Vanacker, Hélène; Buchner, Peter; Noctor, Graham; Foyer, Christine H.

2004-01-01

To investigate the intercellular control of glutathione synthesis and its influence on leaf redox state in response to short-term chilling, genes encoding γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GSH-S) were cloned from maize (Zea mays) and specific antibodies produced. These tools were used to provide the first information on the intercellular distribution of γ-ECS and GSH-S transcript and protein in maize leaves, in both optimal conditions and chilling stress. A 2-d exposure to low growth temperatures (chill) had no effect on leaf phenotype, whereas return to optimal temperatures (recovery) caused extensive leaf bleaching. The chill did not affect total leaf GSH-S transcripts but strongly induced γ-ECS mRNA, an effect reversed during recovery. The chilling-induced increase in γ-ECS transcripts was not accompanied by enhanced total leaf γ-ECS protein or extractable activity. In situ hybridization and immunolocalization of leaf sections showed that γ-ECS and GSH-S transcripts and proteins were found in both the bundle sheath (BS) and the mesophyll cells under optimal conditions. Chilling increased γ-ECS transcript and protein in the BS but not in the mesophyll cells. Increased BS γ-ECS was correlated with a 2-fold increase in both leaf Cys and γ-glutamylcysteine, but leaf total glutathione significantly increased only in the recovery period, when the reduced glutathione to glutathione disulfide ratio decreased 3-fold. Thus, while there was a specific increase in the potential contribution of the BS cells to glutathione synthesis during chilling, it did not result in enhanced leaf glutathione accumulation at low temperatures. Return to optimal temperatures allowed glutathione to increase, particularly glutathione disulfide, and this was associated with leaf chlorosis. PMID:15047902

Perennial peanut (Arachis glabrata Benth.) contains polyphenol oxidase (PPO) and PPO substrates that can reduce post-harvest proteolysis.

PubMed

Sullivan, Michael L; Foster, Jamie L

2013-08-15

Studies of perennial peanut (Arachis glabrata Benth.) suggest its hay and haylage have greater levels of rumen undegraded protein (RUP) than other legume forages such as alfalfa (Medicago sativa L.). Greater RUP can result in more efficient nitrogen utilization by ruminant animals with positive economic and environmental effects. We sought to determine whether, like red clover (Trifolium pretense L.), perennial peanut contains polyphenol oxidase (PPO) and PPO substrates that might be responsible for increased RUP. Perennial peanut extracts contain immunologically detectible PPO protein and high levels of PPO activity (>100 nkatal mg(-1) protein). Addition of caffeic acid (PPO substrate) to perennial peanut extracts depleted of endogenous substrates reduced proteolysis by 90%. Addition of phenolics prepared from perennial peanut leaves to extracts of either transgenic PPO-expressing or control (non-expressing) alfalfa showed peanut phenolics could reduce proteolysis >70% in a PPO-dependent manner. Two abundant likely PPO substrates are present in perennial peanut leaves including caftaric acid. Perennial peanut contains PPO and PPO substrates that together are capable of inhibiting post-harvest proteolysis, suggesting a possible mechanism for increased RUP in this forage. Research related to optimizing the PPO system in other forage crops will likely be applicable to perennial peanut. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Research on Optimal Observation Scale for Damaged Buildings after Earthquake Based on Optimal Feature Space

NASA Astrophysics Data System (ADS)

Chen, J.; Chen, W.; Dou, A.; Li, W.; Sun, Y.

2018-04-01

A new information extraction method of damaged buildings rooted in optimal feature space is put forward on the basis of the traditional object-oriented method. In this new method, ESP (estimate of scale parameter) tool is used to optimize the segmentation of image. Then the distance matrix and minimum separation distance of all kinds of surface features are calculated through sample selection to find the optimal feature space, which is finally applied to extract the image of damaged buildings after earthquake. The overall extraction accuracy reaches 83.1 %, the kappa coefficient 0.813. The new information extraction method greatly improves the extraction accuracy and efficiency, compared with the traditional object-oriented method, and owns a good promotional value in the information extraction of damaged buildings. In addition, the new method can be used for the information extraction of different-resolution images of damaged buildings after earthquake, then to seek the optimal observation scale of damaged buildings through accuracy evaluation. It is supposed that the optimal observation scale of damaged buildings is between 1 m and 1.2 m, which provides a reference for future information extraction of damaged buildings.

Ultrasound-assisted enzymatic hydrolysis for iodinated amino acid extraction from edible seaweed before reversed-phase high performance liquid chromatography-inductively coupled plasma-mass spectrometry.

PubMed

Romarís-Hortas, Vanessa; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

2013-09-27

The combination of reverse phase high performance liquid chromatography (RP-HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) was used for the determination of monoiodotyrosine (MIT) and diiodotyrosine (DIT) in edible seaweed. A sample pre-treatment based on ultrasound assisted enzymatic hydrolysis was optimized for the extraction of these iodinated amino acids. Pancreatin was selected as the most adequate type of enzyme, and parameters affecting the extraction efficiency (pH, temperature, mass of enzyme and extraction time) were evaluated by univariate approaches. In addition, extractable inorganic iodine (iodide) was also quantified by anion exchange high performance liquid chromatography (AE-HPLC) coupled with ICP-MS. The proposed procedure offered limits of detection of 1.1 and 4.3ngg(-1) for MIT and DIT, respectively. Total iodine contents in seaweed, as well as total iodine in enzymatic digests were measured by ICP-MS after microwave assisted alkaline digestion with tetramethylamonium hydroxide (TMAH) for total iodine assessment, and also by treating the pancreatin extracts (extractable total iodine assessment). The optimized procedure was successfully applied to five different types of edible seaweed. The highest total iodine content, and also the highest iodide levels, was found in the brown seaweed Kombu (6646±45μgg(-1)). Regarding iodinated amino acids, Nori (a red seaweed) was by far the one with the highest amount of both species (42±3 and 0.41±0.024μgg(-1) for MIT and DIT, respectively). In general, MIT concentrations were much higher than the amounts of DIT, which suggests that iodine from iodinated proteins in seaweed is most likely bound in the form of MIT residues. Copyright © 2013 Elsevier B.V. All rights reserved.

Optimization of deep eutectic solvent-based ultrasound-assisted extraction of polysaccharides from Dioscorea opposita Thunb.

PubMed

Zhang, Lijin; Wang, Maoshan

2017-02-01

In this study, deep eutectic solvents were proposed for the ultrasound-assisted extraction of polysaccharides from Dioscorea opposita Thunb. Several deep eutectic solvents were prepared for the extraction of polysaccharides, among which the deep eutectic solvent composed of choline chloride and 1,4-butanediol was proved to be suitable for the extraction. Based on the screening of single-factor experiment design and orthogonal experiment design, three experimental factors were optimized for the Box-Behnken experimental design combined with response surface methodology, which gave the optimal extraction conditions: water content of 32.89%(v/v), extraction temperature of 94.00°C, and the extraction time of 44.74min. The optimal extraction conditions could supply higher extraction yield than those of hot water extraction and water-based ultrasound-assisted extraction. Therefore, deep eutectic solvents were an excellent extraction solvent alternative to the extraction of polysaccharides from sample matrices. Copyright © 2016 Elsevier B.V. All rights reserved.

Increased frequency of anti-retina antibodies in asymptomatic patients with chronic t. gondii infection

PubMed Central

Cursino, Sylvia Regina Temer; da Costa, Thaís Boccia; Yamamoto, Joyce Hisae; Meireles, Luciana Regina; Silva, Maria Antonieta Longo Galvão; de Andrade Junior, Heitor Franco

2010-01-01

PURPOSE: To search for anti-retina antibodies that serve as markers for eye disease in uveitis. MATERIALS AND METHODS: Stored sera from patients with uveitis, ocular toxoplasmosis (n = 30) and non-infectious, immune-mediated uveitis (n = 50) and from asymptomatic individuals who were positive (n = 250) and negative (n = 250) for anti-Toxoplasma antibodies were tested. Serum anti-retina IgG was detected by an optimized ELISA using a solid-phase whole human retina extract, bovine S-antigen or interphotoreceptor retinoid-binding protein. RESULTS: Uveitis patients showed a higher mean reactivity to whole human retina extract, interphotoreceptor retinoid-binding protein and S-antigen in comparison to the asymptomatic population. These findings were independent of the uveitis origin and allowed the determination of the lower anti-retina antibody cut-off for the three antigens. Asymptomatic anti-Toxoplasma serum-positive individuals showed a higher frequency of anti-human whole retina extract antibodies in comparison to asymptomatic anti-Toxoplasma serum-negative patients. The bovine S-antigen and interphotoreceptor retinoid-binding protein ELISAs also showed a higher mean reactivity in the uveitis groups compared to the asymptomatic group, but the observed reactivities were lower and overlapped without discrimination. CONCLUSION: We detected higher levels of anti-retina antibodies in uveitis patients and in a small fraction of asymptomatic patients with chronic toxoplasmosis. The presence of anti-retina antibodies in sera might be a marker of eye disease in asymptomatic patients, especially when whole human retina extract is used in a solid-phase ELISA. PMID:21120306

pH-dependence of single-protein adsorption and diffusion at a liquid chromatographic interface.

PubMed

Kisley, Lydia; Poongavanam, Mohan-Vivekanandan; Kourentzi, Katerina; Willson, Richard C; Landes, Christy F

2016-02-01

pH is a common mobile phase variable used to control protein separations due to the tunable nature of amino acid and adsorbent charge. Like other column variables such as column density and ligand loading density, pH is usually optimized empirically. Single-molecule spectroscopy extracts molecular-scale data to provide a framework for mechanistic optimization of pH. The adsorption and diffusion of a model globular protein, α-lactalbumin, was studied by single-molecule microscopy at a silica-aqueous interface analogous to aqueous normal phase and hydrophilic interaction chromatography and capillary electrophoresis interfaces at varied pH. Electrostatic repulsion resulting in free diffusion was observed at pH above the isoelectric point of the protein. In contrast, at low pH strong adsorption and surface diffusion with either no (D ∼ 0.01 μm(2) /s) or translational (D ∼ 0.3 μm(2) /s) motion was observed where the protein likely interacted with the surface through electrostatic, hydrophobic, and hydrogen bonding forces. The fraction of proteins immobilized could be increased by lowering the pH. These results show that retention of proteins at the silica interface cannot be viewed solely as an adsorption/desorption process and that the type of surface diffusion, which ultimately leads to ensemble chromatographic separations, can be controlled by tuning long-range electrostatic and short-range hydrophobic and hydrogen bonding forces with pH. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Central composite rotatable design for investigation of microwave-assisted extraction of okra pod hydrocolloid.

PubMed

Samavati, Vahid

2013-10-01

Microwave-assisted extraction (MAE) technique was employed to extract the hydrocolloid from okra pods (OPH). The optimal conditions for microwave-assisted extraction of OPH were determined by response surface methodology. A central composite rotatable design (CCRD) was applied to evaluate the effects of three independent variables (microwave power (X1: 100-500 W), extraction time (X2: 30-90 min), and extraction temperature (X3: 40-90 °C)) on the extraction yield of OPH. The correlation analysis of the mathematical-regression model indicated that quadratic polynomial model could be employed to optimize the microwave extraction of OPH. The optimal conditions to obtain the highest recovery of OPH (14.911±0.27%) were as follows: microwave power, 395.56 W; extraction time, 67.11 min and extraction temperature, 73.33 °C. Under these optimal conditions, the experimental values agreed with the predicted ones by analysis of variance. It indicated high fitness of the model used and the success of response surface methodology for optimizing OPH extraction. After method development, the DPPH radical scavenging activity of the OPH was evaluated. MAE showed obvious advantages in terms of high extraction efficiency and radical scavenging activity of extract within the shorter extraction time. Copyright © 2013 Elsevier B.V. All rights reserved.

[Optimization of Polysaccharide Extraction from Spirodela polyrrhiza by Plackett-Burman Design Combined with Box-Behnken Response Surface Methodology].

PubMed

Jiang, Zheng; Wang, Hong; Wu, Qi-nan

2015-06-01

To optimize the processing of polysaccharide extraction from Spirodela polyrrhiza. Five factors related to extraction rate of polysaccharide were optimized by the Plackett-Burman design. Based on this study, three factors, including alcohol volume fraction, extraction temperature and ratio of material to liquid, were regarded as investigation factors by Box-Behnken response surface methodology. The effect order of three factors on the extraction rate of polysaccharide from Spirodela polyrrhiza were as follows: extraction temperature, alcohol volume fraction,ratio of material to liquid. According to Box-Behnken response, the best extraction conditions were: alcohol volume fraction of 81%, ratio of material to liquid of 1:42, extraction temperature of 100 degrees C, extraction time of 60 min for four times. Plackett-Burman design and Box-Behnken response surface methodology used to optimize the extraction process for the polysaccharide in this study is effective and stable.

Structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein Rec1-resilin

PubMed Central

Balu, Rajkamal; Knott, Robert; Cowieson, Nathan P.; Elvin, Christopher M.; Hill, Anita J.; Choudhury, Namita R.; Dutta, Naba K.

2015-01-01

Rec1-resilin is the first recombinant resilin-mimetic protein polymer, synthesized from exon-1 of the Drosophila melanogaster gene CG15920 that has demonstrated unusual multi-stimuli responsiveness in aqueous solution. Crosslinked hydrogels of Rec1-resilin have also displayed remarkable mechanical properties including near-perfect rubber-like elasticity. The structural basis of these extraordinary properties is not clearly understood. Here we combine a computational and experimental investigation to examine structural ensembles of Rec1-resilin in aqueous solution. The structure of Rec1-resilin in aqueous solutions is investigated experimentally using circular dichroism (CD) spectroscopy and small angle X-ray scattering (SAXS). Both bench-top and synchrotron SAXS are employed to extract structural data sets of Rec1-resilin and to confirm their validity. Computational approaches have been applied to these experimental data sets in order to extract quantitative information about structural ensembles including radius of gyration, pair-distance distribution function, and the fractal dimension. The present work confirms that Rec1-resilin is an intrinsically disordered protein (IDP) that displays equilibrium structural qualities between those of a structured globular protein and a denatured protein. The ensemble optimization method (EOM) analysis reveals a single conformational population with partial compactness. This work provides new insight into the structural ensembles of Rec1-resilin in solution. PMID:26042819

Structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein Rec1-resilin.

PubMed

Balu, Rajkamal; Knott, Robert; Cowieson, Nathan P; Elvin, Christopher M; Hill, Anita J; Choudhury, Namita R; Dutta, Naba K

2015-06-04

Rec1-resilin is the first recombinant resilin-mimetic protein polymer, synthesized from exon-1 of the Drosophila melanogaster gene CG15920 that has demonstrated unusual multi-stimuli responsiveness in aqueous solution. Crosslinked hydrogels of Rec1-resilin have also displayed remarkable mechanical properties including near-perfect rubber-like elasticity. The structural basis of these extraordinary properties is not clearly understood. Here we combine a computational and experimental investigation to examine structural ensembles of Rec1-resilin in aqueous solution. The structure of Rec1-resilin in aqueous solutions is investigated experimentally using circular dichroism (CD) spectroscopy and small angle X-ray scattering (SAXS). Both bench-top and synchrotron SAXS are employed to extract structural data sets of Rec1-resilin and to confirm their validity. Computational approaches have been applied to these experimental data sets in order to extract quantitative information about structural ensembles including radius of gyration, pair-distance distribution function, and the fractal dimension. The present work confirms that Rec1-resilin is an intrinsically disordered protein (IDP) that displays equilibrium structural qualities between those of a structured globular protein and a denatured protein. The ensemble optimization method (EOM) analysis reveals a single conformational population with partial compactness. This work provides new insight into the structural ensembles of Rec1-resilin in solution.

Structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein Rec1-resilin

NASA Astrophysics Data System (ADS)

Balu, Rajkamal; Knott, Robert; Cowieson, Nathan P.; Elvin, Christopher M.; Hill, Anita J.; Choudhury, Namita R.; Dutta, Naba K.

2015-06-01

Rec1-resilin is the first recombinant resilin-mimetic protein polymer, synthesized from exon-1 of the Drosophila melanogaster gene CG15920 that has demonstrated unusual multi-stimuli responsiveness in aqueous solution. Crosslinked hydrogels of Rec1-resilin have also displayed remarkable mechanical properties including near-perfect rubber-like elasticity. The structural basis of these extraordinary properties is not clearly understood. Here we combine a computational and experimental investigation to examine structural ensembles of Rec1-resilin in aqueous solution. The structure of Rec1-resilin in aqueous solutions is investigated experimentally using circular dichroism (CD) spectroscopy and small angle X-ray scattering (SAXS). Both bench-top and synchrotron SAXS are employed to extract structural data sets of Rec1-resilin and to confirm their validity. Computational approaches have been applied to these experimental data sets in order to extract quantitative information about structural ensembles including radius of gyration, pair-distance distribution function, and the fractal dimension. The present work confirms that Rec1-resilin is an intrinsically disordered protein (IDP) that displays equilibrium structural qualities between those of a structured globular protein and a denatured protein. The ensemble optimization method (EOM) analysis reveals a single conformational population with partial compactness. This work provides new insight into the structural ensembles of Rec1-resilin in solution.

Milk Bottom-Up Proteomics: Method Optimization

PubMed Central

Vincent, Delphine; Ezernieks, Vilnis; Elkins, Aaron; Nguyen, Nga; Moate, Peter J.; Cocks, Benjamin G.; Rochfort, Simone

2016-01-01

Milk is a complex fluid whose proteome displays a diverse set of proteins of high abundance such as caseins and medium to low abundance whey proteins such as ß-lactoglobulin, lactoferrin, immunoglobulins, glycoproteins, peptide hormones, and enzymes. A sample preparation method that enables high reproducibility and throughput is key in reliably identifying proteins present or proteins responding to conditions such as a diet, health or genetics. Using skim milk samples from Jersey and Holstein-Friesian cows, we compared three extraction procedures which have not previously been applied to samples of cows' milk. Method A (urea) involved a simple dilution of the milk in a urea-based buffer, method B (TCA/acetone) involved a trichloroacetic acid (TCA)/acetone precipitation, and method C (methanol/chloroform) involved a tri-phasic partition method in chloroform/methanol solution. Protein assays, SDS-PAGE profiling, and trypsin digestion followed by nanoHPLC-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS) analyses were performed to assess their efficiency. Replicates were used at each analytical step (extraction, digestion, injection) to assess reproducibility. Mass spectrometry (MS) data are available via ProteomeXchange with identifier PXD002529. Overall 186 unique accessions, major and minor proteins, were identified with a combination of methods. Method C (methanol/chloroform) yielded the best resolved SDS-patterns and highest protein recovery rates, method A (urea) yielded the greatest number of accessions, and, of the three procedures, method B (TCA/acetone) was the least compatible of all with a wide range of downstream analytical procedures. Our results also highlighted breed differences between the proteins in milk of Jersey and Holstein-Friesian cows. PMID:26793233

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

PubMed Central

Szymanski, Witold G.; Kierszniowska, Sylwia; Schulze, Waltraud X.

2013-01-01

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures. We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4. PMID:24121251

Expression of rabies glycoprotein and ricin toxin B chain (RGP-RTB) fusion protein in tomato hairy roots: a step towards oral vaccination for rabies.

PubMed

Singh, Ankit; Srivastava, Subhi; Chouksey, Ankita; Panwar, Bhupendra Singh; Verma, Praveen C; Roy, Sribash; Singh, Pradhyumna K; Saxena, Gauri; Tuli, Rakesh

2015-04-01

Transgenic hairy roots of Solanum lycopersicum were engineered to express a recombinant protein containing a fusion of rabies glycoprotein and ricin toxin B chain (rgp-rtxB) antigen under the control of constitutive CaMV35S promoter. Asialofetuin-mediated direct ELISA of transgenic hairy root extracts was performed using polyclonal anti-rabies antibodies (Ab1) and epitope-specific peptidal anti-RGP (Ab2) antibodies which confirmed the expression of functionally viable RGP-RTB fusion protein. Direct ELISA based on asialofetuin-binding activity was used to screen crude protein extracts from five transgenic hairy root lines. Expressions of RGP-RTB fusion protein in different tomato hairy root lines varied between 1.4 and 8 µg in per gram of tissue. Immunoblotting assay of RGP-RTB fusion protein from these lines showed a protein band on monomeric size of ~84 kDa after denaturation. Tomato hairy root line H03 showed highest level of RGP-RTB protein expression (1.14 %) and was used further in bench-top bioreactor for the optimization of scale-up process to produce large quantity of recombinant protein. Partially purified RGP-RTB fusion protein was able to induce the immune response in BALB/c mice after intra-mucosal immunization. In the present investigation, we have not only successfully scaled up the hairy root culture but also established the utility of this system to produce vaccine antigen which subsequently will reduce the total production cost for implementing rabies vaccination programs in developing nations. This study in a way aims to provide consolidated base for low-cost preparation of improved oral vaccine against rabies.

Biorefinery process for protein extraction from oriental mustard (Brassica juncea (L.) Czern.) using ethanol stillage.

PubMed

Ratanapariyanuch, Kornsulee; Tyler, Robert T; Shim, Youn Young; Reaney, Martin Jt

2012-01-12

Large volumes of treated process water are required for protein extraction. Evaporation of this water contributes greatly to the energy consumed in enriching protein products. Thin stillage remaining from ethanol production is available in large volumes and may be suitable for extracting protein rich materials. In this work protein was extracted from ground defatted oriental mustard (Brassica juncea (L.) Czern.) meal using thin stillage. Protein extraction efficiency was studied at pHs between 7.6 and 10.4 and salt concentrations between 3.4 × 10-2 and 1.2 M. The optimum extraction efficiency was pH 10.0 and 1.0 M NaCl. Napin and cruciferin were the most prevalent proteins in the isolate. The isolate exhibited high in vitro digestibility (74.9 ± 0.80%) and lysine content (5.2 ± 0.2 g/100 g of protein). No differences in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability, or amino acid composition were observed between protein extracted with thin stillage and that extracted with NaCl solution. The use of thin stillage, in lieu of water, for protein extraction would decrease the energy requirements and waste disposal costs of the protein isolation and biofuel production processes.

Biorefinery process for protein extraction from oriental mustard (Brassica juncea (L.) Czern.) using ethanol stillage

PubMed Central

2012-01-01

Large volumes of treated process water are required for protein extraction. Evaporation of this water contributes greatly to the energy consumed in enriching protein products. Thin stillage remaining from ethanol production is available in large volumes and may be suitable for extracting protein rich materials. In this work protein was extracted from ground defatted oriental mustard (Brassica juncea (L.) Czern.) meal using thin stillage. Protein extraction efficiency was studied at pHs between 7.6 and 10.4 and salt concentrations between 3.4 × 10-2 and 1.2 M. The optimum extraction efficiency was pH 10.0 and 1.0 M NaCl. Napin and cruciferin were the most prevalent proteins in the isolate. The isolate exhibited high in vitro digestibility (74.9 ± 0.80%) and lysine content (5.2 ± 0.2 g/100 g of protein). No differences in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability, or amino acid composition were observed between protein extracted with thin stillage and that extracted with NaCl solution. The use of thin stillage, in lieu of water, for protein extraction would decrease the energy requirements and waste disposal costs of the protein isolation and biofuel production processes. PMID:22239856

Optimization of papain hydrolysis conditions for release of glycosaminoglycans from the chicken keel cartilage

NASA Astrophysics Data System (ADS)

Le Vien, Nguyen Thi; Nguyen, Pham Bao; Cuong, Lam Duc; An, Trinh Thi Thua; Dao, Dong Thi Anh

2017-09-01

Glycosaminoglycans (GAGs) are natural biocompounds which join to construct cartilage tissuses, it can be extracted from cartilage of sharks, pigs, cows, chickens, etc. GAGs contain a Chondroitin sulfate (CS) content which is a supplement of functional food used for preventing and supporting treatment of arthritis and eye diseases. Therefore, the GAGs extraction from byproducts of the industry of cattle and poultry slaughter to identify the CS content by papain enzyme is necessary. In this study, the optimal hydrolysis conditions were obtained by response surface methodology (RSM). The independent variables were coded as: pH (x1), enzyme concentration (x2), incubation temperature (x3) and hydrolysis time (x4). The results of the analysis of variance (ANOVA) shown that the variables actively affected GAGs content. The optimal conditions of hydrolysis were derived at pH of 7.1, ratio of enzyme per substances of 0.62% w/wpo, temperature of 65°C and hydrolysis time of 230 minutes, GAGs content reached 14.3% of the dry matter of raw material. Analyzes by HPLC revealed that 56.17% of the dry preparations of GAGs were CS compound, were equivalent to 8.11% of the dry matter of chicken keel cartilage. Molecular weight of the dry preparations GAGs was 259.6 kDa. The dry preparations included the contents of moisture 12.2%, protein 8.42%, lipid 0%, ash 10.03% and extracted GAGs 69.35%.

Optimization of conditions for isolation of high quality chitin from shrimp processing raw byproducts using response surface methodology and its characterization.

PubMed

Nidheesh, T; Suresh, P V

2015-06-01

Chitin is one of the most abundant bioactive biopolymer on earth. It is commercially extracted from seafood processing crustacean shell byproducts by harsh thermochemical treatments. The extraction conditions, the source and pretreatment of raw material significantly affect its quality and bioactivity. In this investigation response surface methodology (RSM) has been applied to optimize and evaluate the interaction of variables for extraction of high quality chitin from shrimp processing raw byproducts. Variables such as, concentration of HCl (%, v/v) 4.5 (for wet) and 4.9 (for dry), reaction time 3 h, solid liquid ratio of HCl (w/v) 1:5.5 (for wet) and 1:7.9 (for dry) with two treatments achieved >98 % demineralization of shrimp byproduct. Variables such as, concentration of NaOH 3.6 % (w/v), reaction time 2.5 h, temperature 69.0 ± 1 °C, solid liquid ratio of NaOH 7.4 (w/v) and two treatments accomplished >98 % deproteinization of demineralized byproducts. Significant (p ≤ 0.05-0.001) interactive effects were observed between different variables. Chitin obtained in these conditions had residual content (%, w/w) of ash <0.4 and protein <0.8 and the degree of N-acetylation was >93 % with purity of >98 %. In conclusion, the optimized conditions by RSM can be applied for large scale preparation of high quality chitin from raw shrimp byproduct.

Optimization of β-cyclodextrin-based flavonol extraction from apple pomace using response surface methodology.

PubMed

Parmar, Indu; Sharma, Sowmya; Rupasinghe, H P Vasantha

2015-04-01

The present study investigated five cyclodextrins (CDs) for the extraction of flavonols from apple pomace powder and optimized β-CD based extraction of total flavonols using response surface methodology. A 2(3) central composite design with β-CD concentration (0-5 g 100 mL(-1)), extraction temperature (20-72 °C), extraction time (6-48 h) and second-order quadratic model for the total flavonol yield (mg 100 g(-1) DM) was selected to generate the response surface curves. The optimal conditions obtained were: β-CD concentration, 2.8 g 100 mL(-1); extraction temperature, 45 °C and extraction time, 25.6 h that predicted the extraction of 166.6 mg total flavonols 100 g(-1) DM. The predicted amount was comparable to the experimental amount of 151.5 mg total flavonols 100 g(-1) DM obtained from optimal β-CD based parameters, thereby giving a low absolute error and adequacy of fitted model. In addition, the results from optimized extraction conditions showed values similar to those obtained through previously established solvent based sonication assisted flavonol extraction procedure. To the best of our knowledge, this is the first study to optimize aqueous β-CD based flavonol extraction which presents an environmentally safe method for value-addition to under-utilized bio resources.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Ziyu; Hooker, Brian S.; Anderson, Daniel B.

Optimization of Acidothermus cellulolyticus endoglucanase (E1) gene expression in transgenic potato (Solanum tuberosum L.) was examined in this study, where the E1 coding sequence was transcribed under control of a leaf specific promoter (tomato RbcS-3C) or the Mac promoter (a hybrid promoter of mannopine synthase promoter and cauliflower mosaic virus 35S promoter enhancer region). Average E1 activity in leaf extracts of potato transformants, in which E1 protein was targeted by a chloroplast signal peptide and an apoplast signal peptide were much higher than those by an E1 native signal peptide and a vacuole signal peptide. E1 protein accumulated up tomore » 2.6% of total leaf soluble protein, where E1 gene was under control of the RbcS-3C promoter, alfalfa mosaic virus 5-untranslated leader, and RbcS-2A signal peptide. E1 protein production, based on average E1 activity and E1 protein accumulation in leaf extracts, is higher in potato than those measured previously in transgenic tobacco bearing the same transgene constructs. Comparisons of E1 activity, protein accumulation, and relative mRNA levels showed that E1 expression under control of tomato RbcS-3C promoter was specifically localized in leaf tissues, while E1 gene was expressed in both leaf and tuber tissues under control of Mac promoter. This suggests dual-crop applications in which potato vines serve as enzyme production `bioreactors' while tubers are preserved for culinary applications.« less

Fabrication and evaluation of magnetic phosphodiesterase-5 linked nanoparticles as adsorbent for magnetic dispersive solid-phase extraction of inhibitors from Chinese herbal medicine prior to ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis.

PubMed

Tao, Yi; Gu, Xianghui; Li, Weidong; Cai, Baochang

2018-01-12

In the present study, the preparation of the magnetic phosphodiesterase-5 linked Fe 3 O 4 @ SiO 2 nanoparticles was successfully achieved by amide reaction and the magnetic phosphodiesterase-5 linked Fe 3 O 4 @SiO 2 nanoparticles were evaluated as a new adsorbent for magnetic dispersive solid-phase extraction of ligands from medicinal plant samples before the analysis by UHPLC-Q-TOF/MS. The prepared phosphodiesterase-5 linked Fe 3 O 4 @SiO 2 nanoparticles were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, vibration sample magnetometer and potential laser particle size analyzer. The effects of EDC concentration, incubation time and bead-protein ratio on the amount of immobilized protein were studied. The main experimental parameters affect extraction efficiency of ligands, such as wash times, wash solvents, incubation pH, ion strength and incubation temperature, were investigated and optimized by using echinacoside as a model compound. The absolute recovery of echinacoside was ranged from 98.36%-102.16% in Cistanche tubulosa sample under the optimal extraction conditions. Good linearity was observed in the investigated concentration range of 0.006 mgmL -1 -0.97 mgmL -1 (R 2  = 0.9999). The limit of detection was 0.002 mgmL -1 . The RSDs of within-day and between-day precision were less than 2.3%. Due to the excellent magnetic behavior of Fe 3 O 4 @SiO 2 nanoparticles, the proposed method was shown to be simple and rapid. Remarkably, the magnetic phosphodiesterase-5 linked Fe 3 O 4 @SiO 2 nanoparticles could be recycled for ten times with loss of 10% activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeted Approach for Proteomic Analysis of a Hidden Membrane Protein.

PubMed

Martins-Marques, Tania; Anjo, Sandra I; Ribeiro-Rodrigues, Teresa; Manadas, Bruno; Girao, Henrique

2017-01-01

Given the properties of plasma membrane proteins, namely, their hydrophobicity, low solubility, and high resistance to digestion and extraction, their identification by traditional mass spectrometry (MS) has been a challenging task. Hence, proteomic studies involving the transmembrane protein connexin43 (Cx43) are scarce. Additionally, studies demonstrating the presence of proteins embedded in the lipid bilayer of extracellular vesicles (EVs) are difficult to perform and require specific changes and fine adjustments in the experimental and technical procedure to allow their detection by MS. In this review, we provide a detailed description of the protocol we have used to detect Cx43 in EVs of human peripheral blood. This includes some of the modifications that we have introduced in order to improve the detection of Cx43 in EVs, including an optimization of vesicle isolation, Cx43 purification, MS acquisition data, and further analysis.

One-step extraction of functional recombinant aquaporin Z from inclusion bodies with optimal detergent.

PubMed

Wang, Lili; Zhou, Hu; Li, Zhengjun; Lim, Teck Kwang; Lim, Xin Shan; Lin, Qingsong

2015-11-01

Aquaporins are integral membrane channel proteins found in all kingdoms of life. The Escherichia coli aquaporin Z (AqpZ) has been shown to solely conduct water at high permeability. Functional AqpZ is generally purified from the membrane fraction. However, the quantity of the purified protein is limited. In this study, a new method is developed to achieve high yield of bioactive AqpZ protein. A mild detergent n-dodecyl-β-D-maltopyranoside (DDM) was used to solubilize the over-expressed insoluble AqpZ from inclusion bodies without a refolding process. The recovered AqpZ protein showed high water permeability comparable with AqpZ obtained from the membrane fraction. In this way, the total yield of bioactive AqpZ has been increased greatly, which will facilitate the structural and functional characterization and future applications of AqpZ. Copyright © 2015 Elsevier Inc. All rights reserved.

Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli

PubMed Central

Ashayeri-Panah, Mitra; Eftekhar, Fereshteh; Kazemi, Bahram; Joseph, Joan

2017-01-01

Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E. coli BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting. Results: Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the E. coli cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K 2 HPO 4 , 17 mM KH 2 PO 4 and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient’s serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in E. coli which was successfuly purified. Conclusion: We believe that the purified Rv1733c recombinant protein from M. tuberculosis might be a good candidate for vaccine production against tuberculosis. PMID:29213997

Proteomic analysis of protein deposits on worn daily wear silicone hydrogel contact lenses

PubMed Central

Wei, Xiaojia; Aliwarga, Yulina; Carnt, Nicole A.; Garrett, Qian; Willcox, Mark D.P.

2008-01-01

Purpose Previous studies have demonstrated deposition of tear proteins onto worn contact lenses. In this study, we used proteomic techniques to analyze the protein deposits extracted from worn daily wear silicone hydrogel contact lenses in combination with different lens care solutions. Methods Worn lenses were collected and protein deposits extracted using urea and surfactant. Protein extracts were desalted, concentrated, and then separated using one-dimensional gel electrophoresis. Individual protein components in extracts were identified using liquid chromatography combined with tandem mass spectrometry (LC-MS-MS) after trypsin digestion. Results One-dimensional gel electrophoresis revealed that lysozyme and other small proteins (around 20 kDa) were the most abundant proteins in the extracts. LC-MS-MS revealed a wide array of proteins in lens extracts with lysozyme and lipocalin 1 being the most commonly identified in deposit extracts. Conclusions Worn contact lenses deposit a wide array of proteins from tear film and other sources. Protein deposit profiles varied and were specific for each contact lens material. PMID:18989384

Green ultrasound-assisted extraction of anthocyanin and phenolic compounds from purple sweet potato using response surface methodology

NASA Astrophysics Data System (ADS)

Zhu, Zhenzhou; Guan, Qingyan; Guo, Ying; He, Jingren; Liu, Gang; Li, Shuyi; Barba, Francisco J.; Jaffrin, Michel Y.

2016-01-01

Response surface methodology was used to optimize experimental conditions for ultrasound-assisted extraction of valuable components (anthocyanins and phenolics) from purple sweet potatoes using water as a solvent. The Box-Behnken design was used for optimizing extraction responses of anthocyanin extraction yield, phenolic extraction yield, and specific energy consumption. Conditions to obtain maximal anthocyanin extraction yield, maximal phenolic extraction yield, and minimal specific energy consumption were different; an overall desirability function was used to search for overall optimal conditions: extraction temperature of 68ºC, ultrasonic treatment time of 52 min, and a liquid/solid ratio of 20. The optimized anthocyanin extraction yield, phenolic extraction yield, and specific energy consumption were 4.91 mg 100 g-1 fresh weight, 3.24 mg g-1 fresh weight, and 2.07 kWh g-1, respectively, with a desirability of 0.99. This study indicates that ultrasound-assisted extraction should contribute to a green process for valorization of purple sweet potatoes.

[Efficient extraction of transmembrane proteins using ProteoExtract Transmembrane Protein Extraction Kit].

PubMed

Błachnio, Karina

2010-01-01

Detergents commonly used for solubilization of membrane proteins may be ionic or non-ionic. Exposing membrane proteins to detergents, however, can adversely affect their native structure, which can be a major hindrance for functional studies. This is especially true for proteins with multiple transmembrane domains. The ProteoExtract Transmembrane Protein Extraction Kit (TM-PEK), offered by Merck, provides a detergent-free novel reagents to enable the mild and efficient extraction of proteins containing seven transmembrane domains, such as GPCRs (G-Protein Coupled Receptors) e.g.: Frizzled-4 and CELSR-3, from mammalian cells. The fraction enriched in transmembrane proteins using TM-PEK is directly compatible with enzyme assays, non-denaturing gel electrophoresis, 1- and 2-D SDS-PAGE, MS analysis, Western blotting, immunoprecipitation and ELISA. Unlike many alternatives, TM-PEK extraction procedure does not require sonication, extended rigorous vortexing, ultracentrifugation, or incubation of samples at elevated temperatures--thus minimizing the risk of post-extraction degradation or modifications.

Optimized protocol for quantitative multiple reaction monitoring-based proteomic analysis of formalin-fixed, paraffin embedded tissues

PubMed Central

Kennedy, Jacob J.; Whiteaker, Jeffrey R.; Schoenherr, Regine M.; Yan, Ping; Allison, Kimberly; Shipley, Melissa; Lerch, Melissa; Hoofnagle, Andrew N.; Baird, Geoffrey Stuart; Paulovich, Amanda G.

2016-01-01

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin embedded (FFPE) tissues. While the feasibility of using targeted, multiple reaction monitoring-mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e. 9 processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R2 = 0.94) and immuno-MRM (R2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

Optimization protocol for the extraction of 6-gingerol and 6-shogaol from Zingiber officinale var. rubrum Theilade and improving antioxidant and anticancer activity using response surface methodology.

PubMed

Ghasemzadeh, Ali; Jaafar, Hawa Z E; Rahmat, Asmah

2015-07-30

Analysis and extraction of plant matrices are important processes for the development, modernization, and quality control of herbal formulations. Response surface methodology is a collection of statistical and mathematical techniques that are used to optimize the range of variables in various experimental processes to reduce the number of experimental runs, cost , and time, compared to other methods. Response surface methodology was applied for optimizing reflux extraction conditions for achieving high 6-gingerol and 6-shogaol contents, and high antioxidant activity in Zingiber officinale var. rubrum Theilade . The two-factor central composite design was employed to determine the effects of two independent variables, namely extraction temperature (X1: 50-80 °C) and time (X2: 2-4 h), on the properties of the extracts. The 6-gingerol and 6-shogaol contents were measured using ultra-performance liquid chromatography. The antioxidant activity of the rhizome extracts was determined by means of the 1,1-diphenyl-2-picrylhydrazyl assay. Anticancer activity of optimized extracts against HeLa cancer cell lines was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Increasing the extraction temperature and time induced significant response of the variables. The optimum extraction condition for all responses was at 76.9 °C for 3.4 h. Under the optimum condition, the corresponding predicted response values for 6-gingerol, 6-shogaol, and the antioxidant activity were 2.89 mg/g DW, 1.85 mg/g DW, and 84.3%, respectively. 6-gingerol and 6-shogaol were extracted under optimized condition to check the viability of the models. The values were 2.92 and 1.88 mg/g DW, and 84.0% for 6-gingerol, 6-shogaol, and the antioxidant activity respectively. The experimental values agreed with those predicted, thus indicating suitability of the models employed and the success of RSM in optimizing the extraction condition. With optimizing of reflux extraction anticancer activity of extracts against HeLa cancer cells enhanced about 16.8%. The half inhibition concentration (IC50) value of optimized and unoptimized extract was found at concentration of 20.9 and 38.4 μg/mL respectively. Optimized extract showed more distinct anticancer activities against HeLa cancer cells in a concentration of 40 μg/mL (P < 0.01) without toxicity to normal cells. The results indicated that the pharmaceutical quality of ginger could be improved significantly by optimizing of extraction process using response surface methodology.

Optimization of microwave-assisted extraction conditions for preparing lignan-rich extract from Saraca asoca bark using Box-Behnken design.

PubMed

Mishra, Shikha; Aeri, Vidhu

2016-07-01

Lyoniside is the major constituent of Saraca asoca Linn. (Caesalpiniaceae) bark. There is an immediate need to develop an efficient method to isolate its chemical constituents, since it is a therapeutically important plant. A rapid extraction method for lyoniside based on microwave-assisted extraction of S. asoca bark was developed and optimized using response surface methodology (RSM). Lyoniside was analyzed and quantified by high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV). The extraction solvent ratio (%), material solvent ratio (g/ml) and extraction time (min) were optimized using Box-Behnken design (BBD) to obtain the highest extraction efficiency. The optimal conditions were the use of 1:30 material solvent ratio with 70:30 mixture of methanol:water for 10 min duration. The optimized microwave-assisted extraction yielded 9.4 mg/g of lyoniside content in comparison to reflux extraction under identical conditions which yielded 4.2 mg/g of lyoniside content. Under optimum conditions, the experimental values agreed closely with the predicted values. The analysis of variance (ANOVA) indicated a high goodness-of-fit model and the success of the RSM method for optimizing lyoniside extraction from the bark of S. asoca. All the three variables significantly affected the lyoniside content. Increased polarity of solvent medium enhances the lyoniside yield. The present study shows the applicability of microwave-assisted extraction in extraction of lyoniside from S. asoca bark.

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2.

PubMed

Neviani, E; Boquien, C Y; Monnet, V; Thanh, L P; Gripon, J C

1989-09-01

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.

Yellow Mealworm Protein for Food Purposes - Extraction and Functional Properties

PubMed Central

Zhao, Xue; Vázquez-Gutiérrez, José Luis; Johansson, Daniel P.; Landberg, Rikard; Langton, Maud

2016-01-01

A protocol for extraction of yellow mealworm larvae proteins was established, conditions were evaluated and the resulting protein extract was characterised. The freeze-dried yellow mealworm larvae contained around 33% fat, 51% crude protein and 43% true protein on a dry matter basis. The true protein content of the protein extract was about 75%, with an extraction rate of 70% under optimised extraction conditions using 0.25 M NaOH, a NaOH solution:ethanol defatted worm ratio of 15:1 mL/g, 40°C for 1 h and extraction twice. The protein extract was a good source of essential amino acids. The lowest protein solubility in distilled water solution was found between pH 4 and 5, and increased with either increasing or decreasing pH. Lower solubility was observed in 0.5 M NaCl solution compared with distilled water. The rheological tests indicated that temperature, sample concentration, addition of salt and enzyme, incubation time and pH alterations influenced the elastic modulus of yellow mealworm protein extract (YMPE). These results demonstrate that the functional properties of YMPE can be modified for different food applications. PMID:26840533

Application of response surface methodology to optimize pressurized liquid extraction of antioxidant compounds from sage (Salvia officinalis L.), basil (Ocimum basilicum L.) and thyme (Thymus vulgaris L.).

PubMed

Hossain, M B; Brunton, N P; Martin-Diana, A B; Barry-Ryan, C

2010-12-01

The present study optimized pressurized liquid extraction (PLE) conditions using Dionex ASE® 200, USA to maximize the antioxidant activity [Ferric ion Reducing Antioxidant Power (FRAP)] and total polyphenol content (TP) of the extracts from three spices of Lamiaceae family (sage, basil and thyme). Optimal conditions with regard to extraction temperature (66-129 °C) and solvent concentration (32-88% methanol) were identified using response surface methodology (RSM). For all three spices, results showed that 129 °C was the optimum temperature with regard to antioxidant activity. Optimal methanol concentrations with respect to the antioxidant activity of sage and basil extracts were 58% and 60% respectively. Thyme showed a different trend with regard to methanol concentration and was optimally extracted at 33%. Antioxidant activity yields of the optimal PLE were significantly (p < 0.05) higher than solid/liquid extracts. Predicted models were highly significant (p < 0.05) for both total phenol (TP) and FRAP values in all the spices with high regression coefficients (R(2)) ranging from 0.651 to 0.999.

A comparative study of extraction techniques for maximum recovery of glutamate decarboxylase (GAD) from Aspergillus oryzae NSK

PubMed Central

2013-01-01

Background γ-Amino butyric acid (GABA) is a major inhibitory neurotransmitter of the mammalian central nervous system that plays a vital role in regulating vital neurological functions. The enzyme responsible for producing GABA is glutamate decarboxylase (GAD), an intracellular enzyme that both food and pharmaceutical industries are currently using as the major catalyst in trial biotransformation process of GABA. We have successfully isolated a novel strain of Aspergillus oryzae NSK that possesses a relatively high GABA biosynthesizing capability compared to other reported GABA-producing fungal strains, indicating the presence of an active GAD. This finding has prompted us to explore an effective method to recover maximum amount of GAD for further studies on the GAD’s biochemical and kinetic properties. The extraction techniques examined were enzymatic lysis, chemical permeabilization, and mechanical disruption. Under the GAD activity assay used, one unit of GAD activity is expressed as 1 μmol of GABA produced per min per ml enzyme extract (U/ml) while the specific activity was expressed as U/mg protein. Results Mechanical disruption by sonication, which yielded 1.99 U/mg of GAD, was by far the most effective cell disintegration method compared with the other extraction procedures examined. In contrast, the second most effective method, freeze grinding followed by 10% v/v toluene permeabilization at 25°C for 120 min, yielded only 1.17 U/mg of GAD, which is 170% lower than the sonication method. Optimized enzymatic lysis with 3 mg/ml Yatalase® at 60°C for 30 min was the least effective. It yielded only 0.70 U/mg of GAD. Extraction using sonication was further optimized using a one-variable-at-a-time approach (OVAT). Results obtained show that the yield of GAD increased 176% from 1.99 U/mg to 3.50 U/mg. Conclusion Of the techniques used to extract GAD from A. oryzae NSK, sonication was found to be the best. Under optimized conditions, about 176% of GAD was recovered compared to recovery under non optimized conditions. The high production level of GAD in this strain offers an opportunity to conduct further studies on GABA production at a larger scale. PMID:24321181

Animal component-free Agrobacterium tumefaciens cultivation media for better GMP-compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana.

PubMed

Houdelet, Marcel; Galinski, Anna; Holland, Tanja; Wenzel, Kathrin; Schillberg, Stefan; Buyel, Johannes Felix

2017-04-01

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large-scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal-derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal-derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design-of-experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two-fold increase in OD 600 compared to YEB medium during a 4-L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal-derived components, thus facilitating the GMP-compliant large-scale transient expression of recombinant proteins in plants. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimizing Adipose Tissue Extract Isolation with Stirred Suspension Culture.

PubMed

Zhang, Yan; Yu, Mei; Zhao, Xueyong; Dai, Minjia; Chen, Chang; Tian, Weidong

2018-05-31

Adherent culture which is used to collect adipose tissue extract (ATE) previously brings the problem of inhomogeneity and non-repeatability. Here we aim to extract ATE with stirred suspension culture to speed up the extraction process, stabilize the yield and improve consistent potency metrics of ATE. ATE was collected with adherent culture (ATE-A) and stirred suspension culture (ATE-S) separately. Protein yield and composition were detected by SDS-PAGE while cytokines in ATE were determined with ELISA. The adipogenic and angiogenic potential of ATE were compared by Western blot and qPCR. In addition, HE staining and LDH activity assays were used to analyze the cell viability of adipose tissue cultured with different methods. The yield of ATE-S was consistent while ATE-A varied notably. Characterization of the protein composition and exosome-like vesicles (ELVs) indicated no significant difference between ATE-S and ATE-A. The concentrations of cytokines (VEGF, bFGF and IL-6) showed no significant difference while IGF in ATE-S was higher than that in ATE-A. ATE-S showed upregulated adipogenic and angiogenic potential compared to ATE-A. Morever, stirred suspension culture decreased the LDH activity of ATE while increased the number of viable adipocytes and reduced adipose tissue necrosis. Compared with adherent culture, stirred suspension culture is a reliable, time and labor-saving method to collect ATE, which might be used to improve the downstream applications of ATE.

Gas chromatography-mass spectrometry of biofluids and extracts.

PubMed

Emwas, Abdul-Hamid M; Al-Talla, Zeyad A; Yang, Yang; Kharbatia, Najeh M

2015-01-01

Gas chromatography-mass spectrometry (GC-MS) has been widely used in metabonomics analyses of biofluid samples. Biofluids provide a wealth of information about the metabolism of the whole body and from multiple regions of the body that can be used to study general health status and organ function. Blood serum and blood plasma, for example, can provide a comprehensive picture of the whole body, while urine can be used to monitor the function of the kidneys, and cerebrospinal fluid (CSF) will provide information about the status of the brain and central nervous system (CNS). Different methods have been developed for the extraction of metabolites from biofluids, these ranging from solvent extracts, acids, heat denaturation, and filtration. These methods vary widely in terms of efficiency of protein removal and in the number of metabolites extracted. Consequently, for all biofluid-based metabonomics studies, it is vital to optimize and standardize all steps of sample preparation, including initial extraction of metabolites. In this chapter, recommendations are made of the optimum experimental conditions for biofluid samples for GC-MS, with a particular focus on blood serum and plasma samples.

Sequence-structure relationship study in all-α transmembrane proteins using an unsupervised learning approach.

PubMed

Esque, Jérémy; Urbain, Aurélie; Etchebest, Catherine; de Brevern, Alexandre G

2015-11-01

Transmembrane proteins (TMPs) are major drug targets, but the knowledge of their precise topology structure remains highly limited compared with globular proteins. In spite of the difficulties in obtaining their structures, an important effort has been made these last years to increase their number from an experimental and computational point of view. In view of this emerging challenge, the development of computational methods to extract knowledge from these data is crucial for the better understanding of their functions and in improving the quality of structural models. Here, we revisit an efficient unsupervised learning procedure, called Hybrid Protein Model (HPM), which is applied to the analysis of transmembrane proteins belonging to the all-α structural class. HPM method is an original classification procedure that efficiently combines sequence and structure learning. The procedure was initially applied to the analysis of globular proteins. In the present case, HPM classifies a set of overlapping protein fragments, extracted from a non-redundant databank of TMP 3D structure. After fine-tuning of the learning parameters, the optimal classification results in 65 clusters. They represent at best similar relationships between sequence and local structure properties of TMPs. Interestingly, HPM distinguishes among the resulting clusters two helical regions with distinct hydrophobic patterns. This underlines the complexity of the topology of these proteins. The HPM classification enlightens unusual relationship between amino acids in TMP fragments, which can be useful to elaborate new amino acids substitution matrices. Finally, two challenging applications are described: the first one aims at annotating protein functions (channel or not), the second one intends to assess the quality of the structures (X-ray or models) via a new scoring function deduced from the HPM classification.

Automated Protein Biomarker Analysis: on-line extraction of clinical samples by Molecularly Imprinted Polymers

NASA Astrophysics Data System (ADS)

Rossetti, Cecilia; Świtnicka-Plak, Magdalena A.; Grønhaug Halvorsen, Trine; Cormack, Peter A. G.; Sellergren, Börje; Reubsaet, Léon

2017-03-01

Robust biomarker quantification is essential for the accurate diagnosis of diseases and is of great value in cancer management. In this paper, an innovative diagnostic platform is presented which provides automated molecularly imprinted solid-phase extraction (MISPE) followed by liquid chromatography-mass spectrometry (LC-MS) for biomarker determination using ProGastrin Releasing Peptide (ProGRP), a highly sensitive biomarker for Small Cell Lung Cancer, as a model. Molecularly imprinted polymer microspheres were synthesized by precipitation polymerization and analytical optimization of the most promising material led to the development of an automated quantification method for ProGRP. The method enabled analysis of patient serum samples with elevated ProGRP levels. Particularly low sample volumes were permitted using the automated extraction within a method which was time-efficient, thereby demonstrating the potential of such a strategy in a clinical setting.

Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics

PubMed Central

DeHart, Caroline J.; Schweitzer, Mary H.; Thomas, Paul M.; Kelleher, Neil L.

2016-01-01

Proteomic studies of bone require specialized extraction protocols to demineralize and solubilize proteins from within the bone matrix. Although various protocols exist for bone protein recovery, little is known about how discrete steps in each protocol affect the subset of the bone proteome recovered by mass spectrometry (MS) analyses. Characterizing these different “extractomes” will provide critical data for development of novel and more efficient protein extraction methodologies for fossils. Here, we analyze 22 unique sub-extractions of chicken bone and directly compare individual extraction components for their total protein yield and diversity and coverage of bone proteins identified by MS. We extracted proteins using different combinations and ratios of demineralizing reagents, protein-solubilizing reagents, and post-extraction buffer removal methods, then evaluated tryptic digests from 20 µg aliquots of each fraction by tandem MS/MS on a 12T FT-ICR mass spectrometer. We compared total numbers of peptide spectral matches, peptides, and proteins identified from each fraction, the redundancy of protein identifications between discrete steps of extraction methods, and the sequence coverage obtained for select, abundant proteins. Although both alpha chains of collagen I (the most abundant protein in bone) were found in all fractions, other collagenous and non-collagenous proteins (e.g., apolipoprotein, osteonectin, hemoglobin) were differentially identified. We found that when a standardized amount of extracted proteins was analyzed, extraction steps that yielded the most protein (by weight) from bone were often not the ones that produced the greatest diversity of bone proteins, or the highest degree of protein coverage. Generally, the highest degrees of diversity and coverage were obtained from demineralization fractions, and the proteins found in the subsequent solubilization fractions were highly redundant with those in the previous fraction. Based on these data, we identify future directions and parameters to consider (e.g., proteins targeted, amount of sample required) when applying discrete parts of these protocols to fossils. PMID:27812413

[Study on extraction and purification process of total ginsenosides from Radix Ginseng].

PubMed

Xie, Li-Ling; Ren, Li; Lai, Xian-Sheng; Cao, Jun-Hui; Mo, Quan-Yi; Chen, Wei-Wen

2009-10-01

To optimize the technological parameters of the extraction and purification process of total ginsenosides from Radix Ginseng. With the contents of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1, the orthogonal design was adopted to optimize the extraction process. The purification process was studied by optimizing the elutive ratio of total ginsenosides as the marker. HPLC and spectrophotometer were employed for the study. The optimum conditions were as follows:Using 8 times volume of 75% ethanol extracting for 120 minutes and 2 times, the extraction temperature was 85 degrees C. AB-8 macroporous resin was selected, and the eluant was 4 BV 70% ethanol. The optimal conditions of extracting and purifying the total ginsenosides from Radix Ginseng is feasible.

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

PubMed

Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

2010-10-01

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

Fifteen novel immunoreactive proteins of Chinese virulent Haemophilus parasuis serotype 5 verified by an immunoproteomic assay.

PubMed

Yu, Yanfei; Wu, Guangyan; Zhai, Zhipeng; Yao, Huochun; Lu, Chengping; Zhang, Wei

2015-01-01

Haemophilus parasuis (H. parasuis) is associated with meningitis, polyserositis, polyarthritis and bacterial pneumonia. At present, its prevention and control is difficult because of the lack of suitable subunit vaccines. Nowadays, high-throughput methods, immunoproteomics, are available to screen for more vaccine candidates. A protein extraction method for H. parasuis and two-dimensional electrophoresis (2-DE) were optimized to provide high-resolution profiles covering pH 3 to 10. Twenty immunoreactive spots were excised from gels after strict comparison between 2-DE Western blot membranes and the relevant gels. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and MALDI-TOF-TOF-MS successfully identified 16 different proteins. Fifteen of them were reported as immunoreactive proteins in H. parasuis for the first time. In addition, recombinant HP5-7 (ABC transporter, periplasmic-binding protein) showed immunoreactivity both with hyperimmune rabbit serum and convalescent swine serum. Four recombinants of the 14 successfully expressed genes showed immunoreactivity with hyperimmune rabbit serum.

[Optimization of extraction process for tannins from Geranium orientali-tibeticum by supercritical CO2 method].

PubMed

Xie, Song; Tong, Zhi-Ping; Tan, Rui; Liu, Xiao-Zhen

2014-08-01

In order to optimize extraction process conditions of tannins from Geranium orientali-tibeticum by supercritical CO2, the content of tannins was determined by phosphomolybdium tungsten acid-casein reaction, with extraction pressure, extraction temper- ature and extraction time as factors, the content of tannins from extract of G. orientali-tibeticum as index, technology conditions were optimized by orthogonal test. Optimum technology conditions were as follows: extraction pressure was 25 MPa, extraction temperature was 50 °C, extracted 1.5 h. The content of tannins in extract was 12.91 mg x g(-1), extract rate was 3.67%. The method established could be used for assay the contents of tannin in G. orientali-tibeticum. The circulated extraction was an effective extraction process that was stable and feasible, and that provides a way of the extraction process conditions of tannin from G. orientali-tibeticum.

THE PURIFICATION OF AN ALKALINE PROTEINASE OBTAINED FROM ASPERGILLUS ORYZAE AND THE DETERMINATION OF ITS PROPERTIES.

DTIC Science & Technology

Aspergillopeptidase B, an alkaline protease in A , oryzae extracts, was obtained in highly purified form by conventional fractionation techniques. The...enzyme is a compact protein of 17,900 m.w. with a neutral isoelectric point. It contains no S-containing amino acids, phosphorus or metal ions. It is...composed of a single polypeptide chain with N-terminal glycine and C-terminal alanine residues. The protease activity toward casein is optimal at pH

Imbalanced multi-modal multi-label learning for subcellular localization prediction of human proteins with both single and multiple sites.

PubMed

He, Jianjun; Gu, Hong; Liu, Wenqi

2012-01-01

It is well known that an important step toward understanding the functions of a protein is to determine its subcellular location. Although numerous prediction algorithms have been developed, most of them typically focused on the proteins with only one location. In recent years, researchers have begun to pay attention to the subcellular localization prediction of the proteins with multiple sites. However, almost all the existing approaches have failed to take into account the correlations among the locations caused by the proteins with multiple sites, which may be the important information for improving the prediction accuracy of the proteins with multiple sites. In this paper, a new algorithm which can effectively exploit the correlations among the locations is proposed by using gaussian process model. Besides, the algorithm also can realize optimal linear combination of various feature extraction technologies and could be robust to the imbalanced data set. Experimental results on a human protein data set show that the proposed algorithm is valid and can achieve better performance than the existing approaches.

Dried Blood Spot RNA Transcriptomes Correlate with Transcriptomes Derived from Whole Blood RNA.

PubMed

Reust, Mary J; Lee, Myung Hee; Xiang, Jenny; Zhang, Wei; Xu, Dong; Batson, Tatiana; Zhang, Tuo; Downs, Jennifer A; Dupnik, Kathryn M

2018-05-01

Obtaining RNA from clinical samples collected in resource-limited settings can be costly and challenging. The goals of this study were to 1) optimize messenger RNA extraction from dried blood spots (DBS) and 2) determine how transcriptomes generated from DBS RNA compared with RNA isolated from blood collected in Tempus tubes. We studied paired samples collected from eight adults in rural Tanzania. Venous blood was collected on Whatman 903 Protein Saver cards and in tubes with RNA preservation solution. Our optimal DBS RNA extraction used 8 × 3-mm DBS punches as the starting material, bead beater disruption at maximum speed for 60 seconds, extraction with Illustra RNAspin Mini RNA Isolation kit, and purification with Zymo RNA Concentrator kit. Spearman correlations of normalized gene counts in DBS versus whole blood ranged from 0.887 to 0.941. Bland-Altman plots did not show a trend toward over- or under-counting at any gene size. We report a method to obtain sufficient RNA from DBS to generate a transcriptome. The DBS transcriptome gene counts correlated well with whole blood transcriptome gene counts. Dried blood spots for transcriptome studies could be an option when field conditions preclude appropriate collection, storage, or transport of whole blood for RNA studies.

Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi.

PubMed

Solomon, Kevin V; Henske, John K; Theodorou, Michael K; O'Malley, Michelle A

2016-04-01

Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at -80 °C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long with negligible growth impact. Genomic DNA extraction for these isolates was optimized to yield samples compatible with next generation sequencing platforms (e.g. Illumina, PacBio). Popular DNA isolation kits and precipitation protocols yielded preps that were unsuitable for sequencing due to carbohydrate contaminants from the chitin-rich cell wall and extensive energy reserves of gut fungi. To address this, we identified a proprietary method optimized for hardy plant samples that rapidly yielded DNA fragments in excess of 10 kb with minimal RNA, protein or carbohydrate contamination. Collectively, these techniques serve as fundamental tools to manipulate powerful biomass-degrading gut fungi and improve their accessibility among researchers. Copyright © 2015 Elsevier Ltd. All rights reserved.

Optimization of subcritical water extraction parameters of antioxidant polyphenols from sea buckthorn (Hippophaë rhamnoides L.) seed residue.

PubMed

Gong, Ying; Zhang, Xiaofei; He, Li; Yan, Qiuli; Yuan, Fang; Gao, Yanxiang

2015-03-01

Polyphenols was extracted with subcritical water from the sea buckthorn seed residue (after oil recovery), and the extraction parameters were optimized using response surface methodology (RSM). The independent processing variables were extraction temperature, extraction time and the ratio of water to solid. The optimal extraction parameters for the extracts with highest ABTS radical scavenging activity were 120 °C, 36 min and the water to solid ratio of 20, and the maximize antioxidant capacity value was 32.42 mmol Trolox equivalent (TE)/100 g. Under the optimal conditions, the yield of total phenolics, total flavonoids and proanthocyanidins was 36.62 mg gallic acid equivalents (GAE)/g, 19.98 mg rutin equivalent (RE)/g and 10.76 mg catechin equivalents (CE)/g, respectively.

Optimization of focused ultrasonic extraction of propellant components determined by gas chromatography/mass spectrometry.

PubMed

Fryš, Ondřej; Česla, Petr; Bajerová, Petra; Adam, Martin; Ventura, Karel

2012-09-15

A method for focused ultrasonic extraction of nitroglycerin, triphenyl amine and acetyl tributyl citrate presented in double-base propellant samples following by the gas chromatography/mass spectrometry analysis was developed. A face-centered central composite design of the experiments and response surface modeling was used for optimization of the time, amplitude and sample amount. The dichloromethane was used as the extractant solvent. The optimal extraction conditions with respect to the maximum yield of the lowest abundant compound triphenyl amine were found at the 20 min extraction time, 35% amplitude of ultrasonic waves and 2.5 g of the propellant sample. The results obtained under optimal conditions were compared with the results achieved with validated Soxhlet extraction method, which is typically used for isolation and pre-concentration of compounds from the samples of explosives. The extraction yields for acetyl tributyl citrate using both extraction methods were comparable; however, the yield of ultrasonic extraction of nitroglycerin and triphenyl amine was lower than using Soxhlet extraction. The possible sources of different extraction yields are estimated and discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

The Identification of Proteoglycans and Glycosaminoglycans in Archaeological Human Bones and Teeth

PubMed Central

Coulson-Thomas, Yvette M.; Coulson-Thomas, Vivien J.; Norton, Andrew L.; Gesteira, Tarsis F.; Cavalheiro, Renan P.; Meneghetti, Maria Cecília Z.; Martins, João R.; Dixon, Ronald A.; Nader, Helena B.

2015-01-01

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology. PMID:26107959

[Optimization of extraction technology from Paeoniae Radix Alba using response surface methodology].

PubMed

Jin, Lin; Zhao, Wan-shun; Guo, Qiao-sheng; Zhang, Wen-sheng; Ye, Zheng-liang

2015-08-01

To ensure the stability of chemistry components and the convenience of operation, ultrasound method was chosen to study in this investigation. As the total common peaks area in chromatograms was set to be evaluation index, the influence on the technology caused by extraction time, ethanol concentration and liquid-to-solid ratio was studied by using single factor methodology, and the extraction technology of Paeoniae Radix Alba was optimized by using response surface methodology. The results showed that the extracting results were most affected by ethanol concentration; liquid-to-solid ratio came the second and extraction time thirdly. The optimum ultrasonic-assisted extraction conditions were as follow: the ultrasonic extraction time was 20.06 min, the ethanol concentration in solvent was 72.04%, and the liquid-to-solid ratio was 53.38 mL · g(-1), the predicted value of total common peaks area was 2.1608 x 10(8). Under the extraction conditions after optimization, the total common peaks area was 2.1422 x 10(8), and the relative deviation between the measured and predicted value was 0.86%, so the optimized extraction technology for Paeoniae Radix Alba is suitable and feasible. Besides, for the purpose of extracting more sufficiently and completely, the optimized extraction technology had more advantages than the extraction method recorded in the monogragh of Paeoniae Radix Alba in Chinese Pharmacopoeia, which will come true the assessment and utilization comprehensively.

Optimization of Aqueous Extraction from Kalanchoe pinnata Leaves to Obtain the Highest Content of an Anti-inflammatory Flavonoid using a Response Surface Model.

PubMed

Dos Santos Nascimento, Luana Beatriz; de Aguiar, Paula Fernandes; Leal-Costa, Marcos Vinicius; Coutinho, Marcela Araújo Soares; Borsodi, Maria Paula Gonçalves; Rossi-Bergmann, Bartira; Tavares, Eliana Schwartz; Costa, Sônia Soares

2018-05-01

The medicinal plant Kalanchoe pinnata is a phenolic-rich species used worldwide. The reports on its pharmacological uses have increased by 70% in the last 10 years. The leaves of this plant are the main source of an unusual quercetin-diglycosyl flavonoid (QAR, quercetin arabinopyranosyl rhamnopyranoside), which can be easily extracted using water. QAR possess a strong in vivo anti-inflammatory activity. To optimize the aqueous extraction of QAR from K. pinnata leaves using a three-level full factorial design. After a previous screening design, time (x 1 ) and temperature (x 2 ) were chosen as the two independent variables for optimization. Freeze-dried leaves were extracted with water (20% w/v), at 30°C, 40°C or 50°C for 5, 18 or 30 min. QAR content (determined by HPLC-DAD) and yield of extracts were analyzed. The optimized extracts were also evaluated for cytotoxicity. The optimal heating times for extract yield and QAR content were similar in two-dimensional (2D) surface responses (between 12.8 and 30 min), but their optimal extraction temperatures were ranged between 40°C and 50°C for QAR content and 30°C and 38°C for extract yield. A compromise region for both parameters was at the mean points that were 40°C for the extraction temperature and 18 min for the total time. The optimized process is faster and spends less energy than the previous one (water; 30 min at 55°C); therefore is greener and more attractive for industrial purposes. This is the first report of extraction optimization of this bioactive flavonoid. Copyright © 2018 John Wiley & Sons, Ltd. Copyright © 2018 John Wiley & Sons, Ltd.

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material.

PubMed

Cheng, Yo Ching; Hannaoui, Samia; John, Theodore Ralph; Dudas, Sandor; Czub, Stefanie; Gilch, Sabine

2017-09-29

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with ᵦ-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrP Sc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrP Sc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

[Study on the optimal extraction process of chaihushugan powder].

PubMed

Wang, Chun-yan; Zhang, Wan-ming; Zhang, Dan-shen; An, Fang; Tian, Jia-ming

2009-11-01

To study the optimal extraction process of chaihushugan powder by orthogonal design. RP-HPLC method was developed for the determination of saikosaponin a, ferulic acid, hesperidin and paeoniflorin in chaihushugan powder. The contents of the components and the extraction yield were selected as assessment indices. Four factors were study by L9 (3(4)), including the alcohol concentration, amount of alcohol, duration of extraction and times of extraction. The optimal extracting condition was 80% alcohol consumed as 10 times of crude herb amount, and extracting two times for 90 min each time. This study supplies theoretical base for the development of chaihushugan powder formulation.

Plant Phenolics Extraction from Flos Chrysanthemi: Response Surface Methodology Based Optimization and the Correlation Between Extracts and Free Radical Scavenging Activity.

PubMed

Wu, Yanfang; Wang, Xinsheng; Xue, Jintao; Fan, Enguo

2017-11-01

Huaiju is one of the most famous and widely used Flos Chrysanthemi (FC) for medicinal purposes in China. Although various investigations aimed at phenolics extraction from other FC have been reported, a thorough optimization of the phenolics extraction conditions from Huaiju has not been achieved. This work applied the widely used response surface methodology (RSM) to investigate the effects of 3 independent variables including ethanol concentration (%), extraction time (min), and solvent-to-material ratio (mL/g) on the ultrasound-assisted extraction (UAE) of phenolics from FC. The data suggested the optimal UAE condition was an ethanol concentration of 75.3% and extraction time of 43.5 min, whereas the ratio of solvent to material has no significant effect. When the free radical scavenging ability was used as an indicator for a successful extraction, a similar optimal extraction was achieved with an ethanol concentration of 72.8%, extraction time of 44.3 min, and the ratio of solvent to material was 29.5 mL/g. Furthermore, a moderate correlation between the antioxidant activity of TP extract and the content of extracted phenolic compounds was observed. Moreover, a well consistent of the experimental values under optimal conditions with those predicted values suggests RSM successfully optimized the UAE conditions for phenolics extraction from FC. The work of the research investigated the plant phenolics in Flos Chrysanthemi and antioxidant capacities. These results of this study can support the development of antioxidant additive and relative food. © 2017 The Authors. Journal of Food Science published by Wiley Periodicals, Inc. on behalf of Institute of Food Technologists.

Optimization of extrusion conditions for the production of instant grain amaranth-based porridge flour.

PubMed

Akande, Olamide A; Nakimbugwe, Dorothy; Mukisa, Ivan M

2017-11-01

Malnutrition is one of the foremost causes of death among children below 5 years in developing countries. Development of nutrient-dense food formulations using locally available crops has been proposed as a means to combat this menace. This study optimized the extrusion process for the production of a nutritious amaranth-based porridge flour. Least cost formulations containing grain amaranth, groundnut, iron-rich beans, pumpkin, orange-fleshed sweet potato, carrot, and maize were developed and evaluated by a sensory panel ( n  = 30) for acceptability using the 9-point hedonic scale. Extrusion process of the most acceptable porridge flour was optimized by response surface methodology (RSM). Barrel temperature (130-170°C) and feed moisture content (14%-20%) were the independent variables which significantly ( p  < .05) affected in vitro protein digestibility, vitamin A retention, total polyphenol, phytic content, and iron and zinc extractabilities. Optimization of the extrusion process improved the nutritional quality of the instant flour.

Optimal synthesis and design of the number of cycles in the leaching process for surimi production.

PubMed

Reinheimer, M Agustina; Scenna, Nicolás J; Mussati, Sergio F

2016-12-01

Water consumption required during the leaching stage in the surimi manufacturing process strongly depends on the design and the number and size of stages connected in series for the soluble protein extraction target, and it is considered as the main contributor to the operating costs. Therefore, the optimal synthesis and design of the leaching stage is essential to minimize the total annual cost. In this study, a mathematical optimization model for the optimal design of the leaching operation is presented. Precisely, a detailed Mixed Integer Nonlinear Programming (MINLP) model including operating and geometric constraints was developed based on our previous optimization model (NLP model). Aspects about quality, water consumption and main operating parameters were considered. The minimization of total annual costs, which considered a trade-off between investment and operating costs, led to an optimal solution with lesser number of stages (2 instead of 3 stages) and higher volumes of the leaching tanks comparing with previous results. An analysis was performed in order to investigate how the optimal solution was influenced by the variations of the unitary cost of fresh water, waste treatment and capital investment.

A Comparison of Protein Extraction Methods Suitable for Gel-Based Proteomic Studies of Aphid Proteins

PubMed Central

Cilia, M.; Fish, T.; Yang, X.; Mclaughlin, M.; Thannhauser, T. W.

2009-01-01

Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type. We were unable to ascribe the differences in the proteins extracted to particular physical characteristics, cell location, or biological function. The TCA-acetone extraction yielded the greatest amount of protein from aphid tissues. Each extraction method isolated a unique subset of the aphid proteome. The TCA-acetone method was explored further for its quantitative reliability using 2-D DIGE. Principal component analysis showed that little of the variation in the data was a result of technical issues, thus demonstrating that the TCA-acetone extraction is a reliable method for preparing aphid proteins for a quantitative proteomics experiment. These data suggest that although the TCA-acetone method is a suitable method for quantitative aphid proteomics, a combination of extraction approaches is recommended for increasing proteome coverage when using gel-based separation techniques. PMID:19721822

Application of Box-Behnken design for ultrasonic-assisted extraction of polysaccharides from Paeonia emodi.

PubMed

Ahmad, Ajaz; Alkharfy, Khalid M; Wani, Tanveer A; Raish, Mohammad

2015-01-01

The objective of the present work was to study the ultrasonic assisted extraction and optimization of polysaccharides from Paeonia emodi and evaluation of its anti-inflammatory response. Specifically, the optimization of polysaccharides was carried out using Box-Behnken statistical experimental design. Response surface methodology (RSM) of three factors (extraction temperature, extraction time and liquid solid ratio) was employed to optimize the percentage yield of the polysaccharides. The experimental data were fitted to quadratic response surface models using multiple regression analysis with high coefficient of determination value (R) of 0.9906. The highest polysaccharide yield (8.69%) as per the Derringer's desirability prediction tool was obtained under the optimal extraction condition (extraction temperature 47.03 °C, extraction time 15.68 min, and liquid solid ratio 1.29 ml/g) with a desirability value of 0.98. These optimized values of tested parameters were validated under similar conditions (n = 6), an average of 8.13 ± 2.08% of polysaccharide yield was obtained in an optimized extraction conditions with 93.55% validity. The anti-inflammatory effect of polysaccharides of P. emodi were studied on carrageenan induced paw edema. In vivo results showed that the P. emodi 200mg/kg of polysaccharide extract exhibited strong potential against inflammatory response induced by 1% suspension of carrageenean in normal saline. Copyright © 2014 Elsevier B.V. All rights reserved.

Fractionation and identification of the allergic proteins in Aspergillus species.

PubMed

Falahati, M; Ghanbari, S; Ebrahimi, M; Ghazanfari, M; Bazrafshan, F; Farahyar, S; Falak, R

2016-12-01

Allergy is an undesired immune response to non-pathogenic agents. However, some opportunistic microorganisms such as fungi can also cause allergy. Among those fungi, hyphae form of Aspergillus strains including A. fumigatus , A. flavus , and A. niger could be mentioned. In this study, we aimed to separate allergic proteins from Aspergillus strains and determine their identity. Standard species of Aspergillus strains were cultivated in optimized conditions and the mycelium was separated by centrifugation. The fungal cells were lysed through physical methods such as freeze-thawing and grinding to prepare a suitable protein extract. The protein concentration was measured by Bradford method and the electrophoretic pattern of the extract was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were fractionated by ammonium sulfate precipitation and anion exchange chromatography using fast protein liquid chromatography (FPLC) system. The IgE immunoreactivity of the sensitized patients and controls was studied using the fractionated proteins by enzyme-linked immunosorbent assay (ELISA). Following SDS-PAGE, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes and the strips were blotted with allergic patients' and controls' sera. The immunoreactive bands were excised from colloidal coomassie-stained SDS-PAGE gels and studied by mass spectroscopy methods. Among the studied species, A. fumigatus showed stronger IgE reactivity and more IgE reactive protein bands than others did. The proteins with higher molecular weights showed stronger immunoreactivity in Western blotting. Receiver operating characteristic curve analysis demonstrated a correlation between the results of the applied ELISA methods. One of the most prominent IgE-reactive proteins was confirmed to be 45 kDa mycelia catalase. Our findings confirmed that high molecular weight proteins might play a major role in allergy and IgE reactivity to Aspergillus species. Moreover, the results showed that precipitation and chromatographic methods are applicable for fractionation of fungal proteins such as mycelial catalase.

Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system.

PubMed

Buntru, Matthias; Vogel, Simon; Spiegel, Holger; Schillberg, Stefan

2014-05-03

Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4-5 h, compared to 4-5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5' and 3' untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

Re-evaluation of the reported experimental values of the heat of vaporization of N-methylacetamide

PubMed Central

MacKerell, Alexander D.; Shim, Ji Hyun; Anisimov, Victor M.

2010-01-01

The accuracy of empirical force fields is inherently related to the quality of the target data used for optimization of the model. With the heat of vaporization (ΔHvap) of N-methylacetamide (NMA), a range of values have been reported as target data for optimization of the nonbond parameters associated with the peptide bond in proteins. In the present work, the original experimental data and Antoine constants used for the determination of the ΔHvap of NMA are reanalyzed. Based on this analysis, the wide range of ΔHvap values reported in the literature are shown to be due to incorrect reporting of the temperatures at which the original values were extracted and limitations in the quality of experimental vapor pressure-temperature data over a wide range of temperatures. Taking these problems into account, a consistent ΔHvap value is extracted from three studies for which experimental data are available. This analysis suggests that the most reliable value for ΔHvap is 13.0±0.1 at 410 K for use in force field optimization studies. The present results also indicate that similar analyses, including analysis of Antoine constants alone, may be of utility when reported ΔHvap values are not consistent for a given neat liquid. PMID:20445813

A Workflow for Subsurface Pressure Control in Geological CO2 Storage: Optimization of Brine Extraction

NASA Astrophysics Data System (ADS)

Birkholzer, J. T.; Gonzalez-Nicolas, A.; Cihan, A.

2017-12-01

Industrial-scale injection of CO2 into the subsurface increases the fluid pressure in the reservoir, sometimes to the point that the resulting stress increases must be properly controlled to prevent potential damaging impacts such as fault activation, leakage through abandoned wells, or caprock fracturing. Brine extraction is one approach for managing formation pressure, effective stress, and plume movement in response to CO2 injection. However, the management of the extracted brine adds cost to the carbon capture and sequestration operations; therefore optimizing (minimizing) the extraction volume of brine is of great importance. In this study, we apply an adaptive management approach that optimizes extraction rates of brine for pressure control in an integrated optimization framework involving site monitoring, model calibration, and optimization. We investigate the optimization performance as affected by initial site characterization data and introduction of newly acquired data during the injection phase. More accurate initial reservoir characterization data reduce the risk of pressure buildup damage with better estimations of initial extraction rates, which results in better control of pressure during the overall injection time periods. Results also show that low frequencies of model calibration and optimization with the new data, especially at early injection periods, may lead to optimization problems, either that pressure buildup constraints are violated or excessively high extraction rates are proposed. These optimization problems can be eliminated if more frequent data collection and model calibration are conducted, especially at early injection time periods. Approaches such as adaptive pressure management may constitute an effective tool to manage pressure buildup under uncertain and unknown reservoir conditions by minimizing the brine extraction volumes while not exceeding critical pressure buildups of the reservoir.

Variability of protein content in calcium oxalate monohydrate stones.

PubMed

Williams, James C; Zarse, Chad A; Jackson, Molly E; Witzmann, Frank A; McAteer, James A

2006-08-01

Urinary stones are heterogeneous in their fragility to lithotripter shockwaves. As a first step in gaining a better understanding of the role of matrix in stone fragility, we measured extractible protein in calcium oxalate monohydrate (COM) stones that were extensively characterized by micro-computed tomography (micro CT). Stones were scanned using micro CT (Scanco mCT20, 34 microm). They were ground, and the protein extracted using four methods: 0.25M EDTA, 2% SDS reducing buffer, 9M urea buffer, and 10% acetic acid. Protein was measured using NanoOrange. The SDS extracts were also examined using polyacrylamide electrophoresis (PAGE). Extracted protein was highest with the SDS or urea methods (0.28% +/- 0.13% and 0.24% +/- 0.11%, respectively) and lower using the EDTA method (0.17% +/- 0.05%; P < 0.02). Acetic acid extracted little protein (0.006 +/- 0.002%; P < 0.001). Individual stones were significantly different in extractability of protein by the different methods, and SDS-PAGE revealed different protein patterns for individual stones. Extracted protein did not correlate with X-ray-lucent void percentage, which ranged from 0.06% to 2.8% of stone volume, or with apatite content. Extractible stone-matrix protein differs for individual COM stones, and yield is dependent on the extraction method. The presence of X-ray-lucent voids or minor amounts of apatite in stones did not correlate with protein content. The amounts of protein recovered were much lower than reported by Boyce, showing that these methods extracted only a fraction of the protein bound up in the stones. The results suggest that none of the methods tested will be useful for helping to answer the question of whether matrix content differs among stones of differing fragility to lithotripter shockwaves.

[Optimization of Energy Saving Measures with ABR-MBR Integrated Process].

PubMed

Wu, Peng; Lu, Shuang-jun; Xu, Yue-zhong; Liu, Jie; Shen, Yao-liang

2015-08-01

High energy consumption and membrane fouling are important factors that limit the wide use of membrane bioreactor (MBR). In order to reduce energy consumption and delay the process of membrane fouling, the process of anaerobic baffled reactor (ABR)-MBR was used to treat domestic sewage. The structure of the process and conditions of nitrogen and phosphorus removal were optimized in this study. The results showed that energy consumption was reduced by 43% through optimizing the structure of ABR-MBR process. Meanwhile, the process achieved a high level of COD, NH: -N, TN and TP removal, with the average removal efficiencies of 91%, 85%, 76% and 86%, respectively. In addition, the added particulate media could effectively delay membrane fouling, while the formation process of membrane fouling was changed. The extracted amount of carbohydrates increased while the amount of proteins decreased. Finally, the potential was enhanced for the practical application of MBR.

Soy foods: are they useful for optimal bone health?

PubMed Central

2011-01-01

Numerous studies have investigated the relationship between soy foods, soy protein, or isoflavone extracts and markers of bone health and osteoporosis prevention, and have come to conflicting conclusions. Research on dietary patterns, rather than on specific food ingredients or individual foods, may offer an opportunity for better understanding the role of soy foods in bone health. Evidence is reviewed regarding the question of whether soy foods contribute to a dietary pattern in humans that supports and promotes bone health. Soy foods are associated with improved markers of bone health and improved outcomes, especially among Asian women. Although the optimal amounts and types of soy foods needed to support bone health are not yet clear, dietary pattern evidence suggests that regular consumption of soy foods is likely to be useful for optimal bone health as an integral part of a dietary pattern that is built largely from whole plant foods. PMID:22870487

Soy foods: are they useful for optimal bone health?

PubMed

Lanou, Amy J

2011-12-01

Numerous studies have investigated the relationship between soy foods, soy protein, or isoflavone extracts and markers of bone health and osteoporosis prevention, and have come to conflicting conclusions. Research on dietary patterns, rather than on specific food ingredients or individual foods, may offer an opportunity for better understanding the role of soy foods in bone health. Evidence is reviewed regarding the question of whether soy foods contribute to a dietary pattern in humans that supports and promotes bone health. Soy foods are associated with improved markers of bone health and improved outcomes, especially among Asian women. Although the optimal amounts and types of soy foods needed to support bone health are not yet clear, dietary pattern evidence suggests that regular consumption of soy foods is likely to be useful for optimal bone health as an integral part of a dietary pattern that is built largely from whole plant foods.

Proteomic analysis of tardigrades: towards a better understanding of molecular mechanisms by anhydrobiotic organisms.

PubMed

Schokraie, Elham; Hotz-Wagenblatt, Agnes; Warnken, Uwe; Mali, Brahim; Frohme, Marcus; Förster, Frank; Dandekar, Thomas; Hengherr, Steffen; Schill, Ralph O; Schnölzer, Martina

2010-03-03

Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

PubMed Central

Schokraie, Elham; Hotz-Wagenblatt, Agnes; Warnken, Uwe; Mali, Brahim; Frohme, Marcus; Förster, Frank; Dandekar, Thomas; Hengherr, Steffen; Schill, Ralph O.; Schnölzer, Martina

2010-01-01

Background Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Principal Findings Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. Conclusions The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades. PMID:20224743

Identification of fish species after cooking by SDS-PAGE and urea IEF: a collaborative study.

PubMed

Etienne, M; Jérôme, M; Fleurence, J; Rehbein, H; Kündiger, R; Mendes, R; Costa, H; Pérez-Martín, R; Piñeiro-González, C; Craig, A; Mackie, I; Malmheden Yman, I; Ferm, M; Martínez, I; Jessen, F; Smelt, A; Luten, J

2000-07-01

A collaborative study, to validate the use of SDS-PAGE and urea IEF, for the identification of fish species after cooking has been performed by nine laboratories. By following optimized standard operation procedures, 10 commercially important species (Atlantic salmon, sea trout, rainbow trout, turbot, Alaska pollock, pollack, pink salmon, Arctic char, chum salmon, and New Zealand hake) had to be identified by comparison with 22 reference samples. Some differences in the recoveries of proteins from cooked fish flesh were noted between the urea and the SDS extraction procedures used. Generally, the urea extraction procedure appears to be less efficient than the SDS extraction for protein solubilization. Except for some species belonging to the Salmonidae family (Salmo, Oncorhynchus), both of the analytical techniques tested (urea IEF, SDS-PAGE) enabled identification of the species of the samples to be established. With urea IEF, two laboratories could not differentiate Salmo salar from Salmo trutta. The same difficulties were noted for differentiation between Oncorhynchus gorbuscha and Oncorhynchus keta samples. With SDS-PAGE, three laboratories had some difficulties in identifying the S. trutta samples. However, in the contrast with the previous technique, SDS-PAGE allows the characterization of most of the Oncorhynchus species tested. Only Oncorhynchus mykiss was not clearly recognized by one laboratory. Therefore, SDS-PAGE (Excel gel homogeneous 15%) appears to be better for the identification, after cooking, of fish such as the tuna and salmon species which are characterized by neutral and basic protein bands, and urea IEF (CleanGel) is better for the gadoid species, which are characterized by acid protein bands (parvalbumins). Nevertheless, in contentious cases it is preferable to use both analytical methods.

Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis.

PubMed

Eckert, Catherine; Lecerf, Maxime; Dubost, Lionel; Arthur, Michel; Mesnage, Stéphane

2006-12-01

The major peptidoglycan hydrolase of Enterococcus faecalis, AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is an N-acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of the atlA gene in Escherichia coli or partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms of E. faecalis extracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from an atlA null mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA in E. faecalis extracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan.

Functional Analysis of AtlA, the Major N-Acetylglucosaminidase of Enterococcus faecalis▿

PubMed Central

Eckert, Catherine; Lecerf, Maxime; Dubost, Lionel; Arthur, Michel; Mesnage, Stéphane

2006-01-01

The major peptidoglycan hydrolase of Enterococcus faecalis, AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is an N-acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of the atlA gene in Escherichia coli or partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms of E. faecalis extracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from an atlA null mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA in E. faecalis extracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan. PMID:17041059

Canola Proteins for Human Consumption: Extraction, Profile, and Functional Properties

PubMed Central

Tan, Siong H; Mailer, Rodney J; Blanchard, Christopher L; Agboola, Samson O

2011-01-01

Canola protein isolate has been suggested as an alternative to other proteins for human food use due to a balanced amino acid profile and potential functional properties such as emulsifying, foaming, and gelling abilities. This is, therefore, a review of the studies on the utilization of canola protein in human food, comprising the extraction processes for protein isolates and fractions, the molecular character of the extracted proteins, as well as their food functional properties. A majority of studies were based on proteins extracted from the meal using alkaline solution, presumably due to its high nitrogen yield, followed by those utilizing salt extraction combined with ultrafiltration. Characteristics of canola and its predecessor rapeseed protein fractions such as nitrogen yield, molecular weight profile, isoelectric point, solubility, and thermal properties have been reported and were found to be largely related to the extraction methods. However, very little research has been carried out on the hydrophobicity and structure profiles of the protein extracts that are highly relevant to a proper understanding of food functional properties. Alkaline extracts were generally not very suitable as functional ingredients and contradictory results about many of the measured properties of canola proteins, especially their emulsification tendencies, have also been documented. Further research into improved extraction methods is recommended, as is a more systematic approach to the measurement of desired food functional properties for valid comparison between studies. PMID:21535703

Characterization of N-palmitoylated human growth hormone by in situ liquid-liquid extraction and MALDI tandem mass spectrometry.

PubMed

Sachon, Emmanuelle; Nielsen, Per Franklin; Jensen, Ole Nørregaard

2007-06-01

Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N(epsilon) group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid-liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N-palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N-palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications.

MitoRes: a resource of nuclear-encoded mitochondrial genes and their products in Metazoa.

PubMed

Catalano, Domenico; Licciulli, Flavio; Turi, Antonio; Grillo, Giorgio; Saccone, Cecilia; D'Elia, Domenica

2006-01-24

Mitochondria are sub-cellular organelles that have a central role in energy production and in other metabolic pathways of all eukaryotic respiring cells. In the last few years, with more and more genomes being sequenced, a huge amount of data has been generated providing an unprecedented opportunity to use the comparative analysis approach in studies of evolution and functional genomics with the aim of shedding light on molecular mechanisms regulating mitochondrial biogenesis and metabolism. In this context, the problem of the optimal extraction of representative datasets of genomic and proteomic data assumes a crucial importance. Specialised resources for nuclear-encoded mitochondria-related proteins already exist; however, no mitochondrial database is currently available with the same features of MitoRes, which is an update of the MitoNuc database extensively modified in its structure, data sources and graphical interface. It contains data on nuclear-encoded mitochondria-related products for any metazoan species for which this type of data is available and also provides comprehensive sequence datasets (gene, transcript and protein) as well as useful tools for their extraction and export. MitoRes http://www2.ba.itb.cnr.it/MitoRes/ consolidates information from publicly external sources and automatically annotates them into a relational database. Additionally, it also clusters proteins on the basis of their sequence similarity and interconnects them with genomic data. The search engine and sequence management tools allow the query/retrieval of the database content and the extraction and export of sequences (gene, transcript, protein) and related sub-sequences (intron, exon, UTR, CDS, signal peptide and gene flanking regions) ready to be used for in silico analysis. The tool we describe here has been developed to support lab scientists and bioinformaticians alike in the characterization of molecular features and evolution of mitochondrial targeting sequences. The way it provides for the retrieval and extraction of sequences allows the user to overcome the obstacles encountered in the integrative use of different bioinformatic resources and the completeness of the sequence collection allows intra- and interspecies comparison at different biological levels (gene, transcript and protein).

Comprehensive automation of the solid phase extraction gas chromatographic mass spectrometric analysis (SPE-GC/MS) of opioids, cocaine, and metabolites from serum and other matrices.

PubMed

Lerch, Oliver; Temme, Oliver; Daldrup, Thomas

2014-07-01

The analysis of opioids, cocaine, and metabolites from blood serum is a routine task in forensic laboratories. Commonly, the employed methods include many manual or partly automated steps like protein precipitation, dilution, solid phase extraction, evaporation, and derivatization preceding a gas chromatography (GC)/mass spectrometry (MS) or liquid chromatography (LC)/MS analysis. In this study, a comprehensively automated method was developed from a validated, partly automated routine method. This was possible by replicating method parameters on the automated system. Only marginal optimization of parameters was necessary. The automation relying on an x-y-z robot after manual protein precipitation includes the solid phase extraction, evaporation of the eluate, derivatization (silylation with N-methyl-N-trimethylsilyltrifluoroacetamide, MSTFA), and injection into a GC/MS. A quantitative analysis of almost 170 authentic serum samples and more than 50 authentic samples of other matrices like urine, different tissues, and heart blood on cocaine, benzoylecgonine, methadone, morphine, codeine, 6-monoacetylmorphine, dihydrocodeine, and 7-aminoflunitrazepam was conducted with both methods proving that the analytical results are equivalent even near the limits of quantification (low ng/ml range). To our best knowledge, this application is the first one reported in the literature employing this sample preparation system.

Properties of gelatin film from horse mackerel (Trachurus japonicus) scale.

PubMed

Le, Thuy; Maki, Hiroki; Takahashi, Kigen; Okazaki, Emiko; Osako, Kazufumi

2015-04-01

Optimal conditions for extracting gelatin and preparing gelatin film from horse mackerel scale, such as extraction temperature and time, as well as the protein concentration of film-forming solutions were investigated. Yields of extracted gelatin at 70 °C, 80 °C, and 90 °C for 15 min to 3 h were 1.08% to 3.45%, depending on the extraction conditions. Among the various extraction times and temperatures, the film from gelatin extracted at 70 °C for 1 h showed the highest tensile strength and elongation at break. Horse mackerel scale gelatin film showed the greatly low water vapor permeability (WVP) compared with mammalian or fish gelatin films, maybe due to its containing a slightly higher level of hydrophobic amino acids (total 653 residues per 1000 residues) than that of mammalian, cold-water fish and warm-water fish gelatins. Gelatin films from different preparation conditions showed excellent UV barrier properties at wavelength of 200 nm, although the films were transparent at visible wavelength. As a consequence, it can be suggested that gelatin film from horse mackerel scale extracted at 70 °C for 1 h can be applied to food packaging material due to its lowest WVP value and excellent UV barrier properties. © 2015 Institute of Food Technologists®

Investigating the fate of activated sludge extracellular proteins in sludge digestion using sodium dodecyl sulfate polyacrylamide gel electrophoresis.

PubMed

Park, Chul; Helm, Richard F; Novak, John T

2008-12-01

The fate of activated sludge extracellular proteins in sludge digestion was investigated using three different cation-associated extraction methods and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Extraction methods used were the cation exchange resin (CER) method for extracting calcium (Ca2+) and magnesium (Mg2+), sulfide extraction for removing iron, and base treatment (pH 10.5) for dissolving aluminum. Extracellular polymeric substances extracted were then subjected to SDS-PAGE, and the resultant protein profiles were examined before and after sludge digestion. The SDS-PAGE results showed that three methods led to different SDS-PAGE profiles for both undigested and digested sludges. The results further revealed that CER-extracted proteins remained mainly undegraded in anaerobic digestion, but were degraded in aerobic digestion. While the fate of sulfide- and base-extracted proteins was not clear for aerobic digestion, their changes in anaerobic digestion were elucidated. Most sulfide-extracted proteins were removed by anaerobic digestion, while the increase in protein band intensity and diversity was observed for base-extracted proteins. These results suggest that activated sludge flocs contain different fractions of proteins that are distinguishable by their association with certain cations and that each fraction undergoes different fates in anaerobic and aerobic digestion. The proteins that were resistant to degradation and generated during anaerobic digestion were identified by liquid chromatography tandem mass spectrometry. Protein identification results and their putative roles in activated sludge and anaerobic digestion are discussed in this study.

Protein distribution in lupin protein isolates from Lupinus angustifolius L. prepared by various isolation techniques.

PubMed

Muranyi, Isabel S; Volke, Daniela; Hoffmann, Ralf; Eisner, Peter; Herfellner, Thomas; Brunnbauer, Markus; Koehler, Peter; Schweiggert-Weisz, Ute

2016-09-15

Differences in the protein distribution of various protein isolates from Lupinus angustifolius L. Vitabor were identified as affected by the isolation procedure (alkaline and/or salt-induced extraction followed by isoelectric and/or dilutive precipitation). Protein isolates extracted in alkaline solution showed higher protein yields (26.4-31.7%) compared to salt-induced extraction (19.8-30.0%) or combined alkaline and salt-induced extraction (23.3-25.6%). Chemical variations among the protein isolates especially occurred within the albumins. Protein isolates precipitated isoelectrically showed the highest contents, whereas protein isolates precipitated by dilutive showed the lowest contents of conglutin δ. Furthermore, the alkaline subunits of conglutin α and conglutin γ decreased during alkaline extraction compared to salt-induced extraction. A decrease in protein-bound polar and basic amino acids was shown after protein isolation. In contrast, the amounts of nonpolar, aliphatic, aromatic, hydroxylated and sulfur-rich amino acids were higher in the lupin protein isolates compared to the lupin flakes. However, the functional side chains could not be related to the specific molecular arrangements of the protein isolates, as a similar amino acid composition was found among the protein isolates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Isolation of fish skin and bone gelatin from tilapia (Oreochromis niloticus): Response surface approach

NASA Astrophysics Data System (ADS)

Arpi, N.; Fahrizal; Novita, M.

2018-03-01

In this study, gelatin from fish collagen, as one of halal sources, was extracted from tilapia (Oreochromis niloticus) skin and bone, by using Response Surface Methodology to optimize gelatin extraction conditions. Concentrations of alkaline NaOH and acid HCl, in the pretreatment process, and temperatures in extraction process were chosen as independent variables, while dependent variables were yield, gel strength, and emulsion activity index (EAI). The result of investigation showed that lower NaOH pretreatment concentrations provided proper pH extraction conditions which combine with higher extraction temperatures resulted in high gelatin yield. However, gelatin emulsion activity index increased proportionally to the decreased in NaOH concentrations and extraction temperatures. No significant effect of the three independent variables on the gelatin gel strength. RSM optimization process resulted in optimum gelatin extraction process conditions using alkaline NaOH concentration of 0.77 N, acid HCl of 0.59 N, and extraction temperature of 66.80 °C. The optimal solution formula had optimization targets of 94.38%.

ICan: An Optimized Ion-Current-Based Quantification Procedure with Enhanced Quantitative Accuracy and Sensitivity in Biomarker Discovery

PubMed Central

2015-01-01

The rapidly expanding availability of high-resolution mass spectrometry has substantially enhanced the ion-current-based relative quantification techniques. Despite the increasing interest in ion-current-based methods, quantitative sensitivity, accuracy, and false discovery rate remain the major concerns; consequently, comprehensive evaluation and development in these regards are urgently needed. Here we describe an integrated, new procedure for data normalization and protein ratio estimation, termed ICan, for improved ion-current-based analysis of data generated by high-resolution mass spectrometry (MS). ICan achieved significantly better accuracy and precision, and lower false-positive rate for discovering altered proteins, over current popular pipelines. A spiked-in experiment was used to evaluate the performance of ICan to detect small changes. In this study E. coli extracts were spiked with moderate-abundance proteins from human plasma (MAP, enriched by IgY14-SuperMix procedure) at two different levels to set a small change of 1.5-fold. Forty-five (92%, with an average ratio of 1.71 ± 0.13) of 49 identified MAP protein (i.e., the true positives) and none of the reference proteins (1.0-fold) were determined as significantly altered proteins, with cutoff thresholds of ≥1.3-fold change and p ≤ 0.05. This is the first study to evaluate and prove competitive performance of the ion-current-based approach for assigning significance to proteins with small changes. By comparison, other methods showed remarkably inferior performance. ICan can be broadly applicable to reliable and sensitive proteomic survey of multiple biological samples with the use of high-resolution MS. Moreover, many key features evaluated and optimized here such as normalization, protein ratio determination, and statistical analyses are also valuable for data analysis by isotope-labeling methods. PMID:25285707

Ultrasound Assisted Extraction of Phenolic Compounds from Peaches and Pumpkins

PubMed Central

Altemimi, Ammar; Watson, Dennis G.; Choudhary, Ruplal; Dasari, Mallika R.; Lightfoot, David A.

2016-01-01

The ultrasound-assisted extraction (UAE) method was used to optimize the extraction of phenolic compounds from pumpkins and peaches. The response surface methodology (RSM) was used to study the effects of three independent variables each with three treatments. They included extraction temperatures (30, 40 and 50°C), ultrasonic power levels (30, 50 and 70%) and extraction times (10, 20 and 30 min). The optimal conditions for extractions of total phenolics from pumpkins were inferred to be a temperature of 41.45°C, a power of 44.60% and a time of 25.67 min. However, an extraction temperature of 40.99°C, power of 56.01% and time of 25.71 min was optimal for recovery of free radical scavenging activity (measured by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) reduction). The optimal conditions for peach extracts were an extraction temperature of 41.53°C, power of 43.99% and time of 27.86 min for total phenolics. However, an extraction temperature of 41.60°C, power of 44.88% and time of 27.49 min was optimal for free radical scavenging activity (judged by from DPPH reduction). Further, the UAE processes were significantly better than solvent extractions without ultrasound. By electron microscopy it was concluded that ultrasonic processing caused damage in cells for all treated samples (pumpkin, peach). However, the FTIR spectra did not show any significant changes in chemical structures caused by either ultrasonic processing or solvent extraction. PMID:26885655

Protein profile and protein interaction network of Moniliophthora perniciosa basidiospores.

PubMed

Mares, Joise Hander; Gramacho, Karina Peres; Dos Santos, Everton Cruz; Santiago, André da Silva; Silva, Edson Mário de Andrade; Alvim, Fátima Cerqueira; Pirovani, Carlos Priminho

2016-06-24

Witches' broom, a disease caused by the basidiomycete Moniliophthora perniciosa, is considered to be the most important disease of the cocoa crop in Bahia, an area in the Brazilian Amazon, and also in the other countries where it is found. M. perniciosa germ tubes may penetrate into the host through intact or natural openings in the cuticle surface, in epidermis cell junctions, at the base of trichomes, or through the stomata. Despite its relevance to the fungal life cycle, basidiospore biology has not been extensively investigated. In this study, our goal was to optimize techniques for producing basidiospores for protein extraction, and to produce the first proteomics analysis map of ungerminated basidiospores. We then presented a protein interaction network by using Ustilago maydis as a model. The average pileus area ranged from 17.35 to 211.24 mm(2). The minimum and maximum productivity were 23,200 and 6,666,667 basidiospores per basidiome, respectively. The protein yield in micrograms per million basidiospores were approximately 0.161; 2.307, and 3.582 for germination times of 0, 2, and 4 h after germination, respectively. A total of 178 proteins were identified through mass spectrometry. These proteins were classified according to their molecular function and their involvement in biological processes such as cellular energy production, oxidative metabolism, stress, protein synthesis, and protein folding. Furthermore, to better understand the expression pattern, signaling, and interaction events of spore proteins, we presented an interaction network using orthologous proteins from Ustilago maydis as a model. Most of the orthologous proteins that were identified in this study were not clustered in the network, but several of them play a very important role in hypha development and branching. The quantities of basidiospores 7 × 10(9); 5.2 × 10(8), and 6.7 × 10(8) were sufficient to obtain enough protein mass for the three 2D-PAGE replicates, for the 0, 2, and 4 h-treatments, respectively. The protein extraction method that is based on sedimentation, followed by sonication with SDS-dense buffer, and phenolic extraction, which was utilized in this study, was effective, presenting a satisfactory resolution and reproducibility for M. perniciosa basidiospores. This report constitutes the first comprehensive study of protein expression during the ungerminated stage of the M. perniciosa basidiospore. Identification of the spots observed in the reference gel enabled us to know the main molecular interactions involved in the initial metabolic processes of fungal development.

Effects of controlled gas environments in microbial enhancement of plant protein recovery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mudgett, R.E.; Bajracharya, R.

Controlled gas environments were maintained by a novel aeration system in solid substrate fermentations for enhanced protein recovery from pressed alfalfa residues. High O/sub 2/ pressures stimulated biomass and enzyme production by an Aspergillus species, isolated from alfalfa, which produced cellulase and pectinase activities in growth-associated metabolism. High CO/sub 2/ pressures also stimulated enzyme production, but had less effect on biomass production, as established from the dissimilation of plant solids. Cellulase and pectinase activities were generally related to protein recoveries. Recoveries were greater than or equal to 50% higher than those obtained by mechanical extraction, with maximum recoveries of greatermore » than or equal to 70% of crude protein contents. Protein not recovered at high cellulase and pectinase activities were believed to be in structurally bound forms not amenable to recovery by nonproteolytic enzymes. Buffering at pH 8 and autoclaving of residues prior to fermentation had little effect on protein recoveries. Controlled gas environments are seen to offer an interesting potential for optimizing industrial fermentation processes for the production of microbial enzymes.« less

Polysaccharide extraction from Sphallerocarpus gracilis roots by response surface methodology.

PubMed

Ma, Tingting; Sun, Xiangyu; Tian, Chengrui; Luo, Jiyang; Zheng, Cuiping; Zhan, Jicheng

2016-07-01

The extraction process of Sphallerocarpus gracilis root polysaccharides (SGRP) was optimized using response surface methodology with two methods [hot-water extraction (HWE) and ultrasonic-assisted extraction (UAE)]. The antioxidant activities of SGRP were determined, and the structural features of the untreated materials (HWE residue and UAE residue) and the extracted polysaccharides were compared by scanning electron microscopy. Results showed that the optimal UAE conditions were extraction temperature of 81°C, extraction time of 1.7h, liquid-solid ratio of 17ml/g, ultrasonic power of 300W and three extraction cycles. The optimal HWE conditions were 93°C extraction temperature, 3.6h extraction time, 21ml/g liquid-solid ratio and three extraction cycles. UAE offered a higher extraction yield with a shorter time, lower temperature and a lower solvent consumption compared with HWE, and the extracted polysaccharides possessed a higher antioxidant capacity. Therefore, UAE could be used as an alternative to conventional HWE for SGRP extraction. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of acid- and alkaline-aided extractions on functional and rheological properties of proteins recovered from mechanically separated turkey meat (MSTM).

PubMed

Hrynets, Yuliya; Omana, Dileep A; Xu, Yan; Betti, Mirko

2010-09-01

Functional and rheological characteristics of acid- and alkali-extracted proteins from mechanically separated turkey meat (MSTM) have been investigated. Extractions were carried out at 4 pH values (2.5, 3.5, 10.5, and 11.5). The study demonstrated that alkali and acid extractions resulted in significant (P < 0.0001) decreases of cooking and water loss compared to raw MSTM; however, the cooking loss was found to be similar (P = 0.5699) among the different protein isolates. Proteins extracted at pH 10.5 showed the lowest (P = 0.0249) water loss. Emulsion and foaming properties were found to be slightly higher in alkali-extracted proteins compared to those for acid extractions. The myofibrillar protein fraction showed better ability to form and stabilize emulsions compared to sarcoplasmic proteins. Myofibrillar proteins also showed better foam expansion; however, foam volume stability was similar for both myofibrillar and sarcoplasmic protein fractions. Textural characteristics (hardness, chewiness, springiness, and cohesiveness) of recovered proteins were found to be unaffected (P > 0.05) by different extraction pH. The protein extracted at pH 3.5 formed a highly viscoelastic gel network as evidenced by storage modulus (G') values, whereas the gel formed from proteins extracted at pH 10.5 was found to be the weakest. The work also revealed that acid treatments were more effective for removal of total heme pigments from MSTM. Color characteristics of protein isolates were markedly improved compared to the initial material and tended to be better when subjected to acid extractions. Mechanically separated meat is one of the cheapest sources of protein obtained by grinding meat and bones together and forcing the mixture through a perforated drum. The use of mechanically separated turkey meat (MSTM) for the production of further processed poultry products is limited due to its undesirable color and textural properties. Recovery of proteins from MSTM using pH shifting process will help the poultry processors to get better returns and also create opportunity to produce functional food ingredients.

Optimization of microwave-assisted extraction of analgesic and anti-inflammatory drugs from human plasma and urine using response surface experimental designs.

PubMed

Fernández, Purificación; Fernández, Ana M; Bermejo, Ana M; Lorenzo, Rosa A; Carro, Antonia M

2013-04-01

The performance of microwave-assisted extraction and HPLC with photodiode array detection method for determination of six analgesic and anti-inflammatory drugs from plasma and urine, is described, optimized, and validated. Several parameters affecting the extraction technique were optimized using experimental designs. A four-factor (temperature, phosphate buffer pH 4.0 volume, extraction solvent volume, and time) hybrid experimental design was used for extraction optimization in plasma, and three-factor (temperature, extraction solvent volume, and time) Doehlert design was chosen to extraction optimization in urine. The use of desirability functions revealed the optimal extraction conditions as follows: 67°C, 4 mL phosphate buffer pH 4.0, 12 mL of ethyl acetate and 9 min, for plasma and the same volume of buffer and ethyl acetate, 115°C and 4 min for urine. Limits of detection ranged from 4 to 45 ng/mL in plasma and from 8 to 85 ng/mL in urine. The reproducibility evaluated at two concentration levels was less than 6.5% for both specimens. The recoveries were from 89 to 99% for plasma and from 83 to 99% for urine. The proposed method was successfully applied in plasma and urine samples obtained from analgesic users. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of soft extraction method for structural characterization of boreal forest soil proteins with MALDI-TOF/MS

NASA Astrophysics Data System (ADS)

Kanerva, Sanna; Ketola, Raimo A.; Kitunen, Veikko; Smolander, Aino; Kotiaho, Tapio

2010-05-01

Nitrogen (N) is usually the nutrient restricting productivity in boreal forests. Forest soils contain a great amount of nitrogen, but only a small part of it is in mineral form. Most part of soil N is bound in the structures of different organic compounds such as proteins, peptides, amino acids and more stabilized, refractory compounds. Due to the fact that soil organic N has a very important role in soil nutrient cycling and in plant nutrition, there is a need for more detailed knowledge of its chemistry in soil. Conventional methods to extract and analyze soil organic N are usually very destructive for structures of higher molecular weight organic compounds, such as proteins. The aim of this study was to characterize proteins extracted from boreal forest soil by "soft" extraction methods in order to maintain their molecular structure. The organic layer (F) from birch forest floor containing 78% of organic matter was sieved, freeze dried, pulverized, and extracted with a citrate or phosphate buffer (pH 6 or 8). Sequential extraction with the citrate or phosphate buffer and an SDS buffer (pH 6.8), slightly modified from the method of Chen et al. (2009, Proteomics 9: 4970-4973), was also done. Proteins were purified from the soil extract by extraction with buffered phenol and precipitated with methanol + 0.1M ammonium acetate at -20°C. Characterization of proteins was performed with matrix assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF/MS) and the concentration of total proteins was measured using Bradford's method. Bovine serum albumin (BSA) was used as a positive control in the extractions and as a standard protein in Bradford's method. Our results showed that sequential extraction increased the amount of extracted proteins compared to the extractions without the SDS-buffer; however, it must be noted that the use of SDS-buffer very probably increased denaturization of proteins. Purification of proteins from crude soil extracts by phenol extraction was essential prior to measurement of total proteins; there seemed to be a lot of compounds in crude soil extracts that interfere with the analysis of total proteins, causing overestimation in protein concentration. pH of the buffer solution did not seem to be very crucial for the extractability of soil natural proteins, but at the higher pH, the amount of interfering compounds increased. However, the recovery of BSA added was clearly higher at the higher pH. When the protein precipitates were analyzed with MALDI-TOF/MS, a large curve, most likely formed from wide peaks of several compounds, indicate that most of the compounds in the precipitate were <15 kDa or ~20-50 kDa in molecular weight. It seems that in order to identify individual proteins from mass spectra, a separation of compounds with varying molecular weight is needed before the MALDI-TOF/MS analysis. Due to the fact that a relatively high amount of BSA added was not recovered by the extractions and that the intensity of the signals observed in mass spectra was low, it is questionable whether it is possible to extract soil natural proteins effectively from soils containing a high amount of organic matter without destructing the structures of proteins.

[Studies on extraction process optimization of Panax notogingseng saponins].

PubMed

Qu, Lin-hai; Zheng, Ming; Lou, Yi-jia

2006-06-01

To optimize the conditions for the extraction of panax notogingseng saponins (PNS). After selected extraction solvent and suitable particle, we employed orthogonal experimental design to examine the conditions for the extraction by determination of PNS. Significant effect was observed only in extraction times. The optimum condition for extraction of PNS was to extract panax notogingseng (Burk.) F. H. Chen with 10 times 70% ethanol for 1.5 hours for 3 times.

A new high-performance thin layer chromatography-based assay of detergents and surfactants commonly used in membrane protein studies.

PubMed

Barret, Laurie-Anne; Polidori, Ange; Bonneté, Françoise; Bernard-Savary, Pierre; Jungas, Colette

2013-03-15

The hydrophobic nature of membrane proteins (MPs) necessitates the use of detergents for their extraction, solubilization and purification. Because the concentration of amphiphiles is crucial in the crystallization process, detergent quantification is essential to routine analysis. Here we describe a quantitative high-performance thin-layer chromatography (HPTLC) method we developed for the detection of small quantities of detergent bound to solubilized MPs. After optimization of aqueous deposit conditions, we show that most detergents widely used in membrane protein crystallography display distinctive mobilities in a mixture of dichloromethane, methanol and acetic acid 32:7.6:0.4 (v/v/v). Migration and derivatization conditions were optimized with n-dodecyl-β-D-maltoside (DDM), the most popular detergent for membrane protein crystallization. A linear calibration curve very well fits our data from 0.1 to 1.6 μg of DDM in water with a limit of detection of 0.05 μg. This limit of detection is the best achieved to date for a routine detergent assay, being not modified by the addition of NaCl, commonly used in protein buffers. With these chromatographic conditions, no prior treatment is required to assess the quantities of detergent bound to purified MPs, thus enabling the quantification of close structure detergents via a single procedure. This HPTLC method, which is fast and requires low sample volume, is fully suitable for routine measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Properties of low-fat, low-cholesterol egg yolk prepared by supercritical CO2 extraction.

PubMed

Bringe, N A

1997-01-01

A dry egg yolk ingredient called Eggcellent has 74% less fat and 90% less cholesterol than liquid egg yolks, when reconstituted on an equal protein basis. The phospholipids and proteins are retained, enabling the ingredient to have the taste and texturizing properties of fresh egg yolk. Using the new yolk, it is possible to significantly improve the acceptability of low-fat, low-cholesterol bakery products, scrambled eggs and mayonnaise dressings without losing nutritional claims. The structures and functional properties of egg yolk components and the conditions required to optimize their benefits in foods are reviewed. The lipoproteins of low-fat, low-cholesterol yolk have valuable properties as flavorants, texturizers, foaming agents, emulsifiers, antioxidants, colorants, and nutraceuticals.

Comparison of methods for extracting kafirin proteins from sorghum distillers dried grains with solubles.

PubMed

Wang, Ying; Tilley, Michael; Bean, Scott; Sun, X Susan; Wang, Donghai

2009-09-23

Use of coproducts generated during fermentation is important to the overall economics of biofuel production. The main coproduct from grain-based ethanol production is distillers dried grains with solubles (DDGS). High in protein, DDGS is a potential source of protein for many bioindustrial applications such as adhesives and resins. The objective of this research was to characterize the composition as well as chemical and physical properties of kafirin proteins from sorghum DDGS with various extraction methods including use of acetic acid, HCl-ethanol and NaOH-ethanol under reducing conditions. Extraction conditions affected purity and thermal properties of the extracted kafirin proteins. Extraction yields of 44.2, 24.2, and 56.8% were achieved by using acetic acid, HCl-ethanol and NaOH-ethanol, respectively. Acetic acid and NaOH-ethanol produced protein with higher purity than kafirins extracted with the HCl-ethanol protocol. The acetic acid extraction protocol produced protein with the highest purity, 98.9%. Several techniques were used to evaluate structural, molecular and thermal properties of kairin extracts. FTIR showed alpha-helix dominated in all three samples, with only a small portion of beta-sheet present. Electrophoresis results showed alpha(1), alpha(2) band and beta kafirins were present in all three extracts. Glass transition peaks of the extracts were shown by DSC to be approximately 230 degrees C. Kafirin degraded at 270-290 degrees C. Size exclusion chromatography revealed that the acetic acid and HCl-ethanol based extraction methods tended to extract more high molecular weight protein than the NaOH-ethanol based method. Reversed phase high-performance liquid chromatography showed that the gamma kafirins were found only in extracts from the NaOH-ethanol extraction method.

Absolute quantitation of low abundance plasma APL1β peptides at sub-fmol/mL Level by SRM/MRM without immunoaffinity enrichment.

PubMed

Sano, Shozo; Tagami, Shinji; Hashimoto, Yuuki; Yoshizawa-Kumagaye, Kumiko; Tsunemi, Masahiko; Okochi, Masayasu; Tomonaga, Takeshi

2014-02-07

Selected/multiple reaction monitoring (SRM/MRM) has been widely used for the quantification of specific proteins/peptides, although it is still challenging to quantitate low abundant proteins/peptides in complex samples such as plasma/serum. To overcome this problem, enrichment of target proteins/peptides is needed, such as immunoprecipitation; however, this is labor-intense and generation of antibodies is highly expensive. In this study, we attempted to quantify plasma low abundant APLP1-derived Aβ-like peptides (APL1β), a surrogate marker for Alzheimer's disease, by SRM/MRM using stable isotope-labeled reference peptides without immunoaffinity enrichment. A combination of Cibacron Blue dye mediated albumin removal and acetonitrile extraction followed by C18-strong cation exchange multi-StageTip purification was used to deplete plasma proteins and unnecessary peptides. Optimal and validated precursor ions to fragment ion transitions of APL1β were developed on a triple quadruple mass spectrometer, and the nanoliquid chromatography gradient for peptide separation was optimized to minimize the biological interference of plasma. Using the stable isotope-labeled (SI) peptide as an internal control, absolute concentrations of plasma APL1β peptide could be quantified as several hundred amol/mL. To our knowledge, this is the lowest detection level of endogenous plasma peptide quantified by SRM/MRM.

Antifungal-protein production in maize (Zea mays) suspension cultures.

PubMed

Perri, Fabio; Della Penna, Serena; Rufini, Francesca; Patamia, Maria; Bonito, Mariantonietta; Angiolella, Letizia; Vitali, Alberto

2009-04-01

The growing emergency due to the phenomenon of drug resistance to micro-organisms has pushed forward the search for new potential drug alternatives to those already in use. Plants represent a suitable source of new antifungal molecules, as they produce a series of defensive proteins. Among them are the PRPs (pathogenesis-related proteins), shown to be effective in vitro against human pathogens. An optimized and established cell-suspension culture of maize (Zea mays) was shown to constitutively secrete in the medium a series of PRPs comprising the antifungal protein zeamatin (P33679) with a final yield of approx. 3 mg/litre. The in-vitro-produced zeamatin possessed antifungal activity towards a clinical strain of the human pathogenic yeast Candida albicans, an activity comparable with the one reported for the same protein extracted from maize seeds. Along with zeamatin, other PRPs were expressed: a 9 kDa lipid-transfer protein, a 26 kDa xylanase inhibitor and a new antifungal protein, PR-5. A fast, two-step chromatographic procedure was set up allowing the complete purification of the proteins considered, making this cell line a valuable system for the production of potential antifungal agents in a reliable and easy way.

Targeted Pressure Management During CO 2 Sequestration: Optimization of Well Placement and Brine Extraction

DOE PAGES

Cihan, Abdullah; Birkholzer, Jens; Bianchi, Marco

2014-12-31

Large-scale pressure increases resulting from carbon dioxide (CO 2) injection in the subsurface can potentially impact caprock integrity, induce reactivation of critically stressed faults, and drive CO 2 or brine through conductive features into shallow groundwater. Pressure management involving the extraction of native fluids from storage formations can be used to minimize pressure increases while maximizing CO2 storage. However, brine extraction requires pumping, transportation, possibly treatment, and disposal of substantial volumes of extracted brackish or saline water, all of which can be technically challenging and expensive. This paper describes a constrained differential evolution (CDE) algorithm for optimal well placement andmore » injection/ extraction control with the goal of minimizing brine extraction while achieving predefined pressure contraints. The CDE methodology was tested for a simple optimization problem whose solution can be partially obtained with a gradient-based optimization methodology. The CDE successfully estimated the true global optimum for both extraction well location and extraction rate, needed for the test problem. A more complex example application of the developed strategy was also presented for a hypothetical CO 2 storage scenario in a heterogeneous reservoir consisting of a critically stressed fault nearby an injection zone. Through the CDE optimization algorithm coupled to a numerical vertically-averaged reservoir model, we successfully estimated optimal rates and locations for CO 2 injection and brine extraction wells while simultaneously satisfying multiple pressure buildup constraints to avoid fault activation and caprock fracturing. The study shows that the CDE methodology is a very promising tool to solve also other optimization problems related to GCS, such as reducing ‘Area of Review’, monitoring design, reducing risk of leakage and increasing storage capacity and trapping.« less

Green tea extract impairs meat emulsion properties by disturbing protein disulfide cross-linking.

PubMed

Jongberg, Sisse; Terkelsen, Linda de S; Miklos, Rikke; Lund, Marianne N

2015-02-01

The dose-dependent effects of green tea extract (100, 500, or 1500ppm) on the textural and oxidative stability of meat emulsions were investigated, and compared to a control meat emulsion without extract. All levels of green tea extract inhibited formation of TBARS as a measure for lipid oxidation. Overall protein thiol oxidation and myosin heavy chain (MHC) cross-linking were inhibited by 100ppm green tea extract without jeopardizing the textural stability, while increasing concentrations of extract resulted in reduced thiol concentration and elevated levels of non-reducible protein modifications. Addition of 1500ppm green tea extract was found to modify MHC as evaluated by SDS-PAGE combining both protein staining and specific thiol staining, indicating that protein modifications generated through reactions of green tea phenolic compounds with protein thiols, disrupted the meat emulsion properties leading to reduced water holding capacity and textural stability. Hence, a low dose of green tea extract preserves both the textural and the oxidative stability of the meat proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

Selective extraction and separation of oxymatrine from Sophora flavescens Ait. extract by silica-confined ionic liquid.

PubMed

Bi, Wentao; Tian, Minglei; Row, Kyung Ho

2012-01-01

This study highlighted the application of a two-stepped extraction method for extraction and separation of oxymatrine from Sophora flavescens Ait. extract by utilizing silica-confined ionic liquids as sorbent. The optimized silica-confined ionic liquid was firstly mixed with plant extract to adsorb oxymatrine. Simultaneously, some interference, such as matrine, was removed. The obtained suspension was then added to a cartridge for solid phase extraction. Through these two steps, target compound was adequately separated from interferences with 93.4% recovery. In comparison with traditional solid phase extraction, this method accelerates loading and reduces the use of organic solvents during washing. Moreover, the optimization of loading volume was simplified as optimization of solid/liquid ratio. Copyright © 2011 Elsevier B.V. All rights reserved.

A novel procedure for the extraction of protein deposits from soft hydrophilic contact lenses for analysis.

PubMed

Keith, D; Hong, B; Christensen, M

1997-05-01

A quick, simple, and efficient extraction technique was developed for the removal of protein from soft hydrophilic contact lenses. An extraction solvent consisting of a 50:50 mix of 0.2% trifluoroacetic acid and acetonitrile was used to remove protein from in vitro laboratory-deposited and human-worn contact lenses. The protein removed was analyzed using HPLC, bicinchoninic acid (BCA) analysis, and SDS-PAGE gel electreophoresis. Extraction efficiency for lysozyme from laboratory-deposited Group IV lenses was determined to be approximately 100%. Group IV human-worn contact lenses were extracted and analyzed for lysozyme by HPLC and total protein by bicinchoninic acid (BCA) analysis. Groups I, II, III, and IV contact lenses deposited with an artificial tear protein solution and human-worn lenses were extracted and analyzed by SDS-PAGE gel electreophoresis and micro-BCA. The ACN/TFA procedure offers a simple, quick, and efficient extraction technique for removal of protein from contact lenses for subsequent analysis.

A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

PubMed

Redmile-Gordon, M A; Armenise, E; White, R P; Hirsch, P R; Goulding, K W T

2013-12-01

Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.

Optimization and Technological Development Strategies of an Antimicrobial Extract from Achyrocline alata Assisted by Statistical Design

PubMed Central

Demarque, Daniel P.; Fitts, Sonia Maria F.; Boaretto, Amanda G.; da Silva, Júlio César Leite; Vieira, Maria C.; Franco, Vanessa N. P.; Teixeira, Caroline B.; Toffoli-Kadri, Mônica C.; Carollo, Carlos A.

2015-01-01

Achyrocline alata, known as Jateí-ka-há, is traditionally used to treat several health problems, including inflammations and infections. This study aimed to optimize an active extract against Streptococcus mutans, the main bacteria that causes caries. The extract was developed using an accelerated solvent extraction and chemometric calculations. Factorial design and response surface methodologies were used to determine the most important variables, such as active compound selectivity. The standardized extraction recovered 99% of the four main compounds, gnaphaliin, helipyrone, obtusifolin and lepidissipyrone, which represent 44% of the extract. The optimized extract of A. alata has a MIC of 62.5 μg/mL against S. mutans and could be used in mouth care products. PMID:25710523

Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis.

PubMed

Yu, Daoyong; Huang, Guihong; Xu, Fengxi; Wang, Mengfei; Liu, Shuang; Huang, Fang

2014-06-01

Surfactants play important roles in the preparation, structural, and functional research of membrane proteins, and solubilizing and isolating membrane protein, while keeping their structural integrity and activity intact is complicated. The commercial n-Dodecyl-β-D-maltoside (DDM) and Triton X-100 (TX) were used as solubilizers to extract and purify trimeric photosystem I (PSI) complex, an important photosynthetic membrane protein complex attracting broad interests. With an optimized procedure, TX can be used as an effective surfactant to isolate and purify PSI, as a replace of the much more expensive DDM. A mechanism was proposed to interpret the solubilization process at surfactant concentrations lower than the critical solubilization concentration. PSI-TX and PSI-DDM had identical polypeptide bands, pigment compositions, oxygen consumption, and photocurrent activities. This provides an alternative procedure and paves a way for economical and large-scale trimeric PSI preparation.

Simple, Rapid, and Selective Isolation of 2S Albumins from Allergenic Seeds and Nuts.

PubMed

Hummel, Marlene; Wigger, Tina; Höper, Tessa; Westkamp, Imke; Brockmeyer, Jens

2015-07-08

The 2S albumins belong to the group of seed storage proteins present in different seeds and nuts. Due to their pronounced allergenic potential, which is often associated with severe allergic reactions, this protein family is of special interest in the field of allergen research. Here we present a simple, rapid, and selective method for the purification of 2S albumins directly from allergenic seeds and nuts. We systematically optimized the parameters "buffer system", "extraction temperature", "buffer molarity", and "pH " and were able to achieve 2S albumin purities of about 99% without further purification and demonstrate transferability of this method to nine different allergenic food matrices. Compared to conventional isolation routines, significant reduction of hands-on time and required laboratory equipment is achieved, but nonetheless higher protein yields are obtained. The presented method allows for the rapid purification of different 2S albumins including the corresponding isoforms from natural material.

Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction.

PubMed

Mohammadi, Samira; Esfahani, Bahram Nasr; Moghim, Sharareh; Mirhendi, Hossein; Zaniani, Fatemeh Riyahi; Safaei, Hajieh Ghasemian; Fazeli, Hossein; Salehi, Mahshid

2017-01-01

Nontuberculous mycobacteria (NTM) are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA ® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR) amplification of the heat-shock protein 65 gene with serially diluted DNA samples. The CTAB method showed more positive results at 1:10-1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor.

Pumpkin (Cucurbita maxima) seed proteins: sequential extraction processing and fraction characterization.

PubMed

Rezig, Leila; Chibani, Farhat; Chouaibi, Moncef; Dalgalarrondo, Michèle; Hessini, Kamel; Guéguen, Jacques; Hamdi, Salem

2013-08-14

Seed proteins extracted from Tunisian pumpkin seeds ( Cucurbita maxima ) were investigated for their solubility properties and sequentially extracted according to the Osborne procedure. The solubility of pumpkin proteins from seed flour was greatly influenced by pH changes and ionic strength, with higher values in the alkaline pH regions. It also depends on the seed defatting solvent. Protein solubility was decreased by using chloroform/methanol (CM) for lipid extraction instead of pentane (P). On the basis of differential solubility fractionation and depending on the defatting method, the alkali extract (AE) was the major fraction (42.1 (P), 22.3% (CM)) compared to the salt extract (8.6 (P), 7.5% (CM)). In salt, alkali, and isopropanol extracts, all essential amino acids with the exceptions of threonine and lysine met the minimum requirements for preschool children (FAO/WHO/UNU). The denaturation temperatures were 96.6 and 93.4 °C for salt and alkali extracts, respectively. Pumpkin protein extracts with unique protein profiles and higher denaturation temperatures could impart novel characteristics when used as food ingredients.

Use of an enzyme-assisted method to improve protein extraction from olive leaves.

PubMed

Vergara-Barberán, M; Lerma-García, M J; Herrero-Martínez, J M; Simó-Alfonso, E F

2015-02-15

The improvement of protein extraction from olive leaves using an enzyme-assisted protocol has been investigated. Using a cellulase enzyme (Celluclast® 1.5L), different parameters that affect the extraction process, such as the influence and amount of organic solvent, enzyme amount, pH and extraction temperature and time, were optimised. The influence of these factors was examined using the standard Bradford assay and the extracted proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum extraction parameters were: 30% acetonitrile, 5% (v/v) Celluclast® 1.5L at pH 5.0 and 55°C for 15min. Under these conditions, several protein extracts from olive leaves of different genetic variety (with a total protein amount comprised between 1.87 and 6.64mgg(-1)) were analysed and compared by SDS-PAGE, showing differences in their electrophoretic protein profiles. The developed enzyme-assisted extraction method has shown a faster extraction, higher recovery and reduced solvent usage with respect to the use of the non-enzymatic methods described in literature. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Optimization for supercritical CO2 extraction with response surface methodology and component analysis of Sapindus mukorossi oil].

PubMed

Wu, Yan; Xiao, Xin-yu; Ge, Fa-huan

2012-02-01

To study the extraction conditions of Sapindus mukorossi oil by Supercritical CO2 Extraction and identify its components. Optimized SFE-CO2 Extraction by response surface methodology and used GC-MS to analysie Sapindus mukorossi oil compounds. Established the model of an equation for the extraction rate of Sapindus mukorossi oil by Supercritical CO2 Extraction, and the optimal parameters for the Supercritical CO2 Extraction determined by the equation were: the extraction pressure was 30 MPa, temperature was 40 degrees C; The separation I pressure was 14 MPa, temperature was 45 degrees C; The separation II pressure was 6 MPa, temperature was 40 degrees C; The extraction time was 60 min and the extraction rate of Sapindus mukorossi oil of 17.58%. 22 main compounds of Sapindus mukorossi oil extracted by supercritical CO2 were identified by GC-MS, unsaturated fatty acids were 86.59%. This process is reliable, safe and with simple operation, and can be used for the extraction of Sapindus mukorossi oil.

Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) seed oil.

PubMed

Tian, Yuting; Xu, Zhenbo; Zheng, Baodong; Martin Lo, Y

2013-01-01

The effectiveness of ultrasonic-assisted extraction (UAE) of pomegranate seed oil (PSO) was evaluated using a variety of solvents. Petroleum ether was the most effective for oil extraction, followed by n-hexane, ethyl acetate, diethyl ether, acetone, and isopropanol. Several variables, such as ultrasonic power, extraction temperature, extraction time, and the ratio of solvent volume and seed weight (S/S ratio) were studied for optimization using response surface methodology (RSM). The highest oil yield, 25.11% (w/w), was obtained using petroleum ether under optimal conditions for ultrasonic power, extraction temperature, extraction time, and S/S ratio at 140 W, 40 °C, 36 min, and 10 ml/g, respectively. The PSO yield extracted by UAE was significantly higher than by using Soxhlet extraction (SE; 20.50%) and supercriti cal fluid extraction (SFE; 15.72%). The fatty acid compositions were significantly different among the PSO extracted by Soxhlet extraction, SFE, and UAE, with punicic acid (>65%) being the most dominant using UAE. Copyright © 2012 Elsevier B.V. All rights reserved.

Synthesis of silver nanoparticles by solar irradiation of cell-free Bacillus amyloliquefaciens extracts and AgNO3.

PubMed

Wei, Xuetuan; Luo, Mingfang; Li, Wei; Yang, Liangrong; Liang, Xiangfeng; Xu, Lin; Kong, Peng; Liu, Huizhou

2012-01-01

Silver nanoparticles (AgNPs) were obtained by solar irradiation of cell-free extracts of Bacillusamyloliquefaciens and AgNO3. Light intensity, extract concentration, and NaCl addition influenced the synthesis of AgNPs. Under optimized conditions (solar intensity 70,000 lx, extract concentration 3 mg/mL, and NaCl content 2 mM), 98.23±0.06% of the Ag+ (1 mM) was reduced to AgNPs within 80 min, and the ζ-potential of AgNPs reached -70.84±0.66 mV. TEM (Transmission electron microscopy) and XRD (X-ray diffraction) analysis confirmed that circular and triangular crystalline AgNPs with mean diameter of 14.6 nm were synthesized. Since heat-inactivated extracts also mediated the formation of AgNPs, enzymatic reactions are likely not involved in AgNPs formation. A high absolute ζ-potential value of the AgNPs, possibly caused by interaction with proteins likely explains the high stability of AgNPs suspensions. AgNPs showed antimicrobial activity against Bacillussubtilis and Escherichiacoli in liquid and solid medium. Copyright © 2011 Elsevier Ltd. All rights reserved.

Maltose Neopentyl Glycol-3 (MNG-3) Analogues for Membrane Protein Study

PubMed Central

Cho, Kyung Ho; Husri, Mohd; Amin, Anowarul; Gotfryd, Kamil; Lee, Ho Jin; Go, Juyeon; Kim, Jin Woong; Loland, Claus J.; Guan, Lan; Byrne, Bernadette

2015-01-01

Detergents are typically used to both extract membrane proteins (MPs) from the lipid bilayer and maintain them in solution. However, MPs encapsulated in detergent micelles are often prone to denaturation and aggregation. Thus, development of novel agents with enhanced stabilization characteristics is necessary to advance MP research. Maltose neopentyl glycol-3 (MNG-3) has contributed to >10 crystal structures including G-protein coupled receptors. Here we prepared MNG-3 analogues and characterised their properties using selected MPs. Most MNGs behaved superior to a conventional detergent, n–dodecyl–β–D–maltopyranoside (DDM), in terms of membrane protein stabilization efficacy. Interestingly, optimal stabilization was achieved with different MNG-3 analogues depending on the target MP. The origin for such detergent specificity could be explained by a novel concept: compatibility between detergent hydrophobicity and MP tendency to denature and aggregate. This set of MNGs represents viable alternatives to currently available detergents for handling MPs, and can be also used as tools to estimate MP sensitivity to denaturation and aggregation. PMID:25813698

Maltose neopentyl glycol-3 (MNG-3) analogues for membrane protein study.

PubMed

Cho, Kyung Ho; Husri, Mohd; Amin, Anowarul; Gotfryd, Kamil; Lee, Ho Jin; Go, Juyeon; Kim, Jin Woong; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

2015-05-07

Detergents are typically used to both extract membrane proteins (MPs) from the lipid bilayers and maintain them in solution. However, MPs encapsulated in detergent micelles are often prone to denaturation and aggregation. Thus, the development of novel agents with enhanced stabilization characteristics is necessary to advance MP research. Maltose neopentyl glycol-3 (MNG-3) has contributed to >10 crystal structures including G-protein coupled receptors. Here, we prepared MNG-3 analogues and characterised their properties using selected MPs. Most MNGs were superior to a conventional detergent, n-dodecyl-β-D-maltopyranoside (DDM), in terms of membrane protein stabilization efficacy. Interestingly, optimal stabilization was achieved with different MNG-3 analogues depending on the target MP. The origin for such detergent specificity could be explained by a novel concept: compatibility between detergent hydrophobicity and MP tendency to denature and aggregate. This set of MNGs represents viable alternatives to currently available detergents for handling MPs, and can be also used as tools to estimate MP sensitivity to denaturation and aggregation.

Optimization of acidic extraction of astaxanthin from Phaffia rhodozyma *

PubMed Central

Ni, Hui; Chen, Qi-he; He, Guo-qing; Wu, Guang-bin; Yang, Yuan-fan

2008-01-01

Optimization of a process for extracting astaxanthin from Phaffia rhodozyma by acidic method was investigated, regarding several extraction factors such as acids, organic solvents, temperature and time. Fractional factorial design, central composite design and response surface methodology were used to derive a statistically optimal model, which corresponded to the following optimal condition: concentration of lactic acid at 5.55 mol/L, ratio of ethanol to yeast dry weight at 20.25 ml/g, temperature for cell-disruption at 30 °C, and extraction time for 3 min. Under this condition, astaxanthin and the total carotenoids could be extracted in amounts of 1294.7 μg/g and 1516.0 μg/g, respectively. This acidic method has advantages such as high extraction efficiency, low chemical toxicity and no special requirement of instruments. Therefore, it might be a more feasible and practical method for industrial practice. PMID:18196613

Optimization of ultrasonic-assisted extraction of antioxidant compounds from Guava (Psidium guajava L.) leaves using response surface methodology

PubMed Central

Kong, Fansheng; Yu, Shujuan; Feng, Zeng; Wu, Xinlan

2015-01-01

Objective: To optimization of extraction of antioxidant compounds from guava (Psidium guajava L.) leaves and showed that the guava leaves are the potential source of antioxidant compounds. Materials and Methods: The bioactive polysaccharide compounds of guava leaves (P. guajava L.) were obtained using ultrasonic-assisted extraction. Extraction was carried out according to Box-Behnken central composite design, and independent variables were temperature (20–60°C), time (20–40 min) and power (200–350 W). The extraction process was optimized by using response surface methodology for the highest crude extraction yield of bioactive polysaccharide compounds. Results: The optimal conditions were identified as 55°C, 30 min, and 240 W. 1,1-diphenyl-2-picryl-hydrazyl and hydroxyl free radical scavenging were conducted. Conclusion: The results of quantification showed that the guava leaves are the potential source of antioxidant compounds. PMID:26246720

Optimization of ultrasonic-assisted extraction of antioxidant compounds from Guava (Psidium guajava L.) leaves using response surface methodology.

PubMed

Kong, Fansheng; Yu, Shujuan; Feng, Zeng; Wu, Xinlan

2015-01-01

To optimization of extraction of antioxidant compounds from guava (Psidium guajava L.) leaves and showed that the guava leaves are the potential source of antioxidant compounds. The bioactive polysaccharide compounds of guava leaves (P. guajava L.) were obtained using ultrasonic-assisted extraction. Extraction was carried out according to Box-Behnken central composite design, and independent variables were temperature (20-60°C), time (20-40 min) and power (200-350 W). The extraction process was optimized by using response surface methodology for the highest crude extraction yield of bioactive polysaccharide compounds. The optimal conditions were identified as 55°C, 30 min, and 240 W. 1,1-diphenyl-2-picryl-hydrazyl and hydroxyl free radical scavenging were conducted. The results of quantification showed that the guava leaves are the potential source of antioxidant compounds.

An Optimization-Based Framework for the Transformation of Incomplete Biological Knowledge into a Probabilistic Structure and Its Application to the Utilization of Gene/Protein Signaling Pathways in Discrete Phenotype Classification.

PubMed

Esfahani, Mohammad Shahrokh; Dougherty, Edward R

2015-01-01

Phenotype classification via genomic data is hampered by small sample sizes that negatively impact classifier design. Utilization of prior biological knowledge in conjunction with training data can improve both classifier design and error estimation via the construction of the optimal Bayesian classifier. In the genomic setting, gene/protein signaling pathways provide a key source of biological knowledge. Although these pathways are neither complete, nor regulatory, with no timing associated with them, they are capable of constraining the set of possible models representing the underlying interaction between molecules. The aim of this paper is to provide a framework and the mathematical tools to transform signaling pathways to prior probabilities governing uncertainty classes of feature-label distributions used in classifier design. Structural motifs extracted from the signaling pathways are mapped to a set of constraints on a prior probability on a Multinomial distribution. Being the conjugate prior for the Multinomial distribution, we propose optimization paradigms to estimate the parameters of a Dirichlet distribution in the Bayesian setting. The performance of the proposed methods is tested on two widely studied pathways: mammalian cell cycle and a p53 pathway model.

Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

PubMed

Treder, Krzysztof; Chołuj, Joanna; Zacharzewska, Bogumiła; Babujee, Lavanya; Mielczarek, Mateusz; Burzyński, Adam; Rakotondrafara, Aurélie M

2018-02-01

Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.

Preparation of magnetic core mesoporous shell microspheres with C18-modified interior pore-walls for fast extraction and analysis of phthalates in water samples.

PubMed

Li, Zhongbo; Huang, Danni; Fu, Chinfai; Wei, Biwen; Yu, Wenjia; Deng, Chunhui; Zhang, Xiangmin

2011-09-16

In this study, core-shell magnetic mesoporous microspheres with C18-functionalized interior pore-walls were synthesized through coating Fe(3)O(4) microspheres with a mesoporous inorganic-organic hybrid layer with a n-octadecyltriethoxysilane (C18TES) and tetraethyl orthosilicate (TEOS) as the silica source and cetyltrimethylammonia bromide (CTAB) as a template. The obtained C18-functionalized Fe(3)O(4)@mSiO(2) microspheres possess numerous C18 groups anchored in the interior pore-walls, large surface area (274.7 m(2)/g, high magnetization (40.8 emu/g) and superparamagnetism, uniform mesopores (4.1 nm), which makes them ideal absorbents for simple, fast, and efficient extraction and enrichment of hydrophobic organic compounds in water samples. Several kinds of phthalates were used as the model hydrophobic organic compounds to systematically evaluate the performance of the C18-functionalized Fe(3)O(4)@mSiO(2) microspheres in extracting hydrophobic molecules by using a gas chromatography-mass spectrometry. Various parameters, including eluting solvent, the amounts of absorbents, extraction time and elution time were optimized. Hydrophobic extraction was performed in the interior pore of magnetic mesoporous microspheres, and the materials had the anti-interference ability to macromolecular proteins, which was also investigated in the work. Under the optimized conditions, C18-functionalized Fe(3)O(4)@mSiO(2) microspheres were successfully used to analyze the real water samples. The results indicated that this novel method was fast, convenient and efficient for the target compounds and could avoid being interfered by macromolecules. Copyright © 2011 Elsevier B.V. All rights reserved.

Optimization of extraction conditions for osthol, a melanogenesis inhibitor from Cnidium monnieri fruits.

PubMed

Beom Kim, Seon; Kim, CheongTaek; Liu, Qing; Hee Jo, Yang; Joo Choi, Hak; Hwang, Bang Yeon; Kyum Kim, Sang; Kyeong Lee, Mi

2016-08-01

Coumarin derivatives have been reported to inhibit melanin biosynthesis. The melanogenesis inhibitory activity of osthol, a major coumarin of the fruits of Cnidium monnieri Cusson (Umbelliferae), and optimized extraction conditions for the maximum yield from the isolation of osthol from C. monnieri fruits were investigated. B16F10 melanomas were treated with osthol at concentration of 1, 3, and 10 μM for 72 h. The expression of melanogenesis genes, such as tyrosinase, TRP-1, and TRP-2 was also assessed. For optimization, extraction factors such as extraction solvent, extraction time, and sample/solvent ratio were tested and optimized for maximum yield of osthol using response surface methodology with the Box-Behnken design (BBD). Osthol inhibits melanin content in B16F10 melanoma cells with an IC50 value of 4.9 μM. The melanogenesis inhibitory activity of osthol was achieved not by direct inhibition of tyrosinase activity but by inhibiting melanogenic enzyme expressions, such as tyrosinase, TRP-1, and TRP-2. The optimal condition was obtained as a sample/solvent ratio, 1500 mg/10 ml; an extraction time 30.3 min; and a methanol concentration of 97.7%. The osthol yield under optimal conditions was found to be 15.0 mg/g dried samples, which were well matched with the predicted value of 14.9 mg/g dried samples. These results will provide useful information about optimized extraction conditions for the development of osthol as cosmetic therapeutics to reduce skin hyperpigmentation.

Optimization of ultrasound-assisted extraction (UAE) of phenolic compounds from olive cake.

PubMed

Mojerlou, Zohreh; Elhamirad, Amirhhossein

2018-03-01

The use of ultrasound in ultrasound-assisted extraction (UAE) is one of the main applications of this technology in food industry. This study aimed to optimize UAE conditions for olive cake extract (OCE) through response surface methodology (RSM). The optimal UAE conditions were obtained with extraction temperature of 56 °C, extraction time of 3 min, duty cycle of 0.6 s, and solid to solvent ratio of 3.6%. At the optimum conditions, the total phenolic compounds (TPC) content and antioxidant activity (AA) were measured 4.04 mg/g and 68.9%, respectively. The linear term of temperature had the most effect on TPC content and AA of OCE prepared by UAE. Protocatechuic acid and cinnamic acid were characterized as the highest (19.5%) and lowest (1.6%) phenolic compound measured in OCE extracted by UAE. This research revealed that UAE is an effective method to extract phenolic compounds from olive cake. RSM successfully optimized UAE conditions for OCE.

Effects of ultrahigh pressure extraction on yield and antioxidant activity of chlorogenic acid and cynaroside extracted from flower buds of Lonicera japonica.

PubMed

Hu, Wen; Guo, Ting; Jiang, Wen-Jun; Dong, Guang-Li; Chen, Da-Wei; Yang, Shi-Lin; Li, He-Ran

2015-06-01

The present study was designed to establish and optimize a new method for extracting chlorogenic acid and cynaroside from Lonicera japonica Thunb. through orthogonal experimental designl. A new ultrahigh pressure extraction (UPE) technology was applied to extract chlorogenic acid and cynaroside from L. japonica. The influential factors, including solvent type, ethanol concentration, extraction pressure, time, and temperature, and the solid/liquid ratio, have been studied to optimize the extraction process. The optimal conditions for the UPE were developed by quantitative analysis of the extraction products by HPLC-DAD in comparison with standard samples. In addition, the microstructures of the medicinal materials before and after extraction were studied by scanning electron microscopy (SEM). Furthermore, the extraction efficiency of different extraction methods and the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of the extracts were investigated. The optimal conditions for extracting chlorogenic acid and cynaroside were as follows: ethanol concentration, 60%; extraction pressure, 400 MPa; extraction time, 2 min; extraction temperature, 30 °C; and the solid/liquid ratio, 1 : 50. Under these conditions, the yields of chlorogenic acid and cynaroside were raised to 4.863% and 0.080%, respectively. Compared with other extraction methods, such as heat reflux extraction (HRE), ultrasonic extraction (UE), and Sohxlet extraction (SE), the UPE method showed several advantages, including higher extraction yield, shorter extraction time, lower energy consumption, and higher purity of the extracts. This study could help better utilize L. japonica flower buds as a readily accessible source of natural antioxidants in food and pharmaceutical industries. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Optimized Extraction, Preliminary Characterization, and In Vitro Antioxidant Activity of Polysaccharides from Glycyrrhiza Uralensis Fisch

PubMed Central

Chen, Jie; Li, Wan-chen; Gu, Xin-li

2017-01-01

Background This study performed optimized extraction, preliminary characterization, and in vitro antioxidant activities of polysaccharides from Glycyrrhiza uralensis Fisch. Material/Methods Three parameters (extraction temperature, ratio of water to raw material, and extraction time) were optimized for yields of G. uralensis polysaccharides (GUP) using response surface methodology with Box-Behnken design (BBD). The GUP was purified using DEAE cellulose 32-column chromatography. The main fraction obtained from G. uralensis Fisch was GUP-II, which was composed of rhamnose, arabinose, galactose, and glucose monosaccharide, was screened for antioxidant properties using DP Hand hydroxyl radical scavenging assays. In addition, immunological activity of GUP-II was determined by nitric oxide and lymphocyte proliferation assays. Results Optimization revealed maximum GUP yields with an extraction temperature of 99°C, water: raw material ratio of 15: 1, and extraction duration of 2 h. GUP-II purified from G. uralensis Fisch had good in vitro DPPH and hydroxyl radical scavenging abilities. Immunologically, GUP-II significantly stimulated NO production in RAW 264.7 macrophages, and significantly enhanced LPS-induced lymphocyte proliferation. Conclusions Extraction of GUP from G. uralensis Fisch can be optimized with respect to temperature, extraction period, and ratio of water to material, using response surface methodology. The purified product (GUP-II) possesses excellent antioxidant and immunological activities. PMID:28404983

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals.

PubMed

Colgan, Richard; Atkinson, Christopher J; Paul, Matthew; Hassan, Sally; Drake, Pascal M W; Sexton, Amy L; Santa-Cruz, Simon; James, David; Hamp, Keith; Gutteridge, Colin; Ma, Julian K-C

2010-04-01

Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth temperature. Overall productivity is therefore correlated to biomass production, although other factors such as purification burden, extractability protein stability and quality also need to be considered in the optimal design of cultivation conditions.

Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

PubMed Central

2011-01-01

Background Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His) tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices. Results Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel) for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel) did not work well with respect to specific binding capacity and recovery profile. Conclusions Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also provide helpful hints for those researchers facing same challenge. PMID:21824404

"Fast proteins" with a unique essential amino acid content as an optimal nutrition in the elderly: growing evidence.

PubMed

Gryson, Céline; Walrand, Stéphane; Giraudet, Christophe; Rousset, Paulette; Migné, Carole; Bonhomme, Cécile; Le Ruyet, Pascale; Boirie, Yves

2014-08-01

Adequate protein intake is crucial to maintain body protein content in elderly subjects, but quality of dietary proteins should be also considered since amino acid composition and rate of protein digestion modulate amino acid availability. This study investigates whether the efficacy of optimal protein intake levels for protein retention in the elderly is influenced by protein quality. We investigated the effect of a 10-day adequate-protein (AP) or high-protein (HP) diet together with the protein source as caseins (CAS) or soluble milk proteins (PRO) on whole-body (WB) protein synthesis (PS) and protein breakdown (PB) in 4 groups of healthy elderly men (mean ± SEM: 71.8 ± 24.4 yr). The study consisted of two periods of 4 h each: a post-absorptive period and a postprandial period. The fed state was defined by consumption every 20 min and for 4 h, of either 15 g or 30 g of PRO or CAS. Steady-state WB and splanchnic leucine kinetics were measured using a continuous infusion of L-[1-13C]leucine in the postabsorptive state and L-[1-13C]leucine infusion plus oral L-[5,5,5-2H3]leucine in the postprandial state. WB PS was stimulated by feeding only with HP diets, whereas WB PB corrected for splanchnic extraction showed a similar pattern of post-feeding decrease in all groups. Consequently, net leucine balance was greater in the postprandial state after HP meals than after AP meals, with PRO meals leading to a better postprandial leucine balance (3.63 ± 0.16 μmol kg FFM(-1) min(-1)vs. 2.77 ± 0.21 μmol kg FFM(-1) min(-1) for PRO HP and CAS HP, respectively; P = 0.005). Postprandial protein retention was better improved in elderly men by an increase in protein intake when the protein supplementation was provided as fast-digesting proteins that induce high leucine availability. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

[Study on extraction process of Radix Bupleuri].

PubMed

Zhao, Lei; Liu, Benliang; Wu, Fuxiang; Tao, Lanping; Liu, Jian

2004-10-01

The orthogonal design was used to optimize extraction process of Radix Bupleuri with content of total saponin and yield of the extract as markers. Factors that have been chosen were ethanol concentration, ethanol consumption, extraction times and extraction time. Each factor had three levels. The result showed that the optimum extraction condition was 80% ethanol, 4 times the amount of material, refluxing for 4 times, 60 minutes each time. The optimized process was stable and workable.

Ultrasound-assisted extraction of Mangiferin from Mango (Mangifera indica L.) leaves using response surface methodology.

PubMed

Zou, Tang-Bin; Xia, En-Qin; He, Tai-Ping; Huang, Ming-Yuan; Jia, Qing; Li, Hua-Wen

2014-01-27

Mangiferin is a xanthone widely distributed in higher plants showing antioxidative, antiviral, anticancer, antidiabetic, immunomodulatory, hepatoprotective and analgesic effects. In the present study, an ultrasonic-assisted extraction method was developed for the effective extraction of mangiferin from mango leaves. Some parameters such as ethanol concentration, liquid-to-solid ratio, extraction temperature, and extraction time were optimized by single-factor experiment and response surface methodology. The optimal extraction conditions were 44% ethanol, the liquid-to-solid ratio was 38:1, and extraction for 19.2 min at 60 °C under ultrasound irradiation of 200 W. Under optimal conditions, the yield of mangiferin was 58.46 ± 1.27 mg/g. The results obtained are helpful for the full utilization of mango leaves, and also indicated that ultrasonic-assisted extraction is a very useful method for the extraction of mangiferin from plant materials.

Optimization of Bioactive Ingredient Extraction from Chinese Herbal Medicine Glycyrrhiza glabra: A Comparative Study of Three Optimization Models

PubMed Central

Li, Xiaohong; Zhang, Yuyan

2018-01-01

The ultraviolet spectrophotometric method is often used for determining the content of glycyrrhizic acid from Chinese herbal medicine Glycyrrhiza glabra. Based on the traditional single variable approach, four extraction parameters of ammonia concentration, ethanol concentration, circumfluence time, and liquid-solid ratio are adopted as the independent extraction variables. In the present work, central composite design of four factors and five levels is applied to design the extraction experiments. Subsequently, the prediction models of response surface methodology, artificial neural networks, and genetic algorithm-artificial neural networks are developed to analyze the obtained experimental data, while the genetic algorithm is utilized to find the optimal extraction parameters for the above well-established models. It is found that the optimization of extraction technology is presented as ammonia concentration 0.595%, ethanol concentration 58.45%, return time 2.5 h, and liquid-solid ratio 11.065 : 1. Under these conditions, the model predictive value is 381.24 mg, the experimental average value is 376.46 mg, and the expectation discrepancy is 4.78 mg. For the first time, a comparative study of these three approaches is conducted for the evaluation and optimization of the effects of the extraction independent variables. Furthermore, it is demonstrated that the combinational method of genetic algorithm and artificial neural networks provides a more reliable and more accurate strategy for design and optimization of glycyrrhizic acid extraction from Glycyrrhiza glabra. PMID:29887907

Optimization of Bioactive Ingredient Extraction from Chinese Herbal Medicine Glycyrrhiza glabra: A Comparative Study of Three Optimization Models.

PubMed

Yu, Li; Jin, Weifeng; Li, Xiaohong; Zhang, Yuyan

2018-01-01

The ultraviolet spectrophotometric method is often used for determining the content of glycyrrhizic acid from Chinese herbal medicine Glycyrrhiza glabra . Based on the traditional single variable approach, four extraction parameters of ammonia concentration, ethanol concentration, circumfluence time, and liquid-solid ratio are adopted as the independent extraction variables. In the present work, central composite design of four factors and five levels is applied to design the extraction experiments. Subsequently, the prediction models of response surface methodology, artificial neural networks, and genetic algorithm-artificial neural networks are developed to analyze the obtained experimental data, while the genetic algorithm is utilized to find the optimal extraction parameters for the above well-established models. It is found that the optimization of extraction technology is presented as ammonia concentration 0.595%, ethanol concentration 58.45%, return time 2.5 h, and liquid-solid ratio 11.065 : 1. Under these conditions, the model predictive value is 381.24 mg, the experimental average value is 376.46 mg, and the expectation discrepancy is 4.78 mg. For the first time, a comparative study of these three approaches is conducted for the evaluation and optimization of the effects of the extraction independent variables. Furthermore, it is demonstrated that the combinational method of genetic algorithm and artificial neural networks provides a more reliable and more accurate strategy for design and optimization of glycyrrhizic acid extraction from Glycyrrhiza glabra .

Extraction and characterization of corn germ proteins

USDA-ARS?s Scientific Manuscript database

Our study was conducted to develop methods to extract corn germ protein economically and characterize and identify potential applications of the recovered protein. Protein was extracted from both wet germ and finished (dried) germ using 0.1M NaCl as solvent. The method involved homogenization, sti...

Extraction, composition and functional properties of pennycress (Thlaspi arvense L.) press cake protein

USDA-ARS?s Scientific Manuscript database

This study compared two methods for extracting the protein in pennycress (Thlaspi arvense L.) press cake and determined the composition and functional properties of the protein products. Proteins in pennycress press cake were extracted by using the conventional alkali solubilization-acid precipitati...

The Metaproteome of "Park Grass" soil - a reference for EU soil science

NASA Astrophysics Data System (ADS)

Quinn, Gerry; Dudley, Ed; Doerr, Stefan; Matthews, Peter; Halen, Ingrid; Walley, Richard; Ashton, Rhys; Delmont, Tom; Francis, Lewis; Gazze, Salvatore Andrea; Van Keulen, Geertje

2016-04-01

Soil metaproteomics, the systemic extraction and identification of proteins from a soil, is key to understanding the biological and physical processes that occur within the soil at a molecular level. Until recently, direct extraction of proteins from complex soils have yielded only dozens of protein identifications due to interfering substances, such as humic acids and clay, which co-extract and/or strongly adsorb protein, often causing problems in downstream processing, e.g. mass spectrometry. Furthermore, the current most successful, direct, proteomic extraction protocol favours larger molecular weight and/or heat-stable proteins due to its extraction protocol. We have now developed a novel, faster, direct soil protein extraction protocol which also addressed the problem of interfering substances, while only requiring less than 1 gram of material per extraction. We extracted protein from the 'Genomic Observatory' Park Grass at Rothamsted Research (UK), an ideally suited geographic site as it is the longest (>150 years) continually studied experiment on ungrazed permanent grassland in the world, for which a rich history of environmental/ecological data has been collected, including high quality publically available metagenome DNA sequences. Using this improved methodology, in conjunction with the creation of high quality, curated metagenomic sequence databases, we have been able to significantly improve protein identifications from one soil due to extracting a similar number of proteins that were >90% different when compared to the best current direct protocol. This optimised metaproteomics protocol has now enabled identification of thousands of proteins from one soil, leading therefore to a deeper insight of soil system processes at the molecular scale.

A preliminary study of continuous milk coagulation using Cynara cardunculus flower extract and calf rennet immobilized on magnetic particles.

PubMed

Liburdi, Katia; Emiliani Spinelli, Sara; Benucci, Ilaria; Lombardelli, Claudio; Esti, Marco

2018-01-15

The aim of this study was to develop a bioreactor design for continuous milk coagulation using a biocatalyst composed of immobilized animal and vegetable rennet on aminated magnetic particles, which has been proven to be an appropriate carrier for enzyme immobilization. Calf and vegetable (Cynara cardunculus) rennets were covalently immobilized on CLEA® magnetic supports and the immobilization procedure was optimized in batch mode, by evaluating protein loading, caseinolytic activity and the coagulation properties of skim milk powder and cow's milk. Subsequently the optimal temperature of immobilized coagulant was defined and a technically-friendly enzyme bioreactor was developed in order to carry out a continuous milk coagulation process with the aim of producing soft cheese. Copyright © 2017 Elsevier Ltd. All rights reserved.

Optimization of ultrasonic-assisted extraction of total saponins from Eclipta prostrasta L. using response surface methodology.

PubMed

Hu, Ting; Guo, Yan-Yun; Zhou, Qin-Fan; Zhong, Xian-Ke; Zhu, Liang; Piao, Jin-Hua; Chen, Jian; Jiang, Jian-Guo

2012-09-01

Eclipta prostrasta L. is a traditional Chinese medicine herb, which is rich in saponins and has strong antiviral and antitumor activities. An ultrasonic-assisted extraction (UAE) technique was developed for the fast extraction of saponins from E. prostrasta. The content of total saponins in E. prostrasta was determined using UV/vis spectrophotometric methods. Several influential parameters like ethanol concentration, extraction time, temperature, and liquid/solid ratio were investigated for the optimization of the extraction using single factor and Box-Behnken experimental designs. Extraction conditions were optimized for maximum yield of total saponins in E. prostrasta using response surface methodology (RSM) with 4 independent variables at 3 levels of each variable. Results showed that the optimization conditions for saponins extraction were: ethanol concentration 70%, extraction time 3 h, temperature 70 °C, and liquid/solid ratio 14:1. Corresponding saponins content was 2.096%. The mathematical model developed was found to fit well with the experimental data. Practical Application: Although there are wider applications of Eclipta prostrasta L. as a functional food or traditional medicine due to its various bioactivities, these properties are limited by its crude extracts. Total saponins are the main active ingredient of E. prostrasta. This research has optimized the extraction conditions of total saponins from E. prostrasta, which will provide useful reference information for further studies, and offer related industries with helpful guidance in practice. © 2012 Institute of Food Technologists®

An Optimal Control Method for Maximizing the Efficiency of Direct Drive Ocean Wave Energy Extraction System

PubMed Central

Chen, Zhongxian; Yu, Haitao; Wen, Cheng

2014-01-01

The goal of direct drive ocean wave energy extraction system is to convert ocean wave energy into electricity. The problem explored in this paper is the design and optimal control for the direct drive ocean wave energy extraction system. An optimal control method based on internal model proportion integration differentiation (IM-PID) is proposed in this paper though most of ocean wave energy extraction systems are optimized by the structure, weight, and material. With this control method, the heavy speed of outer heavy buoy of the energy extraction system is in resonance with incident wave, and the system efficiency is largely improved. Validity of the proposed optimal control method is verified in both regular and irregular ocean waves, and it is shown that IM-PID control method is optimal in that it maximizes the energy conversion efficiency. In addition, the anti-interference ability of IM-PID control method has been assessed, and the results show that the IM-PID control method has good robustness, high precision, and strong anti-interference ability. PMID:25152913

An optimal control method for maximizing the efficiency of direct drive ocean wave energy extraction system.

PubMed

Chen, Zhongxian; Yu, Haitao; Wen, Cheng

2014-01-01

The goal of direct drive ocean wave energy extraction system is to convert ocean wave energy into electricity. The problem explored in this paper is the design and optimal control for the direct drive ocean wave energy extraction system. An optimal control method based on internal model proportion integration differentiation (IM-PID) is proposed in this paper though most of ocean wave energy extraction systems are optimized by the structure, weight, and material. With this control method, the heavy speed of outer heavy buoy of the energy extraction system is in resonance with incident wave, and the system efficiency is largely improved. Validity of the proposed optimal control method is verified in both regular and irregular ocean waves, and it is shown that IM-PID control method is optimal in that it maximizes the energy conversion efficiency. In addition, the anti-interference ability of IM-PID control method has been assessed, and the results show that the IM-PID control method has good robustness, high precision, and strong anti-interference ability.

Pectinase hydrolysis of Dendrobium huoshanense polysaccharide and its effect on protein nonenzymatic glycation.

PubMed

Zha, Xue-Qiang; Li, Xiao-Long; Zhang, Hai-Lin; Cui, Shao-Hua; Liu, Jian; Wang, Jun-Hui; Pan, Li-Hua; Luo, Jian-Ping

2013-10-01

The aim of this study was to investigate the inhibitory effects of molecular weight alteration of Dendrobium huoshanense polysaccharide on protein nonenzymatic glycation. For this purpose, one homogeneous active polysaccharide DHPD1 with molecular weight 3.2 kDa was extracted from D. huoshanense. GC analysis showed that DHPD1 was mainly composed of glucose, arabinose, galactose in a molar ratio of 0.023:1.023:0.021 with a trace of mannose and xylose. In order to get DHPD1-derived fragments with different molecular weight, response surface methodology was employed to optimize the enzymatic degradation conditions. The maximum reducing sugar production (0.399 mg/mL) was obtained under an optimal condition including pectinase dosage 126 U/mL, reaction pH 4.46 and reaction temperature 48 °C. By applying this condition, three DHPD1-derived fragments with different molecular weights were obtained through changing the hydrolysis time. Infrared spectroscopy analysis indicated that the backbone structure of DHPD1 was not destroyed by pectinase hydrolysis. Monosaccharide composition analysis showed that pectinase preferred to liberate glucose from DHPD1. The inhibitory action of DHPD1 on protein nonenzymatic glycation reduced with the decrease of molecular weight. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterisation of kiwifruit and asparagus enzyme extracts, and their activities toward meat proteins.

PubMed

Ha, Minh; Bekhit, Alaa El-Din; Carne, Alan; Hopkins, David L

2013-01-15

Two plant enzyme extracts from kiwifruit and asparagus were evaluated for their ability to hydrolyse commercially available substrates and proteins present in both beef connective tissue and topside myofibrillar extracts. The results show significant differences in protease activity depending on the assay used. Protease assays with connective tissue and meat myofibrillar extracts provide a more realistic evaluation of the potential of the enzymes for application in meat tenderization. Overall, the kiwifruit protease extract was found to be more effective at hydrolysing myofibrillar and collagen proteins than the asparagus protease extract. The two protease extracts appeared to target meat myofibrillar and collagen proteins differently, suggesting the potential of a synergistic effect of these proteases in improving the tenderness of specific cuts of meat, based on their intrinsic protein composition. Copyright © 2012 Elsevier Ltd. All rights reserved.

Optimization of ultrasound-assisted extraction of charantin from Momordica charantia fruits using response surface methodology.

PubMed

Ahamad, Javed; Amin, Saima; Mir, Showkat R

2015-01-01

Momordica charantia Linn. (Cucurbitaceae) fruits are well known for their beneficial effects in diabetes that are often attributed to its bioactive component charantin. The aim of the present study is to develop and optimize an efficient protocol for the extraction of charantin from M. charantia fruits. Response surface methodology (RSM) was used for the optimization of ultrasound-assisted extraction (UAE) conditions. RSM was based on a three-level, three-variable Box-Behnken design (BBD), and the studied variables included solid to solvent ratio, extraction temperature, and extraction time. The optimal conditions predicted by the BBD were: UAE with methanol: Water (80:20, v/v) at 46°C for 120 min with solid to solvent ratio of 1:26 w/v, under which the yield of charantin was 3.18 mg/g. Confirmation trials under slightly adjusted conditions yielded 3.12 ± 0.14 mg/g of charantin on dry weight basis of fruits. The result of UAE was also compared with Soxhlet extraction method and UAE was found 2.74-fold more efficient than the Soxhlet extraction for extracting charantin. A facile UAE protocol for a high extraction yield of charantin was developed and validated.

Ultrasound assisted extraction of polysaccharides from Lentinus edodes and its anti-hepatitis B activity in vitro.

PubMed

Zhao, Yong-Ming; Yang, Jian-Ming; Liu, Ying-Hui; Zhao, Ming; Wang, Jin

2018-02-01

The aim of this study was to optimize the extraction process of polysaccharides from the fruiting bodies of Lentinus edodes and investigate its anti-hepatitis B virus activity. The extracting parameters including ultrasonic power (240-320W), extraction temperature (40-60°C) and extraction time (15-25min) was optimized by using three-variable-three-level Box-Behnken design based on the single-factor experiments. Data analysis results showed that the optimal conditions for extracting LEPs were an extraction temperature of 45°C, extraction time of 21min and ultrasonic power of 290W. Under these optimal conditions, the experimental yield of LEPs was 9.75%, a 1.62-fold increase compared with conventional heat water extraction (HWE). In addition, crude polysaccharides were purified to obtain two fractions (LEP-1 and LEP-2). Chemical analysis showed that these components were rich in glucose, arabinose and mannose. Furthermore, HepG2.2.15 cells were used as in vitro models to evaluate their anti-hepatitis B virus (HBV) activity. The results suggest that LEPs possesses potent anti-HBV activity in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

[MALDI-TOF mass spectrometry: Evaluation of the preanalytical phase for identification of molds].

PubMed

Maldonado, Ivana; García Ramírez, Dolores; Striebeck, Pablo; Lafage, Marcelo; Fernández Canigia, Liliana

In order to optimize the identification of molds with MALDI-TOF MS, three protein extraction-methodologies were evaluated against 44 isolates: water extraction (WE), zirconium extraction (ZE) and the provider's recommended method (PRM). Two data bases were compared, Bruker (BK) and Bruker+National Institutes of Health. Considering both databases, results were respectively as follows: correct identification (CI) at gender level, 10 and 16 by WE; 27 and 32 by ZE and 18 and 23 by PRM; CI at species level, 5 and 7 by WE; 17 and 20 by ZE and 11 and 14 by PRM; non-reliable identification, 18 and 12 by WE; 9 and 4 by ZE and by PRM. No peaks were observed in 16 by WE, 8 by ZE and 17 by PRM. ZE showed the best perfomance (p<0.05). Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Combining active learning and semi-supervised learning techniques to extract protein interaction sentences.

PubMed

Song, Min; Yu, Hwanjo; Han, Wook-Shin

2011-11-24

Protein-protein interaction (PPI) extraction has been a focal point of many biomedical research and database curation tools. Both Active Learning and Semi-supervised SVMs have recently been applied to extract PPI automatically. In this paper, we explore combining the AL with the SSL to improve the performance of the PPI task. We propose a novel PPI extraction technique called PPISpotter by combining Deterministic Annealing-based SSL and an AL technique to extract protein-protein interaction. In addition, we extract a comprehensive set of features from MEDLINE records by Natural Language Processing (NLP) techniques, which further improve the SVM classifiers. In our feature selection technique, syntactic, semantic, and lexical properties of text are incorporated into feature selection that boosts the system performance significantly. By conducting experiments with three different PPI corpuses, we show that PPISpotter is superior to the other techniques incorporated into semi-supervised SVMs such as Random Sampling, Clustering, and Transductive SVMs by precision, recall, and F-measure. Our system is a novel, state-of-the-art technique for efficiently extracting protein-protein interaction pairs.

Biorefinery of the macroalgae Ulva lactuca: extraction of proteins and carbohydrates by mild disintegration.

PubMed

Postma, P R; Cerezo-Chinarro, O; Akkerman, R J; Olivieri, G; Wijffels, R H; Brandenburg, W A; Eppink, M H M

2018-01-01

The effect of osmotic shock, enzymatic incubation, pulsed electric field, and high shear homogenization on the release of water-soluble proteins and carbohydrates from the green alga Ulva lactuca was investigated in this screening study. For osmotic shock, both temperature and incubation time had a significant influence on the release with an optimum at 30 °C for 24 h of incubation. For enzymatic incubation, pectinase demonstrated being the most promising enzyme for both protein and carbohydrate release. Pulsed electric field treatment was most optimal at an electric field strength of 7.5 kV cm -1 with 0.05 ms pulses and a specific energy input relative to the released protein as low as 6.6 kWh kg prot -1 . Regarding literature, this study reported the highest protein (~ 39%) and carbohydrate (~ 51%) yields of the four technologies using high shear homogenization. Additionally, an energy reduction up to 86% was achieved by applying a novel two-phase (macrostructure size reduction and cell disintegration) technique.

Antibody mechanisms implicated in digestive disturbances following ingestion of soya protein in calves and piglets.

PubMed Central

Barratt, M E; Strachan, P J; Porter, P

1978-01-01

Serum antibody responses to ingested aqueous alcohol-extracted soya proteins were studied in thirty-six pre-ruminant calves. Characterization of this antibody showed it to be predominantly a complement-fixing IgG1 preciptin. No evidence of tolerance was seen; previously sensitized calves responded to reintroduction of a soya diet with marked increases in antibody levels. The soya antigen was shown to be resistant to proteolysis and, to a lesser degree, to the microbial action of rumen fluid. Biopsy studies showed that the feeding of soya protein resulted in morphological disturbances to the villi and lamina propria of the intestine. Physiological studies by Thirty-Vella loop perfusion in the pig showed that soya protein solutions resulted in significant inhibition of flow rates. The effect was only observed after previous sensitization with the soya antigen. This study shows the necessity of applying immunological criteria to the quality control of soya bean processing in order to ensure that the sensitizing agent is eliminated and the nutritional qualities of soya protein concentrates are optimized. Images FIG. 2a FIG. 2b PMID:565686

An MRM-based workflow for quantifying cardiac mitochondrial protein phosphorylation in murine and human tissue.

PubMed

Lam, Maggie P Y; Scruggs, Sarah B; Kim, Tae-Young; Zong, Chenggong; Lau, Edward; Wang, Ding; Ryan, Christopher M; Faull, Kym F; Ping, Peipei

2012-08-03

The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. This article is part of a Special Issue entitled: Translational Proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

[Studies on extraction process of Radix Platycodi].

PubMed

Wu, Biyuan; Sun, Jun; Jiang, Hongfang

2002-06-01

The orthogonal design was used to optimize the extraction process of Radix Platycodi with content of total saponin and yield of the extract as markers. Factors that have been chosen were alcohol concentration, alcohol consumption, extraction times and extraction time. Each factor has three levels. The result showed that the optimum extraction condition obtained was 70% alcohol, 3 times the amount of material, refluxing for 5 times, 60 minutes each time, the optimized process was stable and workable.

Evaluation of the efficiency of three extraction conditions for the immunochemical detection of allergenic soy proteins in different food matrices.

PubMed

Amponsah, Amma; Nayak, Balunkeswar

2018-04-01

Recent studies have shown the need to improve soy allergen extraction using different extraction conditions to ensure more accurate results in allergen detection. This study investigated some of these extraction conditions to confirm that these methods, especially ultrasound-assisted extraction (UAE) and the use of Laemmli buffer instead of the conventional extraction with phosphate-buffered saline (PBS), could be helpful in improving the extraction step in allergen detection. Higher total soluble protein was obtained in all samples extracted with Laemmli buffer alone and in combination with ultrasound. For immunochemical detection of soy proteins by enzyme-linked immunosorbent assay (ELISA), comparable detection was observed in extracts from all extraction conditions in all commercial samples with the exception of table cracker and veggie burger, where significantly higher detection was seen in extracts from Laemmli buffer only. For the dry mix and cookie samples, the degree of soy protein detection with ELISA varied among the different extraction conditions, but overall, extraction with only Laemmli buffer showed higher detection. Laemmli buffer with conventional extraction and UAE may be better alternatives or additional extraction methods in soy allergen detection. Different food matrices performed differently (whether it was for the recovery of total proteins or detection by ELISA) under different extraction conditions. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

A simplified bioprocess for human alpha-fetoprotein production from inclusion bodies.

PubMed

Leong, Susanna S J; Middelberg, Anton P J

2007-05-01

A simple and effective Escherichia coli (E. coli) bioprocess is demonstrated for the preparation of recombinant human alpha-fetoprotein (rhAFP), a pharmaceutically promising protein that has important immunomodulatory functions. The new rhAFP process employs only unit operations that are easy to scale and validate, and reduces the complexity embedded in existing inclusion body processing methods. A key requirement in the establishment of this process was the attainment of high purity rhAFP prior to protein refolding because (i) rhAFP binds easily to hydrophobic contaminants once refolded, and (ii) rhAFP aggregates during renaturation, in a contaminant- dependent way. In this work, direct protein extraction from cell suspension was coupled with a DNA precipitation-centrifugation step prior to purification using two simple chromatographic steps. Refolding was conducted using a single-step, redox-optimized dilution refolding protocol, with refolding success determined by reversed phase HPLC analysis, ELISA, and circular dichroism spectroscopy. Quantitation of DNA and protein contaminant loads after each unit operation showed that contaminant levels were reduced to levels comparable to traditional flowsheets. Protein microchemical modification due to carbamylation in this urea-based process was identified and minimized, yielding a final refolded and purified product that was significantly purified from carbamylated variants. Importantly, this work conclusively demonstrates, for the first time, that a chemical extraction process can substitute the more complex traditional inclusion body processing flowsheet, without compromising product purity and yield. This highly intensified and simplified process is expected to be of general utility for the preparation of other therapeutic candidates expressed as inclusion bodies. (c) 2006 Wiley Periodicals, Inc.

Evaluation of Three Protein-Extraction Methods for Proteome Analysis of Maize Leaf Midrib, a Compound Tissue Rich in Sclerenchyma Cells.

PubMed

Wang, Ning; Wu, Xiaolin; Ku, Lixia; Chen, Yanhui; Wang, Wei

2016-01-01

Leaf morphology is closely related to the growth and development of maize (Zea mays L.) plants and final kernel production. As an important part of the maize leaf, the midrib holds leaf blades in the aerial position for maximum sunlight capture. Leaf midribs of adult plants contain substantial sclerenchyma cells with heavily thickened and lignified secondary walls and have a high amount of phenolics, making protein extraction and proteome analysis difficult in leaf midrib tissue. In the present study, three protein-extraction methods that are commonly used in plant proteomics, i.e., phenol extraction, TCA/acetone extraction, and TCA/acetone/phenol extraction, were qualitatively and quantitatively evaluated based on 2DE maps and MS/MS analysis using the midribs of the 10th newly expanded leaves of maize plants. Microscopy revealed the existence of substantial amounts of sclerenchyma underneath maize midrib epidermises (particularly abaxial epidermises). The spot-number order obtained via 2DE mapping was as follows: phenol extraction (655) > TCA/acetone extraction (589) > TCA/acetone/phenol extraction (545). MS/MS analysis identified a total of 17 spots that exhibited 2-fold changes in abundance among the three methods (using phenol extraction as a control). Sixteen of the proteins identified were hydrophilic, with GRAVY values ranging from -0.026 to -0.487. For all three methods, we were able to obtain high-quality protein samples and good 2DE maps for the maize leaf midrib. However, phenol extraction produced a better 2DE map with greater resolution between spots, and TCA/acetone extraction produced higher protein yields. Thus, this paper includes a discussion regarding the possible reasons for differential protein extraction among the three methods. This study provides useful information that can be used to select suitable protein extraction methods for the proteome analysis of recalcitrant plant tissues that are rich in sclerenchyma cells.

Optimization of Ultrasound-Assisted Extraction of phenolic compounds and anthocyanins from blueberry (Vaccinium ashei) wine pomace.

PubMed

He, Bo; Zhang, Ling-Li; Yue, Xue-Yang; Liang, Jin; Jiang, Jun; Gao, Xue-Ling; Yue, Peng-Xiang

2016-08-01

Ultrasound-Assisted Extraction (UAE) of total anthocyanins (TA) and phenolics (TP) from Blueberry Wine Pomace (BWP) was optimized using Response Surface Methodology (RSM). A Box-Behnken design was used to predict that the optimized conditions were an extraction temperature of 61.03°C, a liquid-solid ratio of 21.70mL/g and a sonication time of 23.67min. Using the modeled optimized conditions, the predicted and experimental yields of TA and TP were within a 2% difference. The yields of TA and TP obtained through the optimized UAE method were higher than those using a Conventional Solvent Extraction (CSE) method. Seven anthocyanins, namely delphinidin-3-O-glucoside, delphindin-3-O-arabinoside, petunidin-3-O-glucoside, cyanidin-3-O-arabinoside, cyanidin-3-O-glucoside, malvidin-3-O-glucoside and malvidin-3-O-arabinoside, were found in the BWP extract from both the UAE and CSE methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Optimization of antimicrobial activity of flavonoid extracts from pomelo (Citrus grandis) peel as food wrap

NASA Astrophysics Data System (ADS)

Sugumaran, Kamaraj; Zakaria, Nur Zatul-'Iffah; Abdullah, Rozaini; Jalani, Nur Syazana; Zati-Hanani, Sharifah; Ibrahim, Roshita; Zakaria, Zarina

2017-09-01

This study has been carried out to optimize an antimicrobial activity of flavonoid extract from pomelo peels against Staphyloccus aureus (S. aureus). A comparative analysis of total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity were done on two parts of peel which are albedo (inner peel) and flavedo (outer peel). Based on the result obtained, flavedo showed higher TPC, TFC and antioxidant activity (304.20 mg /g, 74.30 mg /g and 46.86 % respectively) when compared to albedo (150.98 mg /g, 52.97 mg /g and 24.70 % respectively). The effects of different extract concentration and pH on inhibition zone of S. aureus were optimized using Research Surface Methodology (RSM). The optimal condition of parameters was obtained based on the maximum zone of S. aureus inhibition at extract concentration of 200 mg/mL and pH of 4.8. The antimicrobial film has been developed by using optimal conditions by incorporating the flavonoid extract into chitosan polymer.

A rapid and efficient SDS-based RNA isolation protocol from different tissues of coffee.

PubMed

Huded, Arun Kumar C; Jingade, Pavankumar; Mishra, Manoj Kumar

2018-03-01

Isolation of high-quality RNA from coffee is challenging because of high level of polysaccharides, polyphenols and other secondary metabolites. In the present study, a rapid and efficient RNA extraction protocol from different tissues of coffee was optimized. Sufficiently high quality and quantity (225.6-454.8 µg/g) of RNA was obtained by using the optimized protocol. The presence of two distinct bands of 28S rRNA and 18S rRNA in agarose gel proved the intactness of the RNA samples. The average spectrophotometric values of the isolated RNA ranged from 1.96 to 2.02 ( A 260/280 ) and 1.95 to 2.14 ( A 260/230 ), indicating the high quality of RNA devoid of polyphenols, polysaccharides and protein contamination. In the optimized protocol, addition of PVPP to the extraction buffer and a brief incubation of samples at 65 °C and subsequent purification with potassium acetate resulted in good-quality RNA isolation. The suitability of RNA for downstream processing was confirmed by PCR amplification with cytochrome c oxidase gene-specific primers. The amplification of a single 392 bp fragment using cDNA and 1.5 kb fragment using genomic DNA samples confirmed the absence of DNA contamination. The present protocol is rapid and yielded good quality and quantity of RNA suitable for functional genomics studies.

Extracting features from protein sequences to improve deep extreme learning machine for protein fold recognition.

PubMed

Ibrahim, Wisam; Abadeh, Mohammad Saniee

2017-05-21

Protein fold recognition is an important problem in bioinformatics to predict three-dimensional structure of a protein. One of the most challenging tasks in protein fold recognition problem is the extraction of efficient features from the amino-acid sequences to obtain better classifiers. In this paper, we have proposed six descriptors to extract features from protein sequences. These descriptors are applied in the first stage of a three-stage framework PCA-DELM-LDA to extract feature vectors from the amino-acid sequences. Principal Component Analysis PCA has been implemented to reduce the number of extracted features. The extracted feature vectors have been used with original features to improve the performance of the Deep Extreme Learning Machine DELM in the second stage. Four new features have been extracted from the second stage and used in the third stage by Linear Discriminant Analysis LDA to classify the instances into 27 folds. The proposed framework is implemented on the independent and combined feature sets in SCOP datasets. The experimental results show that extracted feature vectors in the first stage could improve the performance of DELM in extracting new useful features in second stage. Copyright © 2017 Elsevier Ltd. All rights reserved.

Study of optimal extraction conditions for achieving high yield and antioxidant activity of tomato seed oil

USDA-ARS?s Scientific Manuscript database

Tomato seeds resulting from tomato processing by-product have not been effectively utilized as value-added products. This study investigated the kinetics of oil extraction from tomato seeds and sought to optimize the oil extraction conditions. The oil was extracted by using hexane as solvent for 0 t...

[Optimization of extraction technics of total saponins from Pulsatilla cernua].

PubMed

Li, Hai-Yan; Hao, Ning; Xu, Yong-Nan; Piao, Zhong-Yun

2010-04-01

The extraction condition of total saponins from Pulsatilla cenua by ultrasonic wave was optimized by single factor and orthogonal experiments. The largest absorbency of saponin was intended to be 470 nm by wavelength scan method with the pulchinenoside B4 as control sample, the linear relationship was observed between the absorbency and the content of saponin in the range of 0 - 0.040 mg/mL. The optimal conditions of extraction was as following: 80% of alcohol concentration, 40 min of ultrasonic time, 1: 20 of solid to liquid ratio, 80 W of ultrasonic power and one time for extraction. Among them, alcohol had the most significant effect on the extraction of total saponins. The content of total saponins in Pulsatilla cernua was 4. 32% under the optimal condition. The method developed here is efficient, stable, accurate and repeatable.

Evaluation, prediction and optimization the ultrasound-assisted extraction method using response surface methodology: antioxidant and biological properties of Stachys parviflora L.

PubMed

Bashi, Davoud Salar; Dowom, Samaneh Attaran; Bazzaz, Bibi Sedigheh Fazly; Khanzadeh, Farhad; Soheili, Vahid; Mohammadpour, Ali

2016-05-01

To optimize the extraction method using response surface methodology, extract the phenolic compounds, and identify the antioxidant and biological properties of Stachys parviflora L. extracts. Maceration and ultrasound-assisted extraction (UAE) (4, 7, 10 min treatment time, 40, 70, 100 % high-intensity and 60, 80, 100 % (v v-1) methanol purity) were applied to obtain the extracts. SEM was conducted to provide the microstructure of the extracted plant. MICs (colorimetric assay), MFCs (colony diameter), total phenolic content, total flavonoid content, radical scavenging capacity and extraction efficiency were determined. HPLC analysis was applied to measure the existent phenolic compounds. A quadratic model (4 min treatment time, 74.5 % high-intensity and 74.2 % solvent purity) was suggested as the best (TPC: 20.89 mg GAE g-1 d.m., TFC: 6.22 mg QEs g-1 d.m., DPPH IC50: 21.86 µg ml-1 and EE: 113.65 mg g-1 d.m.) UAE extraction model. The optimized UAE extract was generally more effective against Gram-positive microorganisms (MIC: 10-20; MBC: 10-40 (mg ml-1)) than Gram-negative ones (MIC: 40; MBC: >40 (mg ml-1)). Moreover, it (MGI: 2.32-100 %) revealed more anti-mold activity than maceration (MGI: <28.77 %). Explosive disruption of the cell walls, therefore, enhanced extraction yield by acoustic cavitation, was elucidated using SEM. Caffeic acid, tannic acid, quercetin, trans ferulic acid and rosmarinic acid were determined as the phenolic compounds in the optimized extract. RSM optimization was successfully applied for UAE from S. parviflora. The considerable antioxidant and biological properties were attributed to the phenolic compounds.

Evaluation, prediction and optimization the ultrasound-assisted extraction method using response surface methodology: antioxidant and biological properties of Stachys parviflora L.

PubMed Central

Bashi, Davoud Salar; Dowom, Samaneh Attaran; Bazzaz, Bibi Sedigheh Fazly; Khanzadeh, Farhad; Soheili, Vahid; Mohammadpour, Ali

2016-01-01

Objective(s): To optimize the extraction method using response surface methodology, extract the phenolic compounds, and identify the antioxidant and biological properties of Stachys parviflora L. extracts. Materials and Methods: Maceration and ultrasound-assisted extraction (UAE) (4, 7, 10 min treatment time, 40, 70, 100 % high-intensity and 60, 80, 100 % (v v-1) methanol purity) were applied to obtain the extracts. SEM was conducted to provide the microstructure of the extracted plant. MICs (colorimetric assay), MFCs (colony diameter), total phenolic content, total flavonoid content, radical scavenging capacity and extraction efficiency were determined. HPLC analysis was applied to measure the existent phenolic compounds. Results: A quadratic model (4 min treatment time, 74.5 % high-intensity and 74.2 % solvent purity) was suggested as the best (TPC: 20.89 mg GAE g-1 d.m., TFC: 6.22 mg QEs g-1 d.m., DPPH IC50: 21.86 µg ml-1 and EE: 113.65 mg g-1 d.m.) UAE extraction model. The optimized UAE extract was generally more effective against Gram-positive microorganisms (MIC: 10-20; MBC: 10-40 (mg ml-1)) than Gram-negative ones (MIC: 40; MBC: >40 (mg ml-1)). Moreover, it (MGI: 2.32-100 %) revealed more anti-mold activity than maceration (MGI: <28.77 %). Explosive disruption of the cell walls, therefore, enhanced extraction yield by acoustic cavitation, was elucidated using SEM. Caffeic acid, tannic acid, quercetin, trans ferulic acid and rosmarinic acid were determined as the phenolic compounds in the optimized extract. Conclusion: RSM optimization was successfully applied for UAE from S. parviflora. The considerable antioxidant and biological properties were attributed to the phenolic compounds. PMID:27403260

Use of different sample temperatures in a single extraction procedure for the screening of the aroma profile of plant matrices by headspace solid-phase microextraction.

PubMed

Martendal, Edmar; de Souza Silveira, Cristine Durante; Nardini, Giuliana Stael; Carasek, Eduardo

2011-06-17

This study proposes a new approach to the optimization of the extraction of the volatile fraction of plant matrices using the headspace solid-phase microextraction (HS-SPME) technique. The optimization focused on the extraction time and temperature using a CAR/DVB/PDMS 50/30 μm SPME fiber and 100mg of a mixture of plants as the sample in a 15-mL vial. The extraction time (10-60 min) and temperature (5-60 °C) were optimized by means of a central composite design. The chromatogram was divided into four groups of peaks based on the elution temperature to provide a better understanding of the influence of the extraction parameters on the extraction efficiency considering compounds with different volatilities/polarities. In view of the different optimum extraction time and temperature conditions obtained for each group, a new approach based on the use of two extraction temperatures in the same procedure is proposed. The optimum conditions were achieved by extracting for 30 min with a sample temperature of 60 °C followed by a further 15 min at 5 °C. The proposed method was compared with the optimized conventional method based on a single extraction temperature (45 min of extraction at 50 °C) by submitting five samples to both procedures. The proposed method led to better results in all cases, considering as the response both peak area and the number of identified peaks. The newly proposed optimization approach provided an excellent alternative procedure to extract analytes with quite different volatilities in the same procedure. Copyright © 2011 Elsevier B.V. All rights reserved.

Comparison of Protein Extracts from Various Unicellular Green Sources.

PubMed

Teuling, Emma; Wierenga, Peter A; Schrama, Johan W; Gruppen, Harry

2017-09-13

Photosynthetic unicellular organisms are considered as promising alternative protein sources. The aim of this study is to understand the extent to which these green sources differ with respect to their gross composition and how these differences affect the final protein isolate. Using mild isolation techniques, proteins were extracted and isolated from four different unicellular sources (Arthrospira (spirulina) maxima, Nannochloropsis gaditana, Tetraselmis impellucida, and Scenedesmus dimorphus). Despite differences in protein contents of the sources (27-62% w/w) and in protein extractability (17-74% w/w), final protein isolates were obtained that had similar protein contents (62-77% w/w) and protein yields (3-9% w/w). Protein solubility as a function of pH was different between the sources and in ionic strength dependency, especially at pH < 4.0. Overall, the characterization and extraction protocol used allows a relatively fast and well-described isolation of purified proteins from novel protein sources.

Comparison of Protein Extracts from Various Unicellular Green Sources

PubMed Central

2017-01-01

Photosynthetic unicellular organisms are considered as promising alternative protein sources. The aim of this study is to understand the extent to which these green sources differ with respect to their gross composition and how these differences affect the final protein isolate. Using mild isolation techniques, proteins were extracted and isolated from four different unicellular sources (Arthrospira (spirulina) maxima, Nannochloropsis gaditana, Tetraselmis impellucida, and Scenedesmus dimorphus). Despite differences in protein contents of the sources (27–62% w/w) and in protein extractability (17–74% w/w), final protein isolates were obtained that had similar protein contents (62–77% w/w) and protein yields (3–9% w/w). Protein solubility as a function of pH was different between the sources and in ionic strength dependency, especially at pH < 4.0. Overall, the characterization and extraction protocol used allows a relatively fast and well-described isolation of purified proteins from novel protein sources. PMID:28701042

An effective vacuum assisted extraction method for the optimization of labdane diterpenoids from Andrographis paniculata by response surface methodology.

PubMed

Wang, Ya-Qi; Wu, Zhen-Feng; Ke, Gang; Yang, Ming

2014-12-31

An effective vacuum assisted extraction (VAE) technique was proposed for the first time and applied to extract bioactive components from Andrographis paniculata. The process was carefully optimized by response surface methodology (RSM). Under the optimized experimental conditions, the best results were obtained using a boiling temperature of 65 °C, 50% ethanol concentration, 16 min of extraction time, one extraction cycles and a 12:1 liquid-solid ratio. Compared with conventional ultrasonic assisted extraction and heat reflux extraction, the VAE technique gave shorter extraction times and remarkable higher extraction efficiency, which indicated that a certain degree of vacuum gave the solvent a better penetration of the solvent into the pores and between the matrix particles, and enhanced the process of mass transfer. The present results demonstrated that VAE is an efficient, simple and fast method for extracting bioactive components from A. paniculata, which shows great potential for becoming an alternative technique for industrial scale-up applications.

Response Surface Methodology for Ultrasound-Assisted Extraction of Astaxanthin from Haematococcus pluvialis

PubMed Central

Zou, Tang-Bin; Jia, Qing; Li, Hua-Wen; Wang, Chang-Xiu; Wu, Hong-Fu

2013-01-01

Astaxanthin is a novel carotenoid nutraceutical occurring in many crustaceans and red yeasts. It has exhibited various biological activities including prevention or amelioration of cardiovascular disease, gastric ulcer, hypertension, and diabetic nephropathy. In this study, ultrasound-assisted extraction was developed for the effective extraction of astaxanthin from Haematococcus pluvialis. Some parameters such as extraction solvent, liquid-to-solid ratio, extraction temperature, and extraction time were optimized by single-factor experiment and response surface methodology. The optimal extraction conditions were 48.0% ethanol in ethyl acetate, the liquid-to-solid ratio was 20:1 (mL/g), and extraction for 16.0 min at 41.1 °C under ultrasound irradiation of 200 W. Under optimal conditions, the yield of astaxanthin was 27.58 ± 0.40 mg/g. The results obtained are beneficial for the full utilization of Haematococcus pluvialis, which also indicated that ultrasound-assisted extraction is a very useful method for extracting astaxanthin from marine life. PMID:23697948

Optimization of soy isoflavone extraction with different solvents using the simplex-centroid mixture design.

PubMed

Yoshiara, Luciane Yuri; Madeira, Tiago Bervelieri; Delaroza, Fernanda; da Silva, Josemeyre Bonifácio; Ida, Elza Iouko

2012-12-01

The objective of this study was to optimize the extraction of different isoflavone forms (glycosidic, malonyl-glycosidic, aglycone and total) from defatted cotyledon soy flour using the simplex-centroid experimental design with four solvents of varying polarity (water, acetone, ethanol and acetonitrile). The obtained extracts were then analysed by high-performance liquid chromatography. The profile of the different soy isoflavones forms varied with different extractions solvents. Varying the solvent or mixture used, the extraction of different isoflavones was optimized using the centroid-simplex mixture design. The special cubic model best fitted to the four solvents and its combination for soy isoflavones extraction. For glycosidic isoflavones extraction, the polar ternary mixture (water, acetone and acetonitrile) achieved the best extraction; malonyl-glycosidic forms were better extracted with mixtures of water, acetone and ethanol. Aglycone isoflavones, water and acetone mixture were best extracted and total isoflavones, the best solvents were ternary mixture of water, acetone and ethanol.

Optimized ultrasound-assisted extraction procedure for the analysis of opium alkaloids in papaver plants by cyclodextrin-modified capillary electrophoresis.

PubMed

Fakhari, Ali Reza; Nojavan, Saeed; Ebrahimi, Samad Nejad; Evenhuis, Christopher John

2010-07-01

This study investigated the use of ultrasound-assisted extraction to improve the extraction efficiency of morphine, codeine and thebaine from the papaver plants. Extraction conditions such as type of solvent, temperature, duration, frequency and power level of ultrasonic were optimized and the influences of different parameters on resolution of alkaloids in CE were studied. The optimized condition for CE separation includes a sodium phosphate buffer (100 mM, pH 3.0) containing 5 mM alpha-CD. The optimized extraction conditions for ultrasound-assisted extraction was an extraction time of 1 h, an ultrasonic frequency of 60 kHz with water-methanol (80:20) at 40 degrees C as the extraction solvent. The LOD for alkaloids was found to be 0.1 microg/mL at a signal-to-noise ratio of 3:1. The RSDs for peak areas were in the range of 1.4-4.4%. The amounts of opium alkaloids (mg/100 g dried sample) in four Iranian papaver plants were found to be in the range of 7.8-8.7 (morphine), 5.5-9.5 (codeine) and 1.4-10.4 (thebaine). It should be emphasized that no cleanup of the filtered extract was required; hence, direct determination after extraction drastically simplifies the analytical process.

Optimization and preliminary characterization of venom isolated from 3 medically important jellyfish: the box (Chironex fleckeri), Irukandji (Carukia barnesi), and blubber (Catostylus mosaicus) jellyfish.

PubMed

Wiltshire, C J; Sutherland, S K; Fenner, P J; Young, A R

2000-01-01

To optimize venom extraction and to undertake preliminary biochemical studies of venom from the box jellyfish (Chironex fleckeri), the Irukandji jellyfish (Carukia barnesi), and the blubber jellyfish (Catostylus mosaicus). Lyophilized crude venoms from box jellyfish tentacles and whole Irukandji jellyfish were prepared in water by homogenization, sonication, and rapid freeze thawing. A second technique, consisting of grinding samples with a glass mortar and pestle and using phosphate-buffered saline, was used to prepare crude venom from isolated nematocysts of the box jellyfish, the bells of Irukandji jellyfish, and the oral lobes of blubber jellyfish. Venoms were compared by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot test. Toxicity of some venoms was determined by intravenous median lethal dose assay in mice. Different venom extraction techniques produced significantly different crude venoms for both box and Irukandji jellyfish. Irukandji and blubber venom SDS-PAGE protein profiles were established for the first time. Analysis of Western blot tests revealed that box jellyfish antivenin reacted specifically with the venom of each jellyfish. Toxicity was found in Irukandji jellyfish venom derived by use of the mortar-and-pestle method, but not in the lyophilized venom. Glass mortar-and-pestle grinding and use of an appropriate buffer was found to be a simple and suitable method for the preparation of venom from each jellyfish species studied. This study contributes to biochemical investigations of jellyfish venoms, particularly the venom of the Irukandji jellyfish, for which there are, to our knowledge, no published studies. It also highlights the importance of optimizing venom extraction as the first step toward understanding the complex biological effects of jellyfish venoms.

Extraction of Antioxidant Phenolic Compounds from Brewer’s Spent Grain: Optimization and Kinetics Modeling

PubMed Central

Sologubik, Carlos A.; Fernández, María B.; Manrique, Guillermo D.

2018-01-01

The kinetics of polyphenol extraction from brewer’s spent grain (BSG), using a batch system, ultrasound assistance, and microwave assistance and the evolution of antioxidant capacity of these extracts over time, were studied. The main parameters of extraction employed in the batch system were evaluated, and, by applying response surface analysis, the following optimal conditions were obtained: Liquid/solid ratio of 30:1 mL/g at 80 °C, using 72% (v/v) ethanol:water as the solvent system. Under these optimized conditions, ultrasound assistance demonstrated the highest extraction rate and equilibrium yield, as well as shortest extraction times, followed by microwave assistance. Among the mathematical models used, Patricelli’s model proved the most suitable for describing the extraction kinetics for each method tested, and is therefore able to predict the response values and estimate the extraction rates and potential maximum yields in each case. PMID:29570683

Extraction and Enrichment of Protein from Red and Green Macroalgae.

PubMed

Harnedy, Pádraigín A; FitzGerald, Richard J

2015-01-01

Macroalgae, in particular red and green species, are gaining interest as protein-rich foods for human consumption and sources of proteinaceous biofunctional peptide ingredients. During protein extraction the starting raw material, the cell disruption method utilized and the reagents employed have a major effect on the yield of protein recovered. A method is described herein for extraction and semi-purification of food-grade aqueous and alkaline soluble proteins from red and green macroalgae. Dried milled macroalgae are disrupted by osmotic shock with subsequent removal of aqueous soluble proteins by centrifugation. Alkaline soluble proteins are removed following consecutive treatment of the resultant pellet with an alkaline solution. Aqueous and alkaline soluble proteins are then enriched from the crude extracts by isoelectric precipitation.

Modification of an acetone-sodium dodecyl sulfate disruption method for cellular protein extraction from neuropathogenic Clostridium botulinum

USDA-ARS?s Scientific Manuscript database

An acetone-sodium dodecyl sulfate (SDS) disruption method was used for the extraction of cellular proteins from neurotoxigenic Clostridium botulinum. The amount of protein extracted per gram of dry weight and the protein profile as revealed by polyacrylamide gel electrophoresis (PAGE) was comparabl...

Extraction and functional properties of non-zein proteins in corn germ from wet-milling

USDA-ARS?s Scientific Manuscript database

This study was conducted to develop methods of extracting corn germ protein and characterize and identify potential applications of the recovered protein. Protein was extracted from both wet germ and finished (dried) germ using 0.1M NaCl as solvent. The method involved homogenization, stirring, cent...

Optimization of Ultrasound-Assisted Extraction, HPLC and UHPLC-ESI-Q-TOF-MS/MS Analysis of Main Macamides and Macaenes from Maca (Cultivars of Lepidium meyenii Walp).

PubMed

Chen, Shu-Xiao; Li, Ke-Ke; Pubu, Duoji; Jiang, Si-Ping; Chen, Bin; Chen, Li-Rong; Yang, Zhen; Ma, Chao; Gong, Xiao-Jie

2017-12-10

Ultrasound-assisted extraction (UAE), using petroleum ether as the solvent, was systematically applied to extract main macamides and macaenes from Maca hypocotyls. Extraction yield was related with four variables, including ratio of solution to solid, extraction temperature, extraction time, and extraction power. On the basis of response surface methodology (RSM), the optimal conditions were determined to be the ratio of solution to solid as 10:1 (mL/g), the extraction temperature of 40 °C, the extraction time of 30 min, and the extraction power of 200 W. Based on the optimal extraction method of UAE, the total contents of ten main macamides and two main macaenes of Maca cultivated in twenty different areas of Tibet were analyzed by HPLC and UHPLC-ESI-Q-TOF-MS/MS. This study indicated that UAE was able to effectively extract macamides alkaloids from Maca hypocotyls. Quantitative analysis showed that geographical origins, not ecotypes, played a more important role on the accumulation of active macamides in Maca.

Optimization of ultrasound assisted extraction of bioactive components from brown seaweed Ascophyllum nodosum using response surface methodology.

PubMed

Kadam, Shekhar U; Tiwari, Brijesh K; Smyth, Thomas J; O'Donnell, Colm P

2015-03-01

The objective of this study was to investigate the effect of key extraction parameters of extraction time (5-25 min), acid concentration (0-0.06 M HCl) and ultrasound amplitude (22.8-114 μm) on yields of bioactive compounds (total phenolics, fucose and uronic acid) from Ascophyllumnodosum. Response surface methodology was employed to optimize the extraction variables for bioactive compounds' yield. A second order polynomial model was fitted well to the extraction experimental data with (R(2)>0.79). Extraction yields of 143.12 mgGAE/gdb, 87.06 mg/gdb and 128.54 mg/gdb were obtained for total phenolics, fucose and uronic acid respectively at optimized extraction conditions of extraction time (25 min), acid concentration (0.03 M HCl) and ultrasonic amplitude (114 μm). Mass spectroscopy analysis of extracts show that ultrasound enhances the extraction of high molecular weight phenolic compounds from A. nodosum. This study demonstrates that ultrasound assisted extraction (UAE) can be employed to enhance extraction of bioactive compounds from seaweed. Copyright © 2014 Elsevier B.V. All rights reserved.

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

DOEpatents

Zhang, Jian-Shi; Giometti, Carol S.; Tollaksen, Sandra L.

1989-01-01

After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

Extraction methods of Amaranthus sp. grain oil isolation.

PubMed

Krulj, Jelena; Brlek, Tea; Pezo, Lato; Brkljača, Jovana; Popović, Sanja; Zeković, Zoran; Bodroža Solarov, Marija

2016-08-01

Amaranthus sp. is a fast-growing crop with well-known beneficial nutritional values (rich in protein, fat, dietary fiber, ash, and minerals, especially calcium and sodium, and containing a higher amount of lysine than conventional cereals). Amaranthus sp. is an underexploited plant source of squalene, a compound of high importance in the food, cosmetic and pharmaceutical industries. This paper has examined the effects of the different extraction methods (Soxhlet, supercritical fluid and accelerated solvent extraction) on the oil and squalene yield of three genotypes of Amaranthus sp. grain. The highest yield of the extracted oil (78.1 g kg(-1) ) and squalene (4.7 g kg(-1) ) in grain was obtained by accelerated solvent extraction (ASE) in genotype 16. Post hoc Tukey's HSD test at 95% confidence limit showed significant differences between observed samples. Principal component analysis (PCA) and cluster analysis (CA) were used for assessing the effect of different genotypes and extraction methods on oil and squalene yield, and also the fatty acid composition profile. Using coupled PCA and CA of observed samples, possible directions for improving the quality of product can be realized. The results of this study indicate that it is very important to choose both the right genotype and the right method of extraction for optimal oil and squalene yield. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Optimization of drying process and pressurized liquid extraction for recovery of bioactive compounds from avocado peel by-product.

PubMed

Figueroa, Jorge G; Borrás-Linares, Isabel; Lozano-Sánchez, Jesús; Quirantes-Piné, Rosa; Segura-Carretero, Antonio

2018-04-16

The aim of the present study was to optimize the extraction of phenolic compounds in avocado peel using pressurized liquid extraction (PLE) with GRAS solvents. Response surface methodology (RSM) based on Central Composite Design 2 2 model was used in order to optimize PLE conditions. Moreover, the effect of air drying temperature on the total polyphenol content (TPC) and individual phenolic compounds concentration were evaluated. The quantification of individual compounds was performed by HPLC-DAD-ESI-TOF-MS. The optimized extraction conditions were 200°C as extraction temperature and 1:1 v/v as ethanol/water ratio. Regarding to the effect of drying, the highest TPC was obtained with a drying temperature of 85°C. Forty seven phenolic compounds were quantified in the obtained extracts, showing that phenolic acids found to be the more stables compounds to drying process, while procyanidins were the more thermolabiles analytes. To our knowledge, this is the first available study in which phenolic compounds extraction was optimized using PLE and such amount of phenolic compounds was quantified in avocado peel. These results confirm that PLE represents a powerful tool to obtain avocado peel extracts with high concentration in bioactive compounds suitable for its use in the food, cosmetic or pharmaceutical sector. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Method for Whole Protein Isolation from Human Cranial Bone

PubMed Central

Lyon, Sarah M.; Mayampurath, Anoop; Rogers, M. Rose; Wolfgeher, Donald J.; Fisher, Sean M.; Volchenboum, Samuel L.; He, Tong-Chuan; Reid, Russell R.

2016-01-01

The presence of the dense hydroxyapatite matrix within human bone limits the applicability of conventional protocols for protein extraction. This has hindered the complete and accurate characterization of the human bone proteome thus far, leaving many bone-related disorders poorly understood. We sought to refine an existing method of protein extraction from mouse bone to extract whole proteins of varying molecular weights from human cranial bone. Whole protein was extracted from human cranial suture by mechanically processing samples using a method that limits protein degradation by minimizing heat introduction to proteins. The presence of whole protein was confirmed by western blotting. Mass spectrometry was used to sequence peptides and identify isolated proteins. The data have been deposited to the ProteomeXchange with identifier PXD003215. Extracted proteins were characterized as both intra- and extracellular and had molecular weights ranging from 9.4-629 kDa. High correlation scores among suture protein spectral counts support the reproducibility of the method. Ontology analytics revealed proteins of myriad functions including mediators of metabolic processes and cell organelles. These results demonstrate a reproducible method for isolation of whole protein from human cranial bone, representing a large range of molecular weights, origins and functions. PMID:27677936

Optimization of ultrasound-assisted extraction of phenolic compounds from grapefruit (Citrus paradisi Macf.) leaves via D-optimal design and artificial neural network design with categorical and quantitative variables.

PubMed

Ciğeroğlu, Zeynep; Aras, Ömür; Pinto, Carlos A; Bayramoglu, Mahmut; Kırbaşlar, Ş İsmail; Lorenzo, José M; Barba, Francisco J; Saraiva, Jorge A; Şahin, Selin

2018-03-06

The extraction of phenolic compounds from grapefruit leaves assisted by ultrasound-assisted extraction (UAE) was optimized using response surface methodology (RSM) by means of D-optimal experimental design and artificial neural network (ANN). For this purpose, five numerical factors were selected: ethanol concentration (0-50%), extraction time (15-60 min), extraction temperature (25-50 °C), solid:liquid ratio (50-100 g L -1 ) and calorimetric energy density of ultrasound (0.25-0.50 kW L -1 ), whereas ultrasound probe horn diameter (13 or 19 mm) was chosen as categorical factor. The optimized experimental conditions yielded by RSM were: 10.80% for ethanol concentration; 58.52 min for extraction time; 30.37 °C for extraction temperature; 52.33 g L -1 for solid:liquid ratio; 0.457 kW L -1 for ultrasonic power density, with thick probe type. Under these conditions total phenolics content was found to be 19.04 mg gallic acid equivalents g -1 dried leaf. The same dataset was used to train multilayer feed-forward networks using different approaches via MATLAB, with ANN exhibiting superior performance to RSM (differences included categorical factor in one model and higher regression coefficients), while close values were obtained for the extraction variables under study, except for ethanol concentration and extraction time. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Optimization of extraction of polysaccharides from fruiting body of Cordyceps militaris (L.) link using response surface methodology

NASA Astrophysics Data System (ADS)

Nguyen, Hoang Chinh; Thi, Dinh Huynh Mong; Pham, Dinh Chuong

2018-04-01

Polysaccharides from fruiting body of Cordyceps militaris (L.) Link possess various pharmaceutical activities. In this study, polysaccharides from the fruiting body of C. militaris were extracted with different solvents. Of those solvents tested, distilled water was identified as the most efficient solvent for the extraction, resulting in a significant increase in polysaccharides yield. Response surface methodology was then used to optimize the extraction conditions and establish a reliable mathematical model for prediction. A maximum polysaccharides yield of 11.07% was reached at a ratio of water to raw material of 23.2:1 mL/g, an extraction time of 76 min, and a temperature of 93.6°C. This study indicates that the obtained optimal extraction conditions are an efficient method for extraction of polysaccharides from the fruiting body of C. militaris.

Preparation and mechanical properties of edible rapeseed protein films.

PubMed

Jang, Sung-Ae; Lim, Geum-Ok; Song, Kyung Bin

2011-03-01

Edible films were manufactured from rapeseed oil extraction residues. To prepare rapeseed protein (RP) films, various concentrations of plasticizers and emulsifiers were incorporated into the preparation of a film-forming solution. The optimal conditions for the preparation of the RP film were 2% sorbitol/0.5% sucrose as plasticizer and 1.5% polysorbate 20 as an emulsifier. In addition, RP blend films were prepared. Gelidium corneum or gelatin was added to improve the physical properties of the RP film, and the highest tensile strength value of the films was 53.45 MPa for the 3% RP/4% gelatin film. Our results suggest that the RP-gelatin blend film is suitable for applications in food packaging. Edible RP films prepared in the present investigation can be applied in food packaging.

Effect of Ultrasound in Soybean Protein Extraction

NASA Astrophysics Data System (ADS)

Fukase, Hirokazu; Ohdaira, Etsuzo; Masuzawa, Nobuyoshi; Ide, Masao

1994-05-01

Application of ultrasound for accelerating the extraction of nutriments in food processing has been attempted. However, conditions of exposure to ultrasound were not clear in previous studies. This paper reports on the relationship between the ultrasonic pressure and the amount of extracted protein from soybeans. Experiments were conducted using a beaker, in which the ultrasonic fields were precisely measured. Soybean flakes suspended in water were put in the beaker and placed in a water tank. The amount of extracted protein in water upon ultrasonic exposure was calculated by the Kjeldahl method. It was found that the amount of extracted protein increased in proportion to ultrasonic pressure up to the total amount of soybean protein soluble in water. Furthermore, this paper describes the denaturation of the protein produced by the ultrasonic cavitation.

Information Extraction of High Resolution Remote Sensing Images Based on the Calculation of Optimal Segmentation Parameters

PubMed Central

Zhu, Hongchun; Cai, Lijie; Liu, Haiying; Huang, Wei

2016-01-01

Multi-scale image segmentation and the selection of optimal segmentation parameters are the key processes in the object-oriented information extraction of high-resolution remote sensing images. The accuracy of remote sensing special subject information depends on this extraction. On the basis of WorldView-2 high-resolution data, the optimal segmentation parameters methodof object-oriented image segmentation and high-resolution image information extraction, the following processes were conducted in this study. Firstly, the best combination of the bands and weights was determined for the information extraction of high-resolution remote sensing image. An improved weighted mean-variance method was proposed andused to calculatethe optimal segmentation scale. Thereafter, the best shape factor parameter and compact factor parameters were computed with the use of the control variables and the combination of the heterogeneity and homogeneity indexes. Different types of image segmentation parameters were obtained according to the surface features. The high-resolution remote sensing images were multi-scale segmented with the optimal segmentation parameters. Ahierarchical network structure was established by setting the information extraction rules to achieve object-oriented information extraction. This study presents an effective and practical method that can explain expert input judgment by reproducible quantitative measurements. Furthermore the results of this procedure may be incorporated into a classification scheme. PMID:27362762

Information Extraction of High Resolution Remote Sensing Images Based on the Calculation of Optimal Segmentation Parameters.

PubMed

Zhu, Hongchun; Cai, Lijie; Liu, Haiying; Huang, Wei

2016-01-01

Multi-scale image segmentation and the selection of optimal segmentation parameters are the key processes in the object-oriented information extraction of high-resolution remote sensing images. The accuracy of remote sensing special subject information depends on this extraction. On the basis of WorldView-2 high-resolution data, the optimal segmentation parameters methodof object-oriented image segmentation and high-resolution image information extraction, the following processes were conducted in this study. Firstly, the best combination of the bands and weights was determined for the information extraction of high-resolution remote sensing image. An improved weighted mean-variance method was proposed andused to calculatethe optimal segmentation scale. Thereafter, the best shape factor parameter and compact factor parameters were computed with the use of the control variables and the combination of the heterogeneity and homogeneity indexes. Different types of image segmentation parameters were obtained according to the surface features. The high-resolution remote sensing images were multi-scale segmented with the optimal segmentation parameters. Ahierarchical network structure was established by setting the information extraction rules to achieve object-oriented information extraction. This study presents an effective and practical method that can explain expert input judgment by reproducible quantitative measurements. Furthermore the results of this procedure may be incorporated into a classification scheme.

Supercritical carbon dioxide extraction of seed oil from winter melon (Benincasa hispida) and its antioxidant activity and fatty acid composition.

PubMed

Bimakr, Mandana; Rahman, Russly Abdul; Taip, Farah Saleena; Adzahan, Noranizan Mohd; Sarker, Md Zaidul Islam; Ganjloo, Ali

2013-01-15

In the present study, supercritical carbon dioxide (SC-CO(2)) extraction of seed oil from winter melon (Benincasa hispida) was investigated. The effects of process variables namely pressure (150-300 bar), temperature (40-50 °C) and dynamic extraction time (60-120 min) on crude extraction yield (CEY) were studied through response surface methodology (RSM). The SC-CO(2) extraction process was modified using ethanol (99.9%) as co-solvent. Perturbation plot revealed the significant effect of all process variables on the CEY. A central composite design (CCD) was used to optimize the process conditions to achieve maximum CEY. The optimum conditions were 244 bar pressure, 46 °C temperature and 97 min dynamic extraction time. Under these optimal conditions, the CEY was predicted to be 176.30 mg-extract/g-dried sample. The validation experiment results agreed with the predicted value. The antioxidant activity and fatty acid composition of crude oil obtained under optimized conditions were determined and compared with published results using Soxhlet extraction (SE) and ultrasound assisted extraction (UAE). It was found that the antioxidant activity of the extract obtained by SC-CO(2) extraction was strongly higher than those obtained by SE and UAE. Identification of fatty acid composition using gas chromatography (GC) showed that all the extracts were rich in unsaturated fatty acids with the most being linoleic acid. In contrast, the amount of saturated fatty acids extracted by SE was higher than that extracted under optimized SC-CO(2) extraction conditions.

Ultrasound-assisted extraction of natural antioxidants from the flower of Limonium sinuatum: Optimization and comparison with conventional methods.

PubMed

Xu, Dong-Ping; Zheng, Jie; Zhou, Yue; Li, Ya; Li, Sha; Li, Hua-Bin

2017-02-15

Natural antioxidants are widely used as dietary supplements or food additives. An optimized method of ultrasound-assisted extraction (UAE) was proposed for the effective extraction of antioxidants from the flowers of Limonium sinuatum and evaluated by response surface methodology. In this study, ethanol concentration, ratio of solvent to solid, ultrasonication time and temperature were investigated and optimized using a central composite rotatable design. The optimum extraction conditions were as follows: ethanol concentration, 60%; ratio of solvent to solid, 56.9:1mL/g; ultrasonication time, 9.8min; and temperature, 40°C. Under the optimal UAE conditions, the experimental values (483.01±15.39μmolTrolox/gDW) matched with those predicted (494.13μmolTrolox/gDW) within a 95% confidence level. In addition, the antioxidant activities of UAE were compared with those of conventional maceration and Soxhlet extraction methods, and the ultrasound-assisted extraction could give higher yield of antioxidants and markedly reduce the extraction time. Copyright © 2016 Elsevier Ltd. All rights reserved.

Optimization of enzyme-assisted extraction and characterization of polysaccharides from Hericium erinaceus.

PubMed

Zhu, Yang; Li, Qian; Mao, Guanghua; Zou, Ye; Feng, Weiwei; Zheng, Daheng; Wang, Wei; Zhou, Lulu; Zhang, Tianxiu; Yang, Jun; Yang, Liuqing; Wu, Xiangyang

2014-01-30

The enzyme-assisted extraction (EAE) of polysaccharides from the fruits of Hericium erinaceus was studied. In this study, response surface methodology and the Box-Behnken design based on single-factor and orthogonal experiments were applied to optimize the EAE conditions. The optimal extraction conditions were as follows: a pH of 5.71, a temperature of 52.03°C and a time of 33.79 min. The optimal extraction conditions resulted in the highest H. erinaceus polysaccharides (HEP) yield, with a value 13.46 ± 0.37%, which represented an increase of 67.72% compared to hot water extraction (HWE). The polysaccharides were characterized by FT-IR, SEM, CD, AFM, and GC. The results showed that HEP was composed of mannose, glucose, xylose, and galactose in a molar ratio of 15.16:5.55:4.21:1. The functional groups of the H. erinaceus polysaccharides extracted by HWE and EAE were fundamentally identical but had apparent conformational changes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Antioxidant Compound Extraction from Maqui (Aristotelia chilensis [Mol] Stuntz) Berries: Optimization by Response Surface Methodology

PubMed Central

Quispe-Fuentes, Issis; Vega-Gálvez, Antonio; Campos-Requena, Víctor H.

2017-01-01

The optimum conditions for the antioxidant extraction from maqui berry were determined using a response surface methodology. A three level D-optimal design was used to investigate the effects of three independent variables namely, solvent type (methanol, acetone and ethanol), solvent concentration and extraction time over total antioxidant capacity by using the oxygen radical absorbance capacity (ORAC) method. The D-optimal design considered 42 experiments including 10 central point replicates. A second-order polynomial model showed that more than 89% of the variation is explained with a satisfactory prediction (78%). ORAC values are higher when acetone was used as a solvent at lower concentrations, and the extraction time range studied showed no significant influence on ORAC values. The optimal conditions for antioxidant extraction obtained were 29% of acetone for 159 min under agitation. From the results obtained it can be concluded that the given predictive model describes an antioxidant extraction process from maqui berry.

Extractive cultivation of recombinant Escherichia coli using aqueous two-phase systems for production and separation of intracellular heat shock proteins.

PubMed

Umakoshi, H; Yano, K; Kuboi, R; Komasawa, I

1996-01-01

The extractive cultivation of recombinant Escherichia coli cells to produce, release, and separate heat shock proteins (HSPs; GroEL and GroES) using poly(ethylene glycol) (PEG)/dextran (Dex) aqueous two-phase systems was developed. The growth rate of E. coli OW10/pND5 cells in the PEG/Dex two-phase media was almost the same value as that in the control media. The addition of 0.1 M potassium phosphate salts (KPi) increased the productivity of HSPs with keeping the growth rate of E. coli cells relatively high. The partition coefficients of HSPs were improved to greater values when phosphate salts were added at a concentration of more than 0.1 M. As a result, PEG/Dex systems supplemented with 0.1 M KPi were found to be the optimized two-phase systems for the extractive cultivation of E. coli cells. In the systems, the HSPs were selectively partitioned to the top phase while cells occupied the bottom phase and the interface between the two phases. This integrated process was extended to a semicontinuous operating mode, where the top phase containing the HSPs was recovered following intermittent heating and ultrasonic irradiation. The bottom phase containing cells and cell debris was recycled together with new top phase solution to repeat production and recovery of HSPs.

Production of Trametes pubescens laccase under submerged and semi-solid culture conditions on agro-industrial wastes.

PubMed

Gonzalez, Juan C; Medina, Sandra C; Rodriguez, Alexander; Osma, Johann F; Alméciga-Díaz, Carlos J; Sánchez, Oscar F

2013-01-01

Laccases are copper-containing enzymes involved in the degradation of lignocellulosic materials and used in the treatment of phenol-containing wastewater. In this study we investigated the effect of culture conditions, i.e. submerged or semi-solid, and copper supplementation on laccase production by Trametespubescens grown on coffee husk, soybean pod husk, or cedar sawdust. The highest specific laccase activity was achieved when the culture was conducted under submerged conditions supplemented with copper (5 mM), and using coffee husk as substrate. The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2). Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3. In addition, they showed similar stability and electro-chemical properties. At optimal culture conditions laccase activity was 7.69 ± 0.28 U mg(-1) of protein for the crude extract, and 0.08 ± 0.001 and 2.86 ± 0.05 U mg(-1) of protein for Lac1 and Lac2, respectively. In summary, these results show the potential of coffee husk as an important and economical growth medium to produce laccase, offering a new alternative use for this common agro-industrial byproduct.

Cultivar and Harvest Month Influence the Nutrient Content of Opuntia spp. Cactus Pear Cladode Mucilage Extracts.

PubMed

du Toit, Alba; de Wit, Maryna; Hugo, Arno

2018-04-16

Mucilage extracted from cactus pear cladodes is a hydrocolloid gum. It is a novel, natural, low-kilojoule, cost-effective texture-modifying ingredient in functional food products. Yet, the cultivar with the most optimal nutrient content and the preferred harvest times are as yet unknown. For this reason, mucilage from three Opuntia ficus-indica (Algerian, Morado and Gymno-Carpo) and one Opuntia robusta (Robusta) cultivar were investigated to determine their nutrient content over six months. Nutrients that contribute energy (10.2 kJ/g) were low. The mineral content was high (ash 17.7/100 g), particularly calcium (3.0 g/100 g) and phosphorous (109.5 mg/kg). Low insoluble acid-detergent fibre (1.4 g/kg) and neutral-detergent fibre (2.1 g/kg) values indicated that mucilage was mostly soluble fibre. Calcium oxalate crystals were not detected in dried mucilage. Opuntia robusta powders had higher protein, extractable fat and potassium content, while Opuntia ficus-indica mucilage powders had higher polyunsaturated (Linoleic and α-Linolenic acid) fat content. O. robusta Robusta mucilage, harvested after the fruit harvest (February) had the lowest energy content and the highest mineral and protein content. Mucilage powders were highly soluble, low-kilojoule and mineral-rich. This is a functional ingredient that is produced from an easily cultivated crop, as cactus pears grow in areas with poor soil, extremely high daytime temperatures and limited water supplies.

Production of Trametes pubescens Laccase under Submerged and Semi-Solid Culture Conditions on Agro-Industrial Wastes

PubMed Central

Rodriguez, Alexander; Osma, Johann F.; Alméciga-Díaz, Carlos J.; Sánchez, Oscar F.

2013-01-01

Laccases are copper-containing enzymes involved in the degradation of lignocellulosic materials and used in the treatment of phenol-containing wastewater. In this study we investigated the effect of culture conditions, i.e. submerged or semi-solid, and copper supplementation on laccase production by Trametes pubescens grown on coffee husk, soybean pod husk, or cedar sawdust. The highest specific laccase activity was achieved when the culture was conducted under submerged conditions supplemented with copper (5 mM), and using coffee husk as substrate. The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2). Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3. In addition, they showed similar stability and electro-chemical properties. At optimal culture conditions laccase activity was 7.69±0.28 U mg-1 of protein for the crude extract, and 0.08±0.001 and 2.86±0.05 U mg-1 of protein for Lac1 and Lac2, respectively. In summary, these results show the potential of coffee husk as an important and economical growth medium to produce laccase, offering a new alternative use for this common agro-industrial byproduct. PMID:24019936

Study on extraction process and activity of plant polysaccharides

NASA Astrophysics Data System (ADS)

Ma, Xiaogen; Wang, Xiaojing; Fan, Shuangli; Chen, Jiezhong

2017-10-01

Recent studies have shown that plant polysaccharides have many pharmacological activities, such as hypoglycemic, anti-inflammatory and tumor inhibition. The pharmacological activities of plant polysaccharides were summarized. The extraction methods of plant polysaccharides were discussed. Finally, the extraction process of Herba Taraxaci polysaccharides was optimized by ultrasonic assisted extraction. Through single factor experiments and orthogonal experiment to optimize the optimum extraction process from dandelion polysaccharide, optimum conditions of dandelion root polysaccharide by ultrasonic assisted extraction method for ultrasonic power 320W, temperature 80°C, extraction time 40min, can get higher dandelion polysaccharide extract.

Optimization of microwave-assisted extraction (MAE) of coriander phenolic antioxidants - response surface methodology approach.

PubMed

Zeković, Zoran; Vladić, Jelena; Vidović, Senka; Adamović, Dušan; Pavlić, Branimir

2016-10-01

Microwave-assisted extraction (MAE) of polyphenols from coriander seeds was optimized by simultaneous maximization of total phenolic (TP) and total flavonoid (TF) yields, as well as maximized antioxidant activity determined by 1,1-diphenyl-2-picrylhydrazyl and reducing power assays. Box-Behnken experimental design with response surface methodology (RSM) was used for optimization of MAE. Extraction time (X1 , 15-35 min), ethanol concentration (X2 , 50-90% w/w) and irradiation power (X3 , 400-800 W) were investigated as independent variables. Experimentally obtained values of investigated responses were fitted to a second-order polynomial model, and multiple regression analysis and analysis of variance were used to determine fitness of the model and optimal conditions. The optimal MAE conditions for simultaneous maximization of polyphenol yield and increased antioxidant activity were an extraction time of 19 min, an ethanol concentration of 63% and an irradiation power of 570 W, while predicted values of TP, TF, IC50 and EC50 at optimal MAE conditions were 311.23 mg gallic acid equivalent per 100 g dry weight (DW), 213.66 mg catechin equivalent per 100 g DW, 0.0315 mg mL(-1) and 0.1311 mg mL(-1) respectively. RSM was successfully used for multi-response optimization of coriander seed polyphenols. Comparison of optimized MAE with conventional extraction techniques confirmed that MAE provides significantly higher polyphenol yields and extracts with increased antioxidant activity. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

[Studies on extraction process optimization of patrinia scabra Bunge saponins].

PubMed

Wang, Xue-Xi; Chen, Ru; Li, Shi-Gang; Shen, Wei; Cheng, Wei-Dong; Zhao, Jian-Xiong

2007-05-01

To optimize the conditions for the extraction of Patrinia scabra Bunge saponins. Orthogonal experimental design and ultrasonic method were employed to examine the conditions for the extraction by determination of saponins. The optimun condition for the extraction of Patrinia scabra Bunge saponins was as follows: 65% ethanol for 40 minutes, 55 degrees C and 210 watt of ultrasonic efficinecy. The extraction method of Patrinia scabra Bunge sponins is simple and efficient.

Extraction optimization and nanoencapsulation of jujube pulp and seed for enhancing antioxidant activity.

PubMed

Han, Hye Jung; Lee, Ji-Soo; Park, Sun-Ah; Ahn, Jun-Bae; Lee, Hyeon Gyu

2015-06-01

The aim of this study was to optimize extraction conditions for jujube pulp and seed in order to obtain maximum active ingredient yield and antioxidant activity, as well as to prepare chitosan nanoparticles loaded with jujube pulp and seed extracts for enhancing stability. The extraction conditions, i.e. temperature, time, and ethanol concentration, were optimized at the following respective values: 61.2 °C, 38 h, and 60.4% for pulp, and 58 °C, 34 h, and 59.2% for seed. The jujube nanoparticle size significantly increased with a higher chitosan/sodium tripolyphosphate ratio and extract concentration. Entrapment efficiency was greater than 80% regardless of preparation conditions. The stabilities of jujube pulp and seed extract in terms of total phenolic content and antioxidant activity were effectively enhanced by nanoencapsulation. In conclusion, jujube pulp and seed extracts prepared using optimal conditions could be useful as a natural functional food ingredient with antioxidant activity, and nanoencapsulation can be used to improve the stability of jujube extract. Therefore, these results could be used to promote the utilization of not only jujube pulp but also seed, by product. Copyright © 2015 Elsevier B.V. All rights reserved.

Biochemical characterisation of an allantoate-degrading enzyme from French bean (Phaseolus vulgaris): the requirement of phenylhydrazine.

PubMed

Raso, María José; Muñoz, Alfonso; Pineda, Manuel; Piedras, Pedro

2007-10-01

In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined "in vivo" using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37 degrees C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.

Optimization of an innovative approach involving mechanical activation and acid digestion for the extraction of lithium from lepidolite

NASA Astrophysics Data System (ADS)

Vieceli, Nathália; Nogueira, Carlos A.; Pereira, Manuel F. C.; Durão, Fernando O.; Guimarães, Carlos; Margarido, Fernanda

2018-01-01

The recovery of lithium from hard rock minerals has received increased attention given the high demand for this element. Therefore, this study optimized an innovative process, which does not require a high-temperature calcination step, for lithium extraction from lepidolite. Mechanical activation and acid digestion were suggested as crucial process parameters, and experimental design and response-surface methodology were applied to model and optimize the proposed lithium extraction process. The promoting effect of amorphization and the formation of lithium sulfate hydrate on lithium extraction yield were assessed. Several factor combinations led to extraction yields that exceeded 90%, indicating that the proposed process is an effective approach for lithium recovery.

Preparation, characterization, nanostructures and bio functional analysis of sonicated protein co-precipitates from brewers' spent grain and soybean flour.

PubMed

Alu'datt, Muhammad H; Gammoh, Sana; Rababah, Taha; Almomani, Mohammed; Alhamad, Mohammad N; Ereifej, Khalil; Almajwal, Ali; Tahat, Asma; Hussein, Neveen M; Nasser, Sura Abou

2018-02-01

This investigation was performed to assess the effects of sonication on the structure of protein, extractability of phenolics, and biological properties of isolated proteins and protein co-precipitates prepared from brewers' spent grain and soybean flour. Scanning electron micrographs revealed that the sonicated protein isolates and co-precipitates had different microstructures with fewer aggregates and smaller particles down to the nanometer scale compared to non-sonicated samples. However, the levels of free and bound phenolics extracted from non-sonicated protein isolates and protein co-precipitates increased compared to sonicated samples. The bound phenolics extracted after acid hydrolysis of sonicated protein co-precipitates showed improved ACE inhibitory activity and diminished antioxidant potency compared to non-sonicated samples. However, the free phenolics extracted from sonicated protein co-precipitates showed decreased ACE inhibitory activity and increased antioxidant activities compared to non-sonicated samples. The free and bound phenolics extracted from sonicated protein co-precipitates showed increased alpha-amylase inhibitory activity compared to non-sonicated samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of Culture Conditions on the Production of an Extracellular Protease by Bacillus sp. Isolated from Soil Sample of Lavizan Jungle Park

PubMed Central

Akhavan Sepahy, Abbas; Jabalameli, Leila

2011-01-01

Soil samples of Tehran jungle parks were screened for proteolytic Bacilli. Among eighteen protease producers one of the isolates obtained from Lavizan park, in north east of Tehran, was selected for further experimental studies. This isolate was identified as Bacillus sp. strain CR-179 based on partial sequencing of 16S rRNA. Various nutritional and environmental parameters affected protease production by Bacillus sp. strain CR-179. Protease production by this Bacillus cultivated in liquid cultures reached a maximum at 24 h, with levels of 340.908 U/mL. Starch and maltose were the best substrates for enzyme production while some pure sugars such as fructose, glucose, and sucrose could not influence production of protease. Among various organic nitrogen sources corn steep liquor, which is commercial, was found as the best substrate followed by yeast extract, whey protein, and beef extract. The optimal pH and optimal temperature of enzyme production were 8.0 and 45°C, respectively. Studies on enzymatic characterization revealed that crude protease showed maximum activity at pH 9.0 and 60°C, which is indicating the enzyme to be thermoalkaline protease. PMID:22191016

Optimization of dextran production by Leuconostoc mesenteroides NRRL B-512 using cheap and local sources of carbohydrate and nitrogen.

PubMed

Behravan, Javad; Bazzaz, B Seddigheh Fazly; Salimi, Zohreh

2003-12-01

Using pure components for the fermentation medium in dextran production imposes high costs on the industry. In the present study, the economic production of dextran using local and cheap sources of carbohydrate and nitrogen was investigated. Different concentrations of molasses and wheat-bran extract, after filtration, steam sterilization and pH adjustment, were inoculated with a fresh suspension of Leuconostoc mesenteroides. Cultures were incubated, and then diluted with an equal volume of ethanol. The bacteria were pelleted, and an aliquot of the supernatant was diluted with ethanol and dextran was precipitated. The supernatant was removed and the precipitate was dissolved in a minimal volume of water. Activated charcoal was added and the solution was boiled. The solution was filtered and protein impurities removed by 2-methylbutan-2-ol/chloroform extraction. Dextran was again precipitated with cold ethanol as described above, and the precipitate was dried in a desiccator. Optimum conditions and composition of culture media for dextran production using sugar-beet molasses and wheat bran were determined. The best results were obtained when 20% (w/v) molasses and 15% (w/v) wheat bran were used. The optimal initial pH for dextran production was 7.5.

Curcumin: Synthesis optimization and in silico interaction with cyclin dependent kinase.

PubMed

Ahmed, Mahmood; Abdul Qadir, Muhammad; Imtiaz Shafiq, Muhammad; Muddassar, Muhammad; Hameed, Abdul; Nadeem Arshad, Muhammad; Asiri, Abdullah M

2017-09-01

Curcumin is a natural product with enormous biological potential. In this study, curcumin synthesis was revisited using different reaction solvents, a catalyst (n-butylamine) and a water scavenger [(n-BuO)3B], to develop the optimal procedure for its rapid acquisition. During synthesis, solvent choice was found to be an important parameter for better curcumin yield and high purity. In a typical reaction, acetyl acetone was treated with boron trioxide, followed by condensation with vanillin in the presence of tri-n-butyl borate as water scavenger and n-butylamine as catalyst at 80 °C in ethyl acetate to afford curcumin. Moreover, curcumin was also extracted from turmeric powder and spectroscopic properties such as IR, MS, 1H NMR and 13C NMR with synthetic curcumin were established to identify any impurity. The purity of synthetic and extracted curcumin was also checked by TLC and HPLC-DAD. To computationally assess its therapeutic potential against cyclin dependent kinases (CDKs), curcumin was docked in different isoforms of CDKs. It was observed that it did not dock at the active sites of CDK2 and CDK6. However, it could enter into weak interactions with CDK4 protein.

Comparison of microwave-assisted and conventional extraction of mangiferin from mango (Mangifera indica L.) leaves.

PubMed

Zou, Tangbin; Wu, Hongfu; Li, Huawen; Jia, Qing; Song, Gang

2013-10-01

Mangiferin is the main bioactive component in mango leaves, which possesses anti-inflammatory, antioxidative, antidiabetic, immunomodulatory, and antitumor activities. In the present study, a microwave-assisted extraction method was developed for the extraction of mangiferin from mango leaves. Some parameters such as ethanol concentration, liquid-to-solid ratio, microwave power, and extraction time were optimized by single-factor experiments and response surface methodology. The optimal extraction conditions were 45% ethanol, liquid-to-solid ratio of 30:1 (mL/g), and extraction time of 123 s under microwave irradiation of 474 W. Under optimal conditions, the yield of mangiferin was 36.10 ± 0.72 mg/g, significantly higher than that of conventional extraction. The results obtained are beneficial for the full utilization of mango leaves and also indicate that microwave-assisted extraction is a very useful method for extracting mangiferin from plant materials. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Application of Ionic Liquids in the Microwave-Assisted Extraction of Proanthocyanidins from Larix gmelini Bark

PubMed Central

Yang, Lei; Sun, Xiaowei; Yang, Fengjian; Zhao, Chunjian; Zhang, Lin; Zu, Yuangang

2012-01-01

Ionic liquid based, microwave-assisted extraction (ILMAE) was successfully applied to the extraction of proanthocyanidins from Larix gmelini bark. In this work, in order to evaluate the performance of ionic liquids in the microwave-assisted extraction process, a series of 1-alkyl-3-methylimidazolium ionic liquids with different cations and anions were evaluated for extraction yield, and 1-butyl-3-methylimidazolium bromide was selected as the optimal solvent. In addition, the ILMAE procedure for the proanthocyanidins was optimized and compared with other conventional extraction techniques. Under the optimized conditions, satisfactory extraction yield of the proanthocyanidins was obtained. Relative to other methods, the proposed approach provided higher extraction yield and lower energy consumption. The Larix gmelini bark samples before and after extraction were analyzed by Thermal gravimetric analysis, Fourier-transform infrared spectroscopy and characterized by scanning electron microscopy. The results showed that the ILMAE method is a simple and efficient technique for sample preparation. PMID:22606036

The effect of temperature and bacterial growth phase on protein extraction by means of electroporation.

PubMed

Haberl-Meglič, Saša; Levičnik, Eva; Luengo, Elisa; Raso, Javier; Miklavčič, Damijan

2016-12-01

Different chemical and physical methods are used for extraction of proteins from bacteria, which are used in variety of fields. But on a large scale, many methods have severe drawbacks. Recently, extraction by means of electroporation showed a great potential to quickly obtain proteins from bacteria. Since many parameters are affecting the yield of extracted proteins, our aim was to investigate the effect of temperature and bacterial growth phase on the yield of extracted proteins. At the same time bacterial viability was tested. Our results showed that the temperature has a great effect on protein extraction, the best temperature post treatment being 4°C. No effect on bacterial viability was observed for all temperatures tested. Also bacterial growth phase did not affect the yield of extracted proteins or bacterial viability. Nevertheless, further experiments may need to be performed to confirm this observation, since only one incubation temperature (4°C) and one incubation time before and after electroporation (0.5 and 1h) were tested for bacterial growth phase. Based on our results we conclude that temperature is a key element for bacterial membrane to stay in a permeabilized state, so more proteins flow out of bacteria into surrounding media. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimization of microwave assisted extraction (MAE) and soxhlet extraction of phenolic compound from licorice root.

PubMed

Karami, Zohreh; Emam-Djomeh, Zahra; Mirzaee, Habib Allah; Khomeiri, Morteza; Mahoonak, Alireza Sadeghi; Aydani, Emad

2015-06-01

In present study, response surface methodology was used to optimize extraction condition of phenolic compounds from licorice root by microwave application. Investigated factors were solvent (ethanol 80 %, methanol 80 % and water), liquid/solid ratio (10:1-25:1) and time (2-6 min). Experiments were designed according to the central composite rotatable design. The results showed that extraction conditions had significant effect on the extraction yield of phenolic compounds and antioxidant capacities. Optimal condition in microwave assisted method were ethanol 80 % as solvent, extraction time of 5-6 min and liquid/solid ratio of 12.7/1. Results were compared with those obtained by soxhlet extraction. In soxhlet extraction, Optimum conditions were extraction time of 6 h for ethanol 80 % as solvent. Value of phenolic compounds and extraction yield of licorice root in microwave assisted (MAE), and soxhlet were 47.47 mg/g and 16.38 %, 41.709 mg/g and 14.49 %, respectively. These results implied that MAE was more efficient extracting method than soxhlet.

Tropomyosin and Actin Identified as Major Allergens of the Carpet Clam (Paphia textile) and the Effect of Cooking on Their Allergenicity

PubMed Central

Mohamad Yadzir, Zailatul Hani; Misnan, Rosmilah; Bakhtiar, Faizal; Abdullah, Noormalin; Murad, Shahnaz

2015-01-01

Objectives. To identify the major allergenic proteins of clam (Paphia textile) and to investigate the effect of different cooking methods on the allergenicity of these identified proteins. Methods. Clam protein extracts were separated by denaturing polyacrylamide gel electrophoresis. IgE reactive proteins were then analyzed by immunoblotting with sera from patients with positive skin prick tests (SPT) to the raw clam extract. Mass spectrometry was used to identify the major allergenic proteins of this clam. Results. Raw extract showed 12 protein bands (18–150 kDa). In contrast, fewer protein bands were seen in the boiled extract; those ranging from 40 to 150 kDa were denatured. The protein profiles were similarly altered by frying or roasting. The immunoblots of raw and boiled extracts yielded 10 and 2 IgE-binding proteins, respectively. The fried and roasted extracts showed only a single IgE-binding protein at 37 kDa. Mass spectrometry analysis of the 37 and 42 kDa major allergens indicated that these spots were tropomyosin and actin, respectively. Conclusion. The two major allergens of Paphia textile were identified as the thermostable tropomyosin and a new thermolabile allergen actin. PMID:26413512

Utilization of wild apricot kernel press cake for extraction of protein isolate.

PubMed

Sharma, P C; Tilakratne, B M K S; Gupta, Anil

2010-12-01

The kernels of apricot (Prunus armeniaca) stones are utilized for extraction of oil. The press cake left after extraction of oil was evaluated for preparation of protein isolate for its use in food supplementation. The apricot kernels contained 45-50% oil, 23.6-26.2% protein, 4.2% ash, 5.42% crude fibre, 8.2% carbohydrates and 90 mg HCN/100 g kernels, while press cake obtained after oil extraction contained 34.5% crude protein, which can be utilized for preparation of protein isolates. The method standardized for extraction of protein isolate broadly consisted of boiling the press cake with water in 1:20 (w/v) ratio for 1 h, raising pH to 8 and stirring for a few min followed by filtration, coagulation at pH 4 prior to sieving and pressing of coagulant for overnight and drying followed by grinding which resulted in extraction of about 71.3% of the protein contained in the press cake. The protein isolate contained 68.8% protein, 6.4% crude fat, 0.8% ash, 2.2% crude fibre and 12.7% carbohydrates. Thus the apricot kernel press cake can be utilized for preparation of protein isolate to improve the nutritional status of many food formulations.

Co-expression of hepatitis C virus polytope-HBsAg and p19-silencing suppressor protein in tobacco leaves.

PubMed

Mohammadzadeh, Sara; Roohvand, Farzin; Memarnejadian, Arash; Jafari, Anis; Ajdary, Soheila; Salmanian, Ali-Hatef; Ehsani, Parastoo

2016-01-01

Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition. We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine. A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector. Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants. The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

[Optimization of ultrasonic-assisted extraction of total flavonoids from leaves of the Artocarpus heterophyllus by response surface methodology].

PubMed

Wang, Hong-wu; Liu, Yan-qing; Wang, Yuan-hong

2011-07-01

To investigate the ultrasonic-assisted extract on of total flavonoids from leaves of the Artocarpus heterophyllus. Investigated the effects of ethanol concentration, extraction time, and liquid-solid ratio on flavonoids yield. A 17-run response surface design involving three factors at three levels was generated by the Design-Expert software and experimental data obtained were subjected to quadratic regression analysis to create a mathematical model describing flavonoids extraction. The optimum ultrasonic assisted extraction conditions were: ethanol volume fraction 69.4% and liquid-solid ratio of 22.6:1 for 32 min. Under these optimized conditions, the yield of flavonoids was 7.55 mg/g. The Box-Behnken design and response surface analysis can well optimize the ultrasonic-assisted extraction of total flavonoids from Artocarpus heterophyllus.

Optimal Information Extraction of Laser Scanning Dataset by Scale-Adaptive Reduction

NASA Astrophysics Data System (ADS)

Zang, Y.; Yang, B.

2018-04-01

3D laser technology is widely used to collocate the surface information of object. For various applications, we need to extract a good perceptual quality point cloud from the scanned points. To solve the problem, most of existing methods extract important points based on a fixed scale. However, geometric features of 3D object come from various geometric scales. We propose a multi-scale construction method based on radial basis function. For each scale, important points are extracted from the point cloud based on their importance. We apply a perception metric Just-Noticeable-Difference to measure degradation of each geometric scale. Finally, scale-adaptive optimal information extraction is realized. Experiments are undertaken to evaluate the effective of the proposed method, suggesting a reliable solution for optimal information extraction of object.

Black bean anthocyanin-rich extracts as food colorants: Physicochemical stability and antidiabetes potential

USDA-ARS?s Scientific Manuscript database

Black beans contain anthocyanins that could be used as colorants in foods with associated health benefits. The objective was to optimize anthocyanins extraction from black bean coats and evaluate their physicochemical stability and antidiabetes potential. Optimal extraction conditions were 24% ethan...

Coupling detergent lysis/clean-up methodology with intact protein fractionation for enhanced proteome characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Ritin; Dill, Brian; Chourey, Karuna

2012-01-01

The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four different detergent clean-up methods (Trichloroacetic acid (TCA) precipitation, Chloroform/Methanol/Water (CMW) extraction, commercial detergent removal spin column method (DRS) and filter-aided sample preparation(FASP)) with respect to varying amounts of protein biomass in the samples, and provide efficiency benchmarks with respect to protein, peptide, and spectral identifications for each method. Our results show that for protein limited samples, FASP outperforms the other three clean-up methods, while at high protein amountmore » all the methods are comparable. This information was used in a dual strategy of comparing molecular weight based fractionated and unfractionated lysates from three increasingly complex samples (Escherichia coli, a five microbial isolate mixture, and a natural microbial community groundwater sample), which were all lysed with SDS and cleaned up using FASP. The two approaches complemented each other by enhancing the number of protein identifications by 8%-25% across the three samples and provided broad pathway coverage.« less

An approach of ionic liquids/lithium salts based microwave irradiation pretreatment followed by ultrasound-microwave synergistic extraction for two coumarins preparation from Cortex fraxini.

PubMed

Liu, Zaizhi; Gu, Huiyan; Yang, Lei

2015-10-23

Ionic liquids/lithium salts solvent system was successfully introduced into the separation technique for the preparation of two coumarins (aesculin and aesculetin) from Cortex fraxini. Ionic liquids/lithium salts based microwave irradiation pretreatment followed by ultrasound-microwave synergy extraction (ILSMP-UMSE) procedure was developed and optimized for the sufficient extraction of these two analytes. Several variables which can potentially influence the extraction yields, including pretreatment time and temperature, [C4mim]Br concentration, LiAc content, ultrasound-microwave synergy extraction (UMSE) time, liquid-solid ratio, and UMSE power were optimized by Plackett-Burman design. Among seven variables, UMSE time, liquid-solid ratio, and UMSE power were the statistically significant variables and these three factors were further optimized by Box-Behnken design to predict optimal extraction conditions and find out operability ranges with maximum extraction yields. Under optimum operating conditions, ILSMP-UMSE showed higher extraction yields of two target compounds than those obtained by reference extraction solvents. Method validation studies also evidenced that ILSMP-UMSE is credible for the preparation of two coumarins from Cortex fraxini. This study is indicative of the proposed procedure that has huge application prospects for the preparation of natural products from plant materials. Copyright © 2015 Elsevier B.V. All rights reserved.

Optimizing pressurized liquid extraction of microbial lipids using the response surface method.

PubMed

Cescut, J; Severac, E; Molina-Jouve, C; Uribelarrea, J-L

2011-01-21

Response surface methodology (RSM) was used for the determination of optimum extraction parameters to reach maximum lipid extraction yield with yeast. Total lipids were extracted from oleaginous yeast (Rhodotorula glutinis) using pressurized liquid extraction (PLE). The effects of extraction parameters on lipid extraction yield were studied by employing a second-order central composite design. The optimal condition was obtained as three cycles of 15 min at 100°C with a ratio of 144 g of hydromatrix per 100 g of dry cell weight. Different analysis methods were used to compare the optimized PLE method with two conventional methods (Soxhlet and modification of Bligh and Dyer methods) under efficiency, selectivity and reproducibility criteria thanks to gravimetric analysis, GC with flame ionization detector, High Performance Liquid Chromatography linked to Evaporative Light Scattering Detector (HPLC-ELSD) and thin-layer chromatographic analysis. For each sample, the lipid extraction yield with optimized PLE was higher than those obtained with referenced methods (Soxhlet and Bligh and Dyer methods with, respectively, a recovery of 78% and 85% compared to PLE method). Moreover, the use of PLE led to major advantages such as an analysis time reduction by a factor of 10 and solvent quantity reduction by 70%, compared with traditional extraction methods. Copyright © 2010 Elsevier B.V. All rights reserved.

Optimization of ultrasound-assisted extraction of charantin from Momordica charantia fruits using response surface methodology

PubMed Central

Ahamad, Javed; Amin, Saima; Mir, Showkat R.

2015-01-01

Background: Momordica charantia Linn. (Cucurbitaceae) fruits are well known for their beneficial effects in diabetes that are often attributed to its bioactive component charantin. Objective: The aim of the present study is to develop and optimize an efficient protocol for the extraction of charantin from M. charantia fruits. Materials and Methods: Response surface methodology (RSM) was used for the optimization of ultrasound-assisted extraction (UAE) conditions. RSM was based on a three-level, three-variable Box-Behnken design (BBD), and the studied variables included solid to solvent ratio, extraction temperature, and extraction time. Results: The optimal conditions predicted by the BBD were: UAE with methanol: Water (80:20, v/v) at 46°C for 120 min with solid to solvent ratio of 1:26 w/v, under which the yield of charantin was 3.18 mg/g. Confirmation trials under slightly adjusted conditions yielded 3.12 ± 0.14 mg/g of charantin on dry weight basis of fruits. The result of UAE was also compared with Soxhlet extraction method and UAE was found 2.74-fold more efficient than the Soxhlet extraction for extracting charantin. Conclusions: A facile UAE protocol for a high extraction yield of charantin was developed and validated. PMID:26681889

Building Extraction Based on an Optimized Stacked Sparse Autoencoder of Structure and Training Samples Using LIDAR DSM and Optical Images.

PubMed

Yan, Yiming; Tan, Zhichao; Su, Nan; Zhao, Chunhui

2017-08-24

In this paper, a building extraction method is proposed based on a stacked sparse autoencoder with an optimized structure and training samples. Building extraction plays an important role in urban construction and planning. However, some negative effects will reduce the accuracy of extraction, such as exceeding resolution, bad correction and terrain influence. Data collected by multiple sensors, as light detection and ranging (LIDAR), optical sensor etc., are used to improve the extraction. Using digital surface model (DSM) obtained from LIDAR data and optical images, traditional method can improve the extraction effect to a certain extent, but there are some defects in feature extraction. Since stacked sparse autoencoder (SSAE) neural network can learn the essential characteristics of the data in depth, SSAE was employed to extract buildings from the combined DSM data and optical image. A better setting strategy of SSAE network structure is given, and an idea of setting the number and proportion of training samples for better training of SSAE was presented. The optical data and DSM were combined as input of the optimized SSAE, and after training by an optimized samples, the appropriate network structure can extract buildings with great accuracy and has good robustness.

Advanced Optimal Extraction for the Spitzer/IRS

NASA Astrophysics Data System (ADS)

Lebouteiller, V.; Bernard-Salas, J.; Sloan, G. C.; Barry, D. J.

2010-02-01

We present new advances in the spectral extraction of pointlike sources adapted to the Infrared Spectrograph (IRS) on board the Spitzer Space Telescope. For the first time, we created a supersampled point-spread function of the low-resolution modules. We describe how to use the point-spread function to perform optimal extraction of a single source and of multiple sources within the slit. We also examine the case of the optimal extraction of one or several sources with a complex background. The new algorithms are gathered in a plug-in called AdOpt which is part of the SMART data analysis software.

Bone protein extraction without demineralization using principles from hydroxyapatite chromatography.

PubMed

Cleland, Timothy P; Vashishth, Deepak

2015-03-01

Historically, extraction of bone proteins has relied on the use of demineralization to better retrieve proteins from the extracellular matrix; however, demineralization can be a slow process that restricts subsequent analysis of the samples. Here, we developed a novel protein extraction method that does not use demineralization but instead uses a methodology from hydroxyapatite chromatography where high concentrations of ammonium phosphate and ammonium bicarbonate are used to extract bone proteins. We report that this method has a higher yield than those with previously published small-scale extant bone extractions, with and without demineralization. Furthermore, after digestion with trypsin and subsequent high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis, we were able to detect several extracellular matrix and vascular proteins in addition to collagen I and osteocalcin. Our new method has the potential to isolate proteins within a short period (4h) and provide information about bone proteins that may be lost during demineralization or with the use of denaturing agents. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization of rice starch and protein obtained by a fast alkaline extraction method.

PubMed

Souza, Daiana de; Sbardelotto, Arthur Francisco; Ziegler, Denize Righetto; Marczak, Ligia Damasceno Ferreira; Tessaro, Isabel Cristina

2016-01-15

This study evaluated the characteristics of rice starch and protein obtained by a fast alkaline extraction method on rice flour (RF) derived from broken rice. The extraction was conducted using 0.18% NaOH at 30°C for 30min followed by centrifugation to separate the starch rich and the protein rich fractions. This fast extraction method allowed to obtain an isoelectric precipitation protein concentrate (IPPC) with 79% protein and a starchy product with low protein content. The amino acid content of IPPC was practically unchanged compared to the protein in RF. The proteins of the IPPC underwent denaturation during extraction and some of the starch suffered the cold gelatinization phenomenon, due to the alkaline treatment. With some modifications, the fast method can be interesting in a technological point of view as it enables process cost reduction and useful ingredients obtention to the food and chemical industries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Homology-based Modeling of Rhodopsin-like Family Members in the Inactive State: Structural Analysis and Deduction of Tips for Modeling and Optimization.

PubMed

Pappalardo, Matteo; Rayan, Mahmoud; Abu-Lafi, Saleh; Leonardi, Martha E; Milardi, Danilo; Guccione, Salvatore; Rayan, Anwar

2017-08-01

Modeling G-Protein Coupled Receptors (GPCRs) is an emergent field of research, since utility of high-quality models in receptor structure-based strategies might facilitate the discovery of interesting drug candidates. The findings from a quantitative analysis of eighteen resolved structures of rhodopsin family "A" receptors crystallized with antagonists and 153 pairs of structures are described. A strategy termed endeca-amino acids fragmentation was used to analyze the structures models aiming to detect the relationship between sequence identity and Root Mean Square Deviation (RMSD) at each trans-membrane-domain. Moreover, we have applied the leave-one-out strategy to study the shiftiness likelihood of the helices. The type of correlation between sequence identity and RMSD was studied using the aforementioned set receptors as representatives of membrane proteins and 98 serine proteases with 4753 pairs of structures as representatives of globular proteins. Data analysis using fragmentation strategy revealed that there is some extent of correlation between sequence identity and global RMSD of 11AA width windows. However, spatial conservation is not always close to the endoplasmic side as was reported before. A comparative study with globular proteins shows that GPCRs have higher standard deviation and higher slope in the graph with correlation between sequence identity and RMSD. The extracted information disclosed in this paper could be incorporated in the modeling protocols while using technique for model optimization and refinement. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photothermal stress triggered by near-infrared-irradiated carbon nanotubes up-regulates osteogenesis and mineral deposition in tooth-extracted sockets.

PubMed

Kajiya, Hiroshi; Katsumata, Yuri; Sasaki, Mina; Tsutsumi, Takashi; Kawaguchi, Minoru; Fukushima, Tadao

2015-01-01

The bone regenerative healing process is often prolonged, with a high risk of infection particularly in elderly and diseased patients. A reduction in healing process time usually requires mechanical stress devices, chemical cues, or laser/thermal therapies. Although these approaches have been used extensively for the reduction of bone healing time, the exact mechanisms involved in thermal stress-induced bone regeneration remain unclear. Photothermal stress (PTS) stimulation was carried out using a novel photothermal device, composed of an alginate gel (AG) including carbon nanotubes (CNT-AGs) and their irradiator with near-infrared (NIR) light. We investigated the effects of optimal hyperthermia on osteogenesis, its signalling pathway in vitro and mineral deposition in tooth-extracted sockets in vivo. The PTS (10 min at 42 °C, every day), triggered by NIR-induced CNT, increased the activity of alkaline phosphatase (ALP) in mouse osteoblast MC3T3-E1 cells in a time-dependent manner compared with the non-thermal stress control. PTS significantly induced the expression of osteogenic-related molecules such as ALP, RUNX2 and Osterix in a time-dependent manner with phosphorylated mitogen-activated protein kinases (MAPK). PTS increased the expression of heat shock factor (HSF) 2, but not HSF1, resulting in activation of heat shock protein 27. PTS significantly up-regulated mineral deposition in tooth-extracted sockets in normal and ovariectomised osteoporotic model mice in vivo. Our novel CNT-based PTS up-regulated osteogenesis via activation of heat shock-related molecules, resulting in promotion of mineral deposition in enhanced tooth-extracted sockets.

Use of proteomics for validation of the isolation process of clotting factor IX from human plasma.

PubMed

Clifton, James; Huang, Feilei; Gaso-Sokac, Dajana; Brilliant, Kate; Hixson, Douglas; Josic, Djuro

2010-01-03

The use of proteomic techniques in the monitoring of different production steps of plasma-derived clotting factor IX (pd F IX) was demonstrated. The first step, solid-phase extraction with a weak anion-exchange resin, fractionates the bulk of human serum albumin (HSA), immunoglobulin G, and other non-binding proteins from F IX. The proteins that strongly bind to the anion-exchange resin are eluted by higher salt concentrations. In the second step, anion-exchange chromatography, residual HSA, some proteases and other contaminating proteins are separated. In the last chromatographic step, affinity chromatography with immobilized heparin, the majority of the residual impurities are removed. However, some contaminating proteins still remain in the eluate from the affinity column. The next step in the production process, virus filtration, is also an efficient step for the removal of residual impurities, mainly high molecular weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins are monitored by biochemical and immunochemical methods and by LC-MS/MS and their removal documented. Our methodology is very helpful for further process optimization, rapid identification of target proteins with relatively low abundance, and for the design of subsequent steps for their removal or purification.

A simple purification and activity assay of the coagulant protein from Moringa oleifera seed.

PubMed

Ghebremichael, Kebreab A; Gunaratna, K R; Henriksson, Hongbin; Brumer, Harry; Dalhammar, Gunnel

2005-06-01

Use of extracts from Moringa oleifera (MO) is of great interest for low-cost water treatment. This paper discusses water and salt extraction of a coagulant protein from the seed, purification using ion exchange, its chemical characteristics, coagulation and antimicrobial properties. The coagulant from both extracts is a cationic protein with pI greater than 9.6 and molecular mass less than 6.5 kDa. Mass spectrometric analysis of the purified water extract indicated that it contained at least four homologous proteins, based on MS/MS peptide sequence data. The protein is thermoresistant and remained active after 5h heat treatment at 95 degrees C. The coagulant protein showed both flocculating and antibacterial effects of 1.1--4 log reduction. With samples of high turbidity, the MO extract showed similar coagulation activity as alum. Cecropin A and MO extract were found to have similar flocculation effects for clay and microorganisms. Simple methods for both the purification and assay of MO coagulating proteins are presented, which are necessary for large-scale water treatment applications.

INSOLUBILITY AND ALTERATION OF ALLERGENIC ACTIVITY OF WHEAT PROTEINS IN PROCESSED FOODS.

PubMed

Tanaka, Kajiyo; Kanie, Yuuki; Naitou, Michita; Suzuki, Misa; Umemura, Harue; Tagami, Kazunori; Sakai, Kazunori; Furuta, Tomoko; Yamada, Chikako; Izumi, Hidehiko; Yokooji, Tomoharu; Matsuo, Hiroaki; Ito, Komei

2017-01-01

Food processing causes decomposition, denaturation or polymerization of protein, which may alter an allergic reaction. This study aimed to investigate the insolubility and alteration of wheat allergens in processed foods and the reactivity to patient sera. We extracted proteins from wheat flour, udon and bread using different extracts and conducted SDS-polyacrylamide gel electrophoresis. IgE-immunoblotting was also conducted using sera from children with wheat allergy. Soluble protein was extracted from wheat flour, and gluten fractions were also extracted by adding SDS. However, no proteins were able to be extracted from udon or bread witout severing the disulfide bonds under reducing condition. Only trace amounts of protein were detected in the water after boiling udon noodles. The reactivity of IgE antibody to the extracted protein did not differ among the different processed food types. Wheat allergens became strongly insolubilized after gluten formation and heating. However, the reactivity of IgE antibody to each allergen was not affected by food processing. Further studies are needed for the effects on clinical symptoms.

Starch extraction process coupled to protein recovery from leguminous tuberous roots (Pachyrhizus ahipa).

PubMed

Díaz, Andrea; Dini, Cecilia; Viña, Sonia Z; García, María A

2016-11-05

The objective of this work was to fit together the starch extraction from Pachyrhizus ahipa roots and the recovery of the proteins present in these storage organs, making an improved use of this novel raw material. The replacement of water by buffer PO4(-3)/NaCl as solvent in the first extraction steps improved protein extraction without lowering the starch yield. The starches obtained from the traditional and the proposed methods exhibited some differences in appearance and technological and thermal properties, which were endorsed to the adjustment in the methodology of extraction rather than to the use of buffer as solvent. Thus, P. ahipa starch obtaining procedure could be coupled to protein extraction with a minimum change in the methodology. This innovation did not significantly shift the characteristics of the starch obtained and allowed to obtain a protein yield of 135.7mg BSA equivalent protein/100g of fresh roots. Copyright © 2016 Elsevier Ltd. All rights reserved.

Extraction methods and test techniques for detection of vegetable proteins in meat products. I. Qualitative detection of soya derivatives.

PubMed

Hyslop, N S

1976-06-01

Extracts of 3 soya bean preparations, used commercially in certain countries to replace part of the meat in popular meat products, were made by treatment with (i) sodium dodecyl sulphate, (ii) Triton-X100 or (iii) n-Butanol. Similar extracts were made from beef and pork. All extracts were examined by electrophoretic and immunological techniques. Stained polyacrylamide gels revealed distinctive protein bands after electrophoresis. The migration rates of corresponding bands differed between beef and pork extracts. However, the migration rates of vegetable bands revealed certain similarities, but differed very greatly from those of animal origin. Characteristic fast-migrating S-bands were distinguishable only in extracts of vegetable protein. Immunodiffusion tests, using antisera produced in rabbits against each extract, revealed varying degrees of similarity between extracts of vegetable origin, but the antisera were specific for either vegetable or animal protein.

Extraction methods and test techniques for detection of vegetable proteins in meat products. I. Qualitative detection of soya derivatives.

PubMed Central

Hyslop, N. S.

1976-01-01

Extracts of 3 soya bean preparations, used commercially in certain countries to replace part of the meat in popular meat products, were made by treatment with (i) sodium dodecyl sulphate, (ii) Triton-X100 or (iii) n-Butanol. Similar extracts were made from beef and pork. All extracts were examined by electrophoretic and immunological techniques. Stained polyacrylamide gels revealed distinctive protein bands after electrophoresis. The migration rates of corresponding bands differed between beef and pork extracts. However, the migration rates of vegetable bands revealed certain similarities, but differed very greatly from those of animal origin. Characteristic fast-migrating S-bands were distinguishable only in extracts of vegetable protein. Immunodiffusion tests, using antisera produced in rabbits against each extract, revealed varying degrees of similarity between extracts of vegetable origin, but the antisera were specific for either vegetable or animal protein. Images Plate 1 Plate 2 PMID:819572

[Supercritical CO2 extraction and component analysis of Aesculus wilsonii seed oil].

PubMed

Chen, Guang-Yu; Shi, Zhao-Hua; Li, Hai-Chi; Ge, Fa-Huan; Zhan, Hua-Shu

2013-03-01

To research the optimal extraction process of supercritical CO2 extraction and analyze the component of the oil extracted from Aesculus wilsonii seed. Using the yield of Aesculus wilsonii seed oil as the index, optimized supercritical CO2 extraction parameter by orthogonal experiment methodology and analysed the compounds of Aesculus wilsonii seed oil by GC-MS. The optimal parameters of the supercritical CO2 extraction of the oil extracted from Aesculus wilsoniit seed were determined: the extraction pressure was 28 MPa and the temperature was 38 degrees C, the separation I pressure was 12 MPa and the temperature was 40 degrees C, the separation II pressure was 5 MPa and the temperature was 40 degrees C, the extraction time was 110 min. The average extraction rate of Aesculus wilsonii seed oil was 1.264%. 26 kinds of compounds were identified by GC-MS in Aesculus wilsonii seed oil extracted by supercritical CO2. The main components were fatty acids. Comparing with the petroleum ether extraction, the supercritical CO2 extraction has higher extraction rate, shorter extraction time, more clarity oil. The kinds of fatty acids with high amounts in Aesculus wilsonii seed oil is identical in general, the kinds of fatty acids with low amounts in Aesculus wilsonii seed oil have differences.

Microwave-assisted extraction and mild saponification for determination of organochlorine pesticides in oyster samples.

PubMed

Carro, N; García, I; Ignacio, M-C; Llompart, M; Yebra, M-C; Mouteira, A

2002-10-01

A sample-preparation procedure (extraction and saponification) using microwave energy is proposed for determination of organochlorine pesticides in oyster samples. A Plackett-Burman factorial design has been used to optimize the microwave-assisted extraction and mild saponification on a freeze dried sample spiked with a mixture of aldrin, endrin, dieldrin, heptachlor, heptachorepoxide, isodrin, transnonachlor, p, p'-DDE, and p, p'-DDD. Six variables: solvent volume, extraction time, extraction temperature, amount of acetone (%) in the extractant solvent, amount of sample, and volume of NaOH solution were considered in the optimization process. The results show that the amount of sample is statistically significant for dieldrin, aldrin, p, p'-DDE, heptachlor, and transnonachlor and solvent volume for dieldrin, aldrin, and p, p'-DDE. The volume of NaOH solution is statistically significant for aldrin and p, p'-DDE only. Extraction temperature and extraction time seem to be the main factors determining the efficiency of extraction process for isodrin and p, p'-DDE, respectively. The optimized procedure was compared with conventional Soxhlet extraction.

Improved detection of multiple environmental antibiotics through an optimized sample extraction strategy in liquid chromatography-mass spectrometry analysis.

PubMed

Yi, Xinzhu; Bayen, Stéphane; Kelly, Barry C; Li, Xu; Zhou, Zhi

2015-12-01

A solid-phase extraction/liquid chromatography/electrospray ionization/multi-stage mass spectrometry (SPE-LC-ESI-MS/MS) method was optimized in this study for sensitive and simultaneous detection of multiple antibiotics in urban surface waters and soils. Among the seven classes of tested antibiotics, extraction efficiencies of macrolides, lincosamide, chloramphenicol, and polyether antibiotics were significantly improved under optimized sample extraction pH. Instead of only using acidic extraction in many existing studies, the results indicated that antibiotics with low pK a values (<7) were extracted more efficiently under acidic conditions and antibiotics with high pK a values (>7) were extracted more efficiently under neutral conditions. The effects of pH were more obvious on polar compounds than those on non-polar compounds. Optimization of extraction pH resulted in significantly improved sample recovery and better detection limits. Compared with reported values in the literature, the average reduction of minimal detection limits obtained in this study was 87.6% in surface waters (0.06-2.28 ng/L) and 67.1% in soils (0.01-18.16 ng/g dry wt). This method was subsequently applied to detect antibiotics in environmental samples in a heavily populated urban city, and macrolides, sulfonamides, and lincomycin were frequently detected. Antibiotics with highest detected concentrations were sulfamethazine (82.5 ng/L) in surface waters and erythromycin (6.6 ng/g dry wt) in soils. The optimized sample extraction strategy can be used to improve the detection of a variety of antibiotics in environmental surface waters and soils.

Optimizing Sustainable Geothermal Heat Extraction

NASA Astrophysics Data System (ADS)

Patel, Iti; Bielicki, Jeffrey; Buscheck, Thomas

2016-04-01

Geothermal heat, though renewable, can be depleted over time if the rate of heat extraction exceeds the natural rate of renewal. As such, the sustainability of a geothermal resource is typically viewed as preserving the energy of the reservoir by weighing heat extraction against renewability. But heat that is extracted from a geothermal reservoir is used to provide a service to society and an economic gain to the provider of that service. For heat extraction used for market commodities, sustainability entails balancing the rate at which the reservoir temperature renews with the rate at which heat is extracted and converted into economic profit. We present a model for managing geothermal resources that combines simulations of geothermal reservoir performance with natural resource economics in order to develop optimal heat mining strategies. Similar optimal control approaches have been developed for managing other renewable resources, like fisheries and forests. We used the Non-isothermal Unsaturated-saturated Flow and Transport (NUFT) model to simulate the performance of a sedimentary geothermal reservoir under a variety of geologic and operational situations. The results of NUFT are integrated into the optimization model to determine the extraction path over time that maximizes the net present profit given the performance of the geothermal resource. Results suggest that the discount rate that is used to calculate the net present value of economic gain is a major determinant of the optimal extraction path, particularly for shallower and cooler reservoirs, where the regeneration of energy due to the natural geothermal heat flux is a smaller percentage of the amount of energy that is extracted from the reservoir.

Purification of intact chloroplasts from marine plant Posidonia oceanica suitable for organelle proteomics.

PubMed

Piro, Amalia; Serra, Ilia Anna; Spadafora, Antonia; Cardilio, Monica; Bianco, Linda; Perrotta, Gaetano; Santos, Rui; Mazzuca, Silvia

2015-12-01

Posidonia oceanica is a marine angiosperm, or seagrass, adapted to grow to the underwater life from shallow waters to 50 m depth. This raises questions of how their photosynthesis adapted to the attenuation of light through the water column and leads to the assumption that biochemistry and metabolism of the chloroplast are the basis of adaptive capacity. In the present study, we described a protocol that was adapted from those optimized for terrestrial plants, to extract chloroplasts from as minimal tissue as possible. We obtained the best balance between tissue amount/intact chloroplasts yield using one leaf from one plant. After isopynic separations, the chloroplasts purity and integrity were evaluated by biochemical assay and using a proteomic approach. Chloroplast proteins were extracted from highly purified organelles and resolved by 1DE SDS-PAGE. Proteins were sequenced by nLC-ESI-IT-MS/MS of 1DE gel bands and identified against NCBInr green plant databases, Dr. Zompo database for seagrasses in a local customized dataset. The curated localization of proteins in sub-plastidial compartments (i.e. envelope, stroma and thylakoids) was retrieved in the AT_CHLORO database. This purification protocol and the validation of compartment markers may serve as basis for sub-cellular proteomics in P. oceanica and other seagrasses. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean.

PubMed

Piller, Kenneth J; Clemente, Thomas E; Jun, Sang Mu; Petty, Cynthia C; Sato, Shirley; Pascual, David W; Bost, Kenneth L

2005-09-01

Enterotoxigenic Escherichia coli (ETEC) cause acute diarrhea in humans and farm animals, and can be fatal if the host is left untreated. As a potential alternative to traditional needle vaccination of cattle, we investigated the feasibility of expressing the major K99 fimbrial subunit, FanC, in soybean (Glycine max) for use as an edible subunit vaccine. As a first step in this developmental process, a synthetic version of fanC was optimized for expression in the cytosol and transferred to soybean via Agrobacterium-mediated transformation. Western analysis of T(0) events revealed the presence of a peptide with the expected mobility for FanC in transgenic protein extracts, and immunofluorescense confirmed localization to the cytosol. Two T(0) lines, which accumulated FanC to levels near 0.5% of total soluble protein, were chosen for further molecular characterization in the T(1) and T(2) generations. Mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing synthetic FanC developed significant antibody titers against bacterially derived FanC and produced antigen-specific CD4(+) T lymphocytes, demonstrating the ability of transgenic FanC to function as an immunogen. These experiments are the first to demonstrate the expression and immunogenicity of a model subunit antigen in the soybean system, and mark the first steps toward the development of a K99 edible vaccine to protect against ETEC.

Considerations on the Optimal and Efficient Processing of Information-Bearing Signals

ERIC Educational Resources Information Center

Harms, Herbert Andrew

2013-01-01

Noise is a fundamental hurdle that impedes the processing of information-bearing signals, specifically the extraction of salient information. Processing that is both optimal and efficient is desired; optimality ensures the extracted information has the highest fidelity allowed by the noise, while efficiency ensures limited resource usage. Optimal…

LC-MS determination of steroidal glycosides from Dioscorea deltoidea Wall cell suspension culture: Optimization of pre-LC-MS procedure parameters by Latin Square design.

PubMed

Sarvin, Boris; Fedorova, Elizaveta; Shpigun, Oleg; Titova, Maria; Nikitin, Mikhail; Kochkin, Dmitry; Rodin, Igor; Stavrianidi, Andrey

2018-03-30

In this paper, the ultrasound assisted extraction method for isolation of steroidal glycosides from D. deltoidea plant cell suspension culture with a subsequent HPLC-MS determination was developed. After the organic solvent was selected via a two-factor experiment the optimization via Latin Square 4 × 4 experimental design was carried out for the following parameters: extraction time, organic solvent concentration in extraction solution and the ratio of solvent to sample. It was also shown that the ultrasound assisted extraction method is not suitable for isolation of steroidal glycosides from the D. deltoidea plant material. The results were double-checked using the multiple successive extraction method and refluxing extraction. Optimal conditions for the extraction of steroidal glycosides by the ultrasound assisted extraction method were: extraction time, 60 min; acetonitrile (water) concentration in extraction solution, 50%; the ratio of solvent to sample, 400 mL/g. Also, the developed method was tested on D. deltoidea cell suspension cultures of different terms and conditions of cultivation. The completeness of the extraction was confirmed using the multiple successive extraction method. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of isolation techniques on the characteristics of pigeon pea (Cajanus cajan) protein isolates.

PubMed

Adenekan, Monilola K; Fadimu, Gbemisola J; Odunmbaku, Lukumon A; Oke, Emmanuel K

2018-01-01

In this study, the effect of different isolation techniques on the isolated proteins from pigeon pea was investigated. Water, methanol, ammonium sulfate, and acetone were used for the precipitation of proteins from pigeon pea. Proximate composition, and antinutritional and functional properties of the pigeon pea flour and the isolated proteins were measured. Data generated were statistically analyzed. The proximate composition of the water-extracted protein isolate was moisture 8.30%, protein 91.83%, fat 0.25%, ash 0.05%, and crude fiber 0.05%. The methanol-extracted protein isolate composition was moisture 7.87%, protein 91.83%, fat 0.17%, and ash 0.13%, while crude fiber and carbohydrates were not detected. The composition of the ammonium sulfate-extracted protein isolate was moisture 7.73%, protein 91.73%, fat 0.36, ash 0.13%, and crude fiber 0.67%. The acetone-extracted protein isolate composition was moisture 8.03%, protein 91.50%, ash 0.67%, and fat 0.30%, but crude fiber and carbohydrates were not detected. The isolate precipitated with ammonium sulfate displayed the highest foaming capacity (37.63%) and foaming stability (55.75%). Isolates precipitated with methanol and acetone had the highest water absorption capacity (160%). Pigeon pea protein isolates extracted with methanol and ammonium sulfate had the highest oil absorption capacity of 145%. Protein isolates recovered through acetone and methanol had the highest emulsifying capacity of 2.23% and emulsifying stability of 91.47%, respectively. The proximate composition of the recovered protein isolates were of high purity. This shows the efficiency of the extraction techniques. The isolates had desirable solubility index. All the isolation techniques brought significant impact on the characteristics of the isolated pigeon pea protein.

Optimization extraction of polysaccharide from Tunisian Zizyphus lotus fruit by response surface methodology: Composition and antioxidant activity.

PubMed

Mkadmini Hammi, Khaoula; Hammami, Majdi; Rihouey, Christophe; Le Cerf, Didier; Ksouri, Riadh; Majdoub, Hatem

2016-12-01

Response surface methodology using a Box-Behnken design was employed to optimize extraction temperature, extraction time and ratio of water to material to obtain a maximum polysaccharide yield with high uronic acid content and antioxidant property from edible Zizyphus lotus fruit. The optimal conditions were: extraction time of 3h 15min, extraction temperature of 91.2°C and water to solid ratio of 39mL/g. Under these conditions, the experimental extraction yield, uronic acid content and 2,2-diphenyl-1-picrylhydrazyl scavenging ability (IC50) were 18.88%, 41.89 and 0.518mg/mL, respectively. Chemical analysis revealed that the extract was composed of 97.92% carbohydrate of which 41.89% is uronic acid. The extracted polysaccharides, with an average molecular weight of 2720kDa, are composed of arabinose, rhamnose, glucose, fructose, galactose and xylose. Moreover, the polysaccharides exhibited a significant reducing power and anti-lipid peroxidation activities. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Optimal extraction of effective constituents from Aralia elata by central composite design and response surface methodology].

PubMed

Lv, Shao-Wa; Liu, Dong; Hu, Pan-Pan; Ye, Xu-Yan; Xiao, Hong-Bin; Kuang, Hai-Xue

2010-03-01

To optimize the process of extracting effective constituents from Aralia elata by response surface methodology. The independent variables were ethanol concentration, reflux time and solvent fold, the dependent variable was extraction rate of total saponins in Aralia elata. Linear or no-linear mathematic models were used to estimate the relationship between independent and dependent variables. Response surface methodology was used to optimize the process of extraction. The prediction was carried out through comparing the observed and predicted values. Regression coefficient of binomial fitting complex model was as high as 0.9617, the optimum conditions of extraction process were 70% ethanol, 2.5 hours for reflux, 20-fold solvent and 3 times for extraction. The bias between observed and predicted values was -2.41%. It shows the optimum model is highly predictive.

Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes.

PubMed

Debaize, Lydie; Jakobczyk, Hélène; Rio, Anne-Gaëlle; Gandemer, Virginie; Troadec, Marie-Bérengère

2017-01-01

Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients' cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field.

Extraction of extracellular polymeric substances from aerobic granule with compact interior structure.

PubMed

Adav, Sunil S; Lee, Duu-Jong

2008-06-15

Extracellular polymeric substances (EPS) were extracted from aerobic granules of compact interior structure using seven extraction methods. Ultrasound followed by the chemical reagents formamide and NaOH outperformed other methods in extracting EPS from aerobic granules of compact interior. The collected EPS revealed no contamination by intracellular substances and consisted mainly of proteins, polysaccharides, humic substances and lipids. The quantity of extracted proteins exhibited a weak correlation with quantity of extracted carbohydrates but no correlation with quantity of extracted humic substances. The total polysaccharides/total proteins (PN/PS) ratios for sludge flocs were approximately 0.9 regardless of extraction method. Protein content was significantly enriched in the granules, producing a PN/PS ratio of 3.4-6.2. This experimental result correlated with observations using excitation-emission matrix (EEM) and confocal laser scanning microscope technique. However, detailed study disproved the use of EEM results as a quantitative index of extracted EPS from sludge flocs or from granules.

Effects of cholesterol depletion on membrane nanostructure in MCF-7 cells by atomic force microscopy

NASA Astrophysics Data System (ADS)

Wang, Yuhua; Jiang, Ningcheng; Shi, Aisi; Zheng, Liqin; Yang, Hongqin; Xie, Shusen

2017-02-01

The cell membrane is composed of phospholipids, glycolipids, cholesterol and proteins that are dynamic and heterogeneous distributed in the bilayer structure and many researches have showed that the plasma membrane in eukaryotic cells contains microdomains termed "lipid raft" in which cholesterol, sphingolipids and specific membrane proteins are enriched. Cholesterol extraction induced lipid raft disruption is one of the most widely used methods for lipid raft research and MβCD is a type of solvent to extract the cholesterol from cell membranes. In this study, the effect of MβCD treatment on the membrane nanostructure in MCF-7 living cells was investigated by atomic force microscopy. Different concentrations of MβCD were selected to deplete cholesterol for 30 min and the viability of cells was tested by MTT assay to obtain the optimal concentration. Then the nanostructure of the cell membrane was detected. The results show that an appropriate concentration of MβCD can induce the alteration of cell membranes nanostructure and the roughness of membrane surface decreases significantly. This may indicate that microdomains of the cell membrane disappear and the cell membrane appears more smoothly. Cholesterol can affect nanostructure and inhomogeneity of the plasma membrane in living cells.

Algal Proteins: Extraction, Application, and Challenges Concerning Production

PubMed Central

Bleakley, Stephen; Hayes, Maria

2017-01-01

Population growth combined with increasingly limited resources of arable land and fresh water has resulted in a need for alternative protein sources. Macroalgae (seaweed) and microalgae are examples of under-exploited “crops”. Algae do not compete with traditional food crops for space and resources. This review details the characteristics of commonly consumed algae, as well as their potential for use as a protein source based on their protein quality, amino acid composition, and digestibility. Protein extraction methods applied to algae to date, including enzymatic hydrolysis, physical processes, and chemical extraction and novel methods such as ultrasound-assisted extraction, pulsed electric field, and microwave-assisted extraction are discussed. Moreover, existing protein enrichment methods used in the dairy industry and the potential of these methods to generate high value ingredients from algae, such as bioactive peptides and functional ingredients are discussed. Applications of algae in human nutrition, animal feed, and aquaculture are examined. PMID:28445408

The effects of thermal treatments on protein profiles of Macrobrachium rosenbergii (giant river prawn)

NASA Astrophysics Data System (ADS)

Sockalingam, Komathi; Misnan, Rosmilah; Yadzir, Zailatul Hani Mohd

2017-05-01

Prawn allergy is certainly the most frequent cause of allergic reactions in countries where this crustacean is a popular dish of seafood. The aim of this study was to determine the protein profiles of giant river prawn which scientifically known as Macrobrachium rosenbergii. Raw and cooked extracts (boiled, steamed and fried) of prawn samples were prepared and then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 27 protein bands between 6 to 207 kDa were detected in the SDS-PAGE gel of raw extracts while boiled, steamed and fried extracts revealed fewer protein bands. Steamed and boiled prawns presented higher numbers of protein bands compared to fried prawn. A prominent heat-resistant band between 32 to 38 kDa was seen in all extracts, might hypothesized to be tropomyosin. Other prominent bands between 17 to 20 kDa were also seen in all treated prawn extracts while bands of 24 to 27 kDa were seen in steamed and boiled prawn extracts. These positions are consistent with the known shellfish allergens myosin light chain, sacroplasmic calcium binding protein and troponin C respectively. Several other heat-sensitive protein bands at various molecular weights were also not detected in boiled, steamed and fried extracts of this prawn. This study showed that M. rosenbergii contains numerous heat-sensitive and heat-resistant proteins, which may play an important role in prawn allergy.

Effect of Psidium cattleianum leaf extract on Streptococcus mutans viability, protein expression and acid production.

PubMed

Brighenti, F L; Luppens, S B I; Delbem, A C B; Deng, D M; Hoogenkamp, M A; Gaetti-Jardim, E; Dekker, H L; Crielaard, W; ten Cate, J M

2008-01-01

Plants naturally produce secondary metabolites that can be used as antimicrobials. The aim of this study was to assess the effects of Psidium cattleianum leaf extract on Streptococcus mutans. The extract (100%) was obtained by decoction of 100 g of leaves in 600 ml of deionized water. To assess killing, S. mutans biofilms were treated with water (negative control) or various extract dilutions [100, 50, 25% (v/v) in water] for 5 or 60 min. To evaluate the effect on protein expression, biofilms were exposed to water or 1.6% (v/v) extract for 120 min, proteins were extracted and submitted to 2-dimensional difference gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry. The effect of 1.6% (v/v) extract on acid production was determined by pH measurements and compared to a water control. Viability was similar after 5 min of treatment with the 100% extract or 60 min with the 50% extract (about 0.03% survival). There were no differences in viability between the biofilms exposed to the 25 or 50% extract after 60 min of treatment (about 0.02% survival). Treatment with the 1.6% extract significantly changed protein expression. The abundance of 24 spots was decreased compared to water (p < 0.05). The extract significantly inhibited acid production (p < 0.05). It is concluded that P. cattleianum leaf extract kills S. mutans grown in biofilms when applied at high concentrations. At low concentrations it inhibits S. mutans acid production and reduces the expression of proteins involved in general metabolism, glycolysis and lactic acid production. (c) 2008 S. Karger AG, Basel

Comparison of salting-out and sugaring-out liquid-liquid extraction methods for the partition of 10-hydroxy-2-decenoic acid in royal jelly and their co-extracted protein content.

PubMed

Tu, Xijuan; Sun, Fanyi; Wu, Siyuan; Liu, Weiyi; Gao, Zhaosheng; Huang, Shaokang; Chen, Wenbin

2018-01-15

Homogeneous liquid-liquid extraction (h-LLE) has been receiving considerable attention as a sample preparation method due to its simple and fast partition of compounds with a wide range of polarities. To better understand the differences between the two h-LLE extraction approaches, salting-out assisted liquid-liquid extraction (SALLE) and sugaring-out assisted liquid-liquid extraction (SULLE), have been compared for the partition of 10-hydroxy-2-decenoic acid (10-HDA) from royal jelly, and for the co-extraction of proteins. Effects of the amount of phase partition agents and the concentration of acetonitrile (ACN) on the h-LLE were discussed. Results showed that partition efficiency of 10-HDA depends on the phase ratio in both SALLE and SULLE. Though the partition triggered by NaCl and glucose is less efficient than MgSO 4 in the 50% (v/v) ACN-water mixture, their extraction yields can be improved to be similar with that in MgSO 4 SALLE by increasing the initial concentration of ACN in the ACN-water mixture. The content of co-extracted protein was correlated with water concentration in the obtained upper phase. MgSO 4 showed the largest protein co-extraction at the low concentration of salt. Glucose exhibited a large protein co-extraction in the high phase ratio condition. Furthermore, NaCl with high initial ACN concentration is recommended because it produced high extraction yield for 10-HDA and the lowest amount of co-extracted protein. These observations would be valuable for the sample preparation of royal jelly. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction

PubMed Central

Mohammadi, Samira; Esfahani, Bahram Nasr; Moghim, Sharareh; Mirhendi, Hossein; Zaniani, Fatemeh Riyahi; Safaei, Hajieh Ghasemian; Fazeli, Hossein; Salehi, Mahshid

2017-01-01

Background: Nontuberculous mycobacteria (NTM) are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. Materials and Methods: The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR) amplification of the heat-shock protein 65 gene with serially diluted DNA samples. Results: The CTAB method showed more positive results at 1:10–1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. Conclusions: According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor. PMID:29279831

Enhancement of yield, nutritional and nutraceutical properties of two common bean cultivars following the application of seaweed extract (Ecklonia maxima).

PubMed

Kocira, Anna; Świeca, Michał; Kocira, Sławomir; Złotek, Urszula; Jakubczyk, Anna

2018-03-01

In the present study, application of Ecklonia maxima extract (Kelpak SL - a water soluble concentrate) was optimized and its impact on yield, nutraceutical and nutritional potential of Phaseolus vulgaris L. (var. Aura and Toska) was measured. The study was carried out in 2012 and 2013 in Poland. During the growing season, 0.2% and 0.4% solution of Kelpak SL was applied by single and double spraying of plants. These four treatments with Kelpak SL were compared with the control, where no biostimulator was applied. Kelpak SL treatments stimulated the yield of both cultivars studied. The application of E. maxima extract had no effect on the content of starch, free sugars or proteins in seeds of either of the tested cultivars. The highest level of phenolics was found for double sprayed Toska plants. All the tested variants of Kelpak SL application significantly increased the content of anthocyanins in the seeds. Also, both the reducing power and antiradical ability of Aura seeds were elevated in all the studied treatments. E. maxima extract is a natural, environmentally friendly and safe preparation increasing the yield and nutraceutical quality of beans without any negative effect on their nutritional quality.

Extraction Optimization for Obtaining Artemisia capillaris Extract with High Anti-Inflammatory Activity in RAW 264.7 Macrophage Cells

PubMed Central

Jang, Mi; Jeong, Seung-Weon; Kim, Bum-Keun; Kim, Jong-Chan

2015-01-01

Plant extracts have been used as herbal medicines to treat a wide variety of human diseases. We used response surface methodology (RSM) to optimize the Artemisia capillaris Thunb. extraction parameters (extraction temperature, extraction time, and ethanol concentration) for obtaining an extract with high anti-inflammatory activity at the cellular level. The optimum ranges for the extraction parameters were predicted by superimposing 4-dimensional response surface plots of the lipopolysaccharide- (LPS-) induced PGE2 and NO production and by cytotoxicity of A. capillaris Thunb. extracts. The ranges of extraction conditions used for determining the optimal conditions were extraction temperatures of 57–65°C, ethanol concentrations of 45–57%, and extraction times of 5.5–6.8 h. On the basis of the results, a model with a central composite design was considered to be accurate and reliable for predicting the anti-inflammation activity of extracts at the cellular level. These approaches can provide a logical starting point for developing novel anti-inflammatory substances from natural products and will be helpful for the full utilization of A. capillaris Thunb. The crude extract obtained can be used in some A. capillaris Thunb.-related health care products. PMID:26075271

Response Surface Methodology Optimization of Ultrasonic-Assisted Extraction of Acer Truncatum Leaves for Maximal Phenolic Yield and Antioxidant Activity.

PubMed

Yang, Lingguang; Yin, Peipei; Fan, Hang; Xue, Qiang; Li, Ke; Li, Xiang; Sun, Liwei; Liu, Yujun

2017-02-04

This study is the first to report the use of response surface methodology to improve phenolic yield and antioxidant activity of Acer truncatum leaves extracts (ATLs) obtained by ultrasonic-assisted extraction. The phenolic composition in ATLs extracted under the optimized conditions were characterized by UPLC-QTOF-MS/MS. Solvent and extraction time were selected based on preliminary experiments, and a four-factors-three-levels central composite design was conducted to optimize solvent concentration ( X ₁), material-to-liquid ratio ( X ₂), ultrasonic temperature ( X ₃) and power ( X ₄) for an optimal total phenol yield ( Y ₁) and DPPH• antioxidant activity ( Y ₂). The results showed that the optimal combination was ethanol:water ( v : v ) 66.21%, material-to-liquid ratio 1:15.31 g/mL, ultrasonic bath temperature 60 °C, power 267.30 W, and time 30 min with three extractions, giving a maximal total phenol yield of 7593.62 mg gallic acid equivalent/100 g d.w. and a maximal DPPH• antioxidant activity of 74,241.61 μmol Trolox equivalent/100 g d.w. Furthermore, 22 phenolics were first identified in ATL extract obtained under the optimized conditions, indicating that gallates, gallotannins, quercetin, myricetin and chlorogenic acid derivatives were the main phenolic components in ATL. What's more, a gallotannins pathway existing in ATL from gallic acid to penta- O -galloylglucoside was proposed. All these results provide practical information aiming at full utilization of phenolics in ATL, together with fundamental knowledge for further research.

Intracellular coagulation inhibits the extraction of proteins from Prochloron

NASA Technical Reports Server (NTRS)

Fall, R.; Lewin, R. A.; Fall, L. R.

1983-01-01

Protein extraction from the prokaryotic alga Prochloron LP (isolated from the ascidian host Lissoclinum patella) was complicated by an irreversible loss of cell fragility in the isolated algae. Accompanying this phenomenon, which is termed intracellular coagulation, was a redistribution of thylakoids around the cell periphery, a loss of photosynthetic O2 production, and a drastic decrease in the extractability of cell proteins. Procedures are described for the successful preparation and transport of cell extracts yielding the enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase as well as other soluble proteins.

Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements.

PubMed

Qian, Chen; Hettich, Robert L

2017-07-07

The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient cycling. Liquid chromatography coupled to high-performance mass spectrometry provides a powerful approach to characterize soil microbiomes; however, the limited microbial biomass and the presence of abundant interferences in soil samples present major challenges to proteome extraction and subsequent MS measurement. To this end, we have designed an experimental method to improve microbial proteome measurement by removing the soil-borne humic substances coextraction from soils. Our approach employs an in situ detergent-based microbial lysis/TCA precipitation coupled to an additional cleanup step involving acidified precipitation and filtering at the peptide level to remove most of the humic acid interferences prior to proteolytic peptide measurement. The novelty of this approach is an integration to exploit two different characteristics of humic acids: (1) Humic acids are insoluble in acidic solution but should not be removed at the protein level, as undesirable protein removal may also occur. Rather it is better to leave the humics acids in the samples until the peptide level, at which point the significant differential solubility of humic acids versus peptides at low pH can be exploited very efficiently. (2) Most of the humic acids have larger molecule weights than the peptides. Therefore, filtering a pH 2 to 3 peptide solution with a 10 kDa filter will remove most of the humic acids. This method is easily interfaced with normal proteolytic processing approaches and provides a reliable and straightforward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or biasing protein identification in mass spectrometry. In general, this humic acid removal step is universal and can be adopted by any workflow to effectively remove humic acids to avoid them negatively competing with peptides for binding with reversed-phase resin or ionization in the electrospray.

Nutrient-Specific Foraging in Invertebrate Predators

NASA Astrophysics Data System (ADS)

Mayntz, David; Raubenheimer, David; Salomon, Mor; Toft, Søren; Simpson, Stephen J.

2005-01-01

Many herbivores and omnivores adjust their food selection behavior to regulate the intake of multiple nutrients. Carnivores, however, are generally assumed to optimize the rate of prey capture rather than select prey according to nutrient composition. We showed experimentally that invertebrate predators can forage selectively for protein and lipids to redress specific nutritional imbalances. This selection can take place at different stages of prey handling: The predator may select among foods of different nutritional composition, eat more of a prey if it is rich in nutrients that the predator is deficient in, or extract specific nutrients from a single prey item.

Literature mining of protein-residue associations with graph rules learned through distant supervision.

PubMed

Ravikumar, Ke; Liu, Haibin; Cohn, Judith D; Wall, Michael E; Verspoor, Karin

2012-10-05

We propose a method for automatic extraction of protein-specific residue mentions from the biomedical literature. The method searches text for mentions of amino acids at specific sequence positions and attempts to correctly associate each mention with a protein also named in the text. The methods presented in this work will enable improved protein functional site extraction from articles, ultimately supporting protein function prediction. Our method made use of linguistic patterns for identifying the amino acid residue mentions in text. Further, we applied an automated graph-based method to learn syntactic patterns corresponding to protein-residue pairs mentioned in the text. We finally present an approach to automated construction of relevant training and test data using the distant supervision model. The performance of the method was assessed by extracting protein-residue relations from a new automatically generated test set of sentences containing high confidence examples found using distant supervision. It achieved a F-measure of 0.84 on automatically created silver corpus and 0.79 on a manually annotated gold data set for this task, outperforming previous methods. The primary contributions of this work are to (1) demonstrate the effectiveness of distant supervision for automatic creation of training data for protein-residue relation extraction, substantially reducing the effort and time involved in manual annotation of a data set and (2) show that the graph-based relation extraction approach we used generalizes well to the problem of protein-residue association extraction. This work paves the way towards effective extraction of protein functional residues from the literature.

Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.

PubMed

Bertolde, F Z; Almeida, A-A F; Silva, F A C; Oliveira, T M; Pirovani, C P

2014-07-04

Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

Development of green betaine-based deep eutectic solvent aqueous two-phase system for the extraction of protein.

PubMed

Li, Na; Wang, Yuzhi; Xu, Kaijia; Huang, Yanhua; Wen, Qian; Ding, Xueqin

2016-05-15

Six kinds of new type of green betaine-based deep eutectic solvents (DESs) have been synthesized. Deep eutectic solvent aqueous two-phase systems (DES-ATPS) were established and successfully applied in the extraction of protein. Betaine-urea (Be-U) was selected as the suitable extractant. Single factor experiments were carried out to determine the optimum conditions of the extraction process, such as the salt concentration, the mass of DES, the separation time, the amount of protein, the temperature and the pH value. The extraction efficiency could achieve to 99.82% under the optimum conditions. Mixed sample and practical sample analysis were discussed. The back extraction experiment was implemented and the back extraction efficiency could reach to 32.66%. The precision experiment, repeatability experiment and stability experiment were investigated. UV-vis, FT-IR and circular dichroism (CD) spectra confirmed that the conformation of protein was not changed during the process of extraction. The mechanisms of extraction were researched by dynamic light scattering (DLS), the measurement of the conductivity and transmission electron microscopy (TEM). DES-protein aggregates and embraces phenomenon play considerable roles in the separation process. All of these results indicated that betaine-based DES-ATPS may provide a potential substitute new method for the separation of proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

Ligand Extraction Properties of the GM2 Activator Protein and Its Interactions with Lipid Vesicles

PubMed Central

Ran, Yong; Fanucci, Gail E.

2009-01-01

Abstract The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles. PMID:19580763

Ligand extraction properties of the GM2 activator protein and its interactions with lipid vesicles.

PubMed

Ran, Yong; Fanucci, Gail E

2009-07-08

The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles.

Optimization of physicochemical and textural properties of pizza cheese fortified with soybean oil and carrot extract.

PubMed

Motevalizadeh, Ehsan; Mortazavi, Seyed Ali; Milani, Elnaz; Hooshmand-Dalir, Moosa Al-Reza

2018-03-01

Response surface methodology (RSM) was used to optimize pizza cheese containing carrot extract. The effects of two important independent variables including soybean oil (5%-20%) and carrot extract (5%-20%) were studied on physicochemical and textural properties of pizza cheese containing carrot extract. According to the results, RSM was successfully used for optimizing formulation of pizza cheese containing carrot juice. Results of this study revealed that oil (A), carrot (B), AB, square term of carrot (B 2 ), B, AB, square term of oil (A 2 ), B 2 , AB, AB, A 2 B, A 2 , A 2 , A, A 2 , A 2 , AB, and AB 2 had the most effect on moisture, acidity, stretch, L*, a*, b*, hardness, meltability, springiness, peroxide value (PV), cohesiveness, chewiness, gumminess, fracture force, adhesiveness force, stiffness, flavor, and overall acceptability, respectively. A formulation upon 20% oil and 10.88% carrot extract was found as the optimal formulation for pizza cheese containing carrot extract. At the optimal formulation, PV, L*, a*, b*, meltability, stretch, cohesiveness, springiness, gumminess, chewiness, adhesive force, flavor, texture, and overall acceptability at the optimum formulation were measured 2.23, 82.51, -3.69, 18.05, 17.86, 85.61, 0.41, 7.874, 23.7, 0.27, 0.61, 3.50, 3.95, and 3.65, respectively.

Evaluation and optimization of microbial DNA extraction from fecal samples of wild Antarctic bird species

PubMed Central

Eriksson, Per; Mourkas, Evangelos; González-Acuna, Daniel; Olsen, Björn; Ellström, Patrik

2017-01-01

ABSTRACT Introduction: Advances in the development of nucleic acid-based methods have dramatically facilitated studies of host–microbial interactions. Fecal DNA analysis can provide information about the host’s microbiota and gastrointestinal pathogen burden. Numerous studies have been conducted in mammals, yet birds are less well studied. Avian fecal DNA extraction has proved challenging, partly due to the mixture of fecal and urinary excretions and the deficiency of optimized protocols. This study presents an evaluation of the performance in avian fecal DNA extraction of six commercial kits from different bird species, focusing on penguins. Material and methods: Six DNA extraction kits were first tested according to the manufacturers’ instructions using mallard feces. The kit giving the highest DNA yield was selected for further optimization and evaluation using Antarctic bird feces. Results: Penguin feces constitute a challenging sample type: most of the DNA extraction kits failed to yield acceptable amounts of DNA. The QIAamp cador Pathogen kit (Qiagen) performed the best in the initial investigation. Further optimization of the protocol resulted in good yields of high-quality DNA from seven bird species of different avian orders. Conclusion: This study presents an optimized approach to DNA extraction from challenging avian fecal samples. PMID:29152162

Batch and Continuous Ultrasound Assisted Extraction of Boldo Leaves (Peumus boldus Mol.).

PubMed

Petigny, Loïc; Périno-Issartier, Sandrine; Wajsman, Joël; Chemat, Farid

2013-03-12

Vegetal extracts are widely used as primary ingredients for various products from creams to perfumes in the pharmaceutical, nutraceutic and cosmetic industries. Having concentrated and active extract is essential, as the process must extract as much soluble material as possible in a minimum time, using the least possible volume of solvent. The boldo leaves extract is of great interest for the industry as it holds a great anti-oxidant activity due to high levels of flavonoids and alkaloids such as boldine. Ultrasound Assisted Extraction (UAE) has been used to improve the efficiency of the plant extraction, reducing extraction time, increasing the concentration of the extract with the same amount of solvent and plant material. After a preliminary study, a response surface method has been used to optimize the extraction of soluble material from the plant. The results provided by the statistical analysis revealed that the optimized conditions were: sonication power 23 W/cm2 for 40 min and a temperature of 36 °C. The optimized parameters of the UAE provide a better extraction compared to a conventional maceration in terms of process time (30 min instead of 120 min), higher yield, more energy saving, cleanliness, safety and product quality.

Batch and Continuous Ultrasound Assisted Extraction of Boldo Leaves (Peumus boldus Mol.)

PubMed Central

Petigny, Loïc; Périno-Issartier, Sandrine; Wajsman, Joël; Chemat, Farid

2013-01-01

Vegetal extracts are widely used as primary ingredients for various products from creams to perfumes in the pharmaceutical, nutraceutic and cosmetic industries. Having concentrated and active extract is essential, as the process must extract as much soluble material as possible in a minimum time, using the least possible volume of solvent. The boldo leaves extract is of great interest for the industry as it holds a great anti-oxidant activity due to high levels of flavonoids and alkaloids such as boldine. Ultrasound Assisted Extraction (UAE) has been used to improve the efficiency of the plant extraction, reducing extraction time, increasing the concentration of the extract with the same amount of solvent and plant material. After a preliminary study, a response surface method has been used to optimize the extraction of soluble material from the plant. The results provided by the statistical analysis revealed that the optimized conditions were: sonication power 23 W/cm2 for 40 min and a temperature of 36 °C. The optimized parameters of the UAE provide a better extraction compared to a conventional maceration in terms of process time (30 min instead of 120 min), higher yield, more energy saving, cleanliness, safety and product quality. PMID:23481637

Electrochemical detection of cardiac biomarker myoglobin using polyphenol as imprinted polymer receptor.

PubMed

Ribeiro, J A; Pereira, C M; Silva, A F; Sales, M Goreti F

2017-08-15

An electrochemical biosensor was developed by merging the features of Molecular Imprinting technique and Screen-Printed Electrode (SPE) for the simple and fast screening of cardiac biomarker myoglobin (Myo) in point-of-care (POC). The MIP artificial receptor for Myo was prepared by electrooxidative polymerization of phenol (Ph) on a AuSPE in the presence of Myo as template molecule. The choice of the most effective protein extraction procedure from the various extraction methods tested (mildly acidic/basic solutions, pure/mixed organic solvents, solutions containing surfactants and enzymatic digestion methods), and the optimization of the thickness of the polymer film was carefully undertaken in order to improve binding characteristics of Myo to the imprinted polymer receptor and increase the sensitivity of the MIP biosensor. The film thickness was optimized by adjusting scan rate and the number of cycles during cyclic voltammetric electropolymerization of Ph. The thickness of the polyphenol nanocoating of only few nanometres (∼4.4 nm), and similar to the protein diameter, brought in significant improvements in terms of sensor sensitivity. The binding affinity of MIP receptor film was estimated by fitting the experimental data to Freundlich isotherm and a ∼8 fold increase in the binding affinity of Myo to the imprinted polymer (K F = 0.119 ± 0.002 ng -1  mL) when compared to the non-imprinted polymer (K F  = 0.015 ± 0.002 ng -1  mL) which demonstrated excellent (re)binding affinity for the imprinted protein. The incubation of the Myo MIP receptor modified electrode with increasing concentration of protein (from 0.001 ng mL -1 to 100 μg mL -1 ) resulted in a decrease of the ferro/ferricyanide redox current. LODs of 2.1 and 14 pg mL -1 were obtained from calibration curves built in neutral buffer and diluted artificial serum, respectively, using SWV technique, enabling the detection of the protein biomarker at clinically relevant levels. The prepared MIP biosensor was applied to the determination of Myo spiked serum samples with satisfactory results. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karlstad, M.D.; DeMichele, S.J.; Istfan, N.

The effects of burn and first-pass splanchnic leucine extraction (FPE) on protein kinetics and energy expenditure were assessed by measuring O/sub 2/ consumption, CO/sub 2/ production, nitrogen balance, leucine kinetics, and tissue fractional protein synthetic rates (FSR-%/day) in enterally fed rats. Anesthetized male rats (200 g) were scalded on their dorsum with boiling water (25-30% body surface area) and enterally fed isovolemic diets that provided 60 kcal/day and 2.4 g of amino acids/day for 3 days. Controls were not burned. An intravenous or intragastric infusion of L-(1-/sup 14/C)leucine was used to assess protein kinetics on day 3. FPE was takenmore » as the ratio of intragastric to intravenous plasma leucine specific activity. There was a 69% reduction in cumulative nitrogen balance (P less than 0.001) and a 17-19% increase in leucine oxidation (P less than 0.05) and total energy expenditure (P less than 0.01) in burned rats. A 15% decrease in plasma leucine clearance (P less than 0.05) was accompanied by a 20% increase in plasma (leucine) (P less than 0.01) in burned rats. Burn decreased rectus muscle FSR from 5.0 +/- 0.4 to 3.5 +/- 0.5 (P less than 0.05) and increased liver FSR from 19.0 +/- 0.5 to 39.2 +/- 3.4 (P less than 0.01). First pass extraction of dietary leucine by the splanchnic bed was 8% in controls and 26% in burned rats. Leucine kinetics corrected for FPE showed increased protein degradation with burn that was not evident without FPE correction. This hypermetabolic burn model can be useful in the design of enteral diets that optimize rates of protein synthesis and degradation.« less

Effects of surfactant and salt species in reverse micellar forward extraction efficiency of isoflavones with enriched protein from soy flour.

PubMed

Zhao, Xiaoyan; Wei, Zhiyi; Du, Fangling; Zhu, Junqing

2010-11-01

Suitability of reverse micelles of anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT) and sodium dodecyl sulfate (SDS), cationic surfactant hexadecyl trimethyl ammonium bromide (CTAB) and nonionic surfactant polyoxyethylene p-t-octylphenol (TritonX-100) in organic solvent isooctane for extraction of soy isoflavone-enriching proteins was investigated. The results showed that the order of combined isoflavone contents was SDS>CTAB>Triton X-100>AOT, while the order of protein recovery was SDS>AOT>TritonX-100>CTAB. As compared with ACN-HCl extraction, the total amount of isoflavones was lower than reverse micellar extraction. Ion strength was one of the important conditions to control extraction of isoflavone-enriching proteins with AOT reversed micelles. For the six salt systems, KNO(3), KCl, MgCl(2), CaCl(2), NaCl, and Na(2)SO(4), extracted fraction of isoflavone-enriching proteins was measured. Salt solutions greatly influenced the extraction efficiency of isoflavones in an order of KNO(3)>MgCl(2)>CaCl(2)>KCl>NaCl>Na(2)SO(4), while protein in an order of MgCl(2)>CaCl(2)>NaCl>KNO(3)>Na(2)SO(4)>KCl.

Fed batch fermentation and purification strategy for high yield production of Brucella melitensis recombinant Omp 28 kDa protein and its application in disease diagnosis.

PubMed

Karothia, B S; Athmaram, T N; D, Thavaselvam; Ashu, Kumar; Tiwari, Sapna; Singh, Anil K; Sathyaseelan, K; Gopalan, N

2013-07-01

Brucellosis is a disease caused by bacteria belonging to the genus Brucella. It affects cattle, goat, sheep, dog and humans. The serodiagnosis of brucellosis involves detection of antibodies generated against the LPS or whole cell bacterial extracts, however these tests lack sensitivity and specificity. The present study was performed to optimize the culture condition for the production of recombinant Brucella melitensis outer membrane protein 28 kDa protein in E.coli via fed batch fermentation. Expression was induced with 1.5mM isopropyl β thiogalactoside and the expressed recombinant protein was purified using Ni-NTA affinity chromatography. After fed-batch fermentation the dry cell weight of 17.81 g/L and a purified protein yield of 210.10 mg/L was obtained. The purified Brucella melitensis recombinant Omp 28 kDa protein was analyzed through SDS- poly acrylamide gel electrophoresis and western blotting. The obtained recombinant protein was evaluated for its diagnostic application through Indirect ELISA using brucellosis suspected human sera samples. Our results clearly indicate that recombinant Omp28 produced via fed batch fermentation has immense potential as a diagnostic reagent that could be employed in sero monitoring of brucellosis.

Preparation and reactions of an iodinated imidoester reagent with actin and alpha-actinin.

PubMed

Bright, G R; Spooner, B S

1983-06-01

The chemical iodination of an imidoester (methyl-p-hydroxybenzimidate, Wood et al. (1975) Anal. Biochem. 68, 339) and subsequent coupling of iodinated imidoester (IIE) to protein is an indirect method of iodinating proteins that is specific for the epsilon amino group of lysine residues and maintains the positive charge on the amino group at physiological pH. Purification of the IIE from chloramine-T and free iodine by benzene extraction eliminates the need for isoelectric precipitation and produces a more time- and cost-efficient IIE preparation and purification protocol. The separation of free from protein-bound label by chromatography, using centrifugal elution, provides a separation method that is rapid and efficient, without the generation of large volumes of radioactive wastes characteristic of conventional chromatographic and dialysis methods. To optimize the parameters of labeling protein with IIE, a systematic assessment of the effects of pH, reactant concentrations, and reaction time was made using purified cardiac actin and gizzard alpha-actinin. The parameters were defined to achieve an average labeling ratio of one IIE per protein polypeptide. The data demonstrate that both proteins appear to be labeled at the same rate and define several determining factors that limit the rate and extent of IIE incorporation into protein.

Correction of protein metabolic disorders by composite extract of Musa paradisiaca and Coccinia indica in streptozotocin-induced diabetic albino rat: an approach through the pancreas.

PubMed

Mallick, Chhanda; De, Debasis; Ghosh, Debidas

2009-04-01

The study focused on the ability of the extracts of Musa paradisiaca and Coccinia indica on protein metabolic disorders in streptozotocin (STZ)-induced diabetes. Wistar strain rats were divided into 6 groups as control, control + composite extract treated, STZ-induced diabetes, diabetic + composite extract treated, composite extract-pretreated diabetes, and composite extract-pretreated diabetes + composite extract treated. Protein metabolic status was assessed by serum levels of urea, uric acid, albumin, and creatinine along with urine urea and albumin levels. Diabetic therapeutic ability was assessed by blood glucose, glycated hemoglobin, and serum insulin levels. Histology of the pancreas, liver, and kidney was evaluated. Indices of protein metabolic disorders were deviated from control in STZ-induced diabetes, which were protected significantly after the treatment of composite extract of M. paradisiaca and C. indica. This protection was more prominent when the extract-pretreated animals were subjected to diabetes induction by STZ. The composite extract has a protective therapeutic effect against diabetes through beta-cell regeneration capacity.

Salting-out extraction of allicin from garlic (Allium sativum L.) based on ethanol/ammonium sulfate in laboratory and pilot scale.

PubMed

Li, Fenfang; Li, Qiao; Wu, Shuanggen; Tan, Zhijian

2017-02-15

Salting-out extraction (SOE) based on lower molecular organic solvent and inorganic salt was considered as a good substitute for conventional polymers aqueous two-phase extraction (ATPE) used for the extraction of some bioactive compounds from natural plants resources. In this study, the ethanol/ammonium sulfate was screened as the optimal SOE system for the extraction and preliminary purification of allicin from garlic. Response surface methodology (RSM) was developed to optimize the major conditions. The maximum extraction efficiency of 94.17% was obtained at the optimized conditions for routine use: 23% (w/w) ethanol concentration and 24% (w/w) salt concentration, 31g/L loaded sample at 25°C with pH being not adjusted. The extraction efficiency had no obvious decrease after amplification of the extraction. This ethanol/ammonium sulfate SOE is much simpler, cheaper, and effective, which has the potentiality of scale-up production for the extraction and purification of other compounds from plant resources. Copyright © 2016 Elsevier Ltd. All rights reserved.

The optimization of phenolic compounds extraction from cactus pear (Opuntia ficus-indica) skin in a reflux system using response surface methodology.

PubMed

Jorge, Aguirre Joya; Heliodoro, De La Garza Toledo; Alejandro, Zugasti Cruz; Ruth, Belmares Cerda; Noé, Aguilar Cristóbal

2013-06-01

To extract, quantify, and evaluate the phenolic content in Opuntia ficus-indica skin for their antioxidant capacity with three different methods (ABTS, DPPH, and lipid oxidation) and to optimize the extraction conditions (time, temperature and ethanol concentration) in a reflux system. The extraction process was done using a reflux system. A San Cristobal II experimental design with three variables and three levels was used. The variables evaluated were time of extraction (h), concentration of ethanol (%, v/v) and temperature (°C). The extraction process was optimized using a response surface methodology. It was observed that at higher temperature more phenolic compounds were extracted, but the antioxidant capacity was decreased. The optimum conditions for phenolic compounds extraction and antioxidant capacity mixing the three methods were as follows: 45% of ethanol, 80 °C and 2 hours of extraction. Values obtained in our results are little higher that other previously reported. It can be concluded the by-products of Opuntia ficus-indica represent a good source of natural antioxidants with possible applications in food, cosmetics or drugs industries.

Optimization of supercritical fluid extraction and HPLC identification of wedelolactone from Wedelia calendulacea by orthogonal array design.

PubMed

Patil, Ajit A; Sachin, Bhusari S; Wakte, Pravin S; Shinde, Devanand B

2014-11-01

The purpose of this work is to provide a complete study of the influence of operational parameters of the supercritical carbon dioxide assisted extraction (SC CO2E) on yield of wedelolactone from Wedelia calendulacea Less., and to find an optimal combination of factors that maximize the wedelolactone yield. In order to determine the optimal combination of the four factors viz. operating pressure, temperature, modifier concentration and extraction time, a Taguchi experimental design approach was used: four variables (three levels) in L9 orthogonal array. Wedelolactone content was determined using validated HPLC methodology. Optimum extraction conditions were found to be as follows: extraction pressure, 25 MPa; temperature, 40 °C; modifier concentration, 10% and extraction time, 90 min. Optimum extraction conditions demonstrated wedelolactone yield of 8.01 ± 0.34 mg/100 g W. calendulacea Less. Pressure, temperature and time showed significant (p < 0.05) effect on the wedelolactone yield. The supercritical carbon dioxide extraction showed higher selectivity than the conventional Soxhlet assisted extraction method.

Optimization of supercritical fluid extraction and HPLC identification of wedelolactone from Wedelia calendulacea by orthogonal array design

PubMed Central

Patil, Ajit A.; Sachin, Bhusari S.; Wakte, Pravin S.; Shinde, Devanand B.

2013-01-01

The purpose of this work is to provide a complete study of the influence of operational parameters of the supercritical carbon dioxide assisted extraction (SC CO2E) on yield of wedelolactone from Wedelia calendulacea Less., and to find an optimal combination of factors that maximize the wedelolactone yield. In order to determine the optimal combination of the four factors viz. operating pressure, temperature, modifier concentration and extraction time, a Taguchi experimental design approach was used: four variables (three levels) in L9 orthogonal array. Wedelolactone content was determined using validated HPLC methodology. Optimum extraction conditions were found to be as follows: extraction pressure, 25 MPa; temperature, 40 °C; modifier concentration, 10% and extraction time, 90 min. Optimum extraction conditions demonstrated wedelolactone yield of 8.01 ± 0.34 mg/100 g W. calendulacea Less. Pressure, temperature and time showed significant (p < 0.05) effect on the wedelolactone yield. The supercritical carbon dioxide extraction showed higher selectivity than the conventional Soxhlet assisted extraction method. PMID:25687584

Validation of a pressurized solvent extraction and GC-NCI-MS method for the low level determination of 40 polybrominated diphenyl ethers in mothers' milk.

PubMed

Lacorte, Silvia; Guillamon, Míriam

2008-08-01

This study reports an efficient method for the determination of 40 PBDEs from mono- to hepta-brominated in human milk. Pressurized liquid extraction (PLE) was optimized to recover PBDEs in a quantitative way using 1g of freeze dried milk. Due to the great amount of coextracted compounds, the clean-up step was optimized using alumina SPE disposable cartridges of 2 and 5 g. Whereas 2g alumina SPE failed in providing good extraction yields, all PBDEs were satisfactorily recovered using 5 g alumina SPE cartridges. Detection was performed with gas chromatography coupled to mass spectrometry with negative chemical ionization and method detection limits ranged from 0.01 to 0.05 microg kg(-1) wet weight (ww) with a good intra and inter-day variation (coefficient of variation lower than 13.4%). This method was validated by participating in an interlaboratory exercise from Quasimeme (United Kingdom), where a standard solution containing seven congeners and a certified unspiked mothers' milk were analyzed. In the standard mixture, levels between 96.915 and 570.172 microg l(-1) were quantified, whereas in certified mothers' milk, BDEs 47, 99, 100 and 154 were detected at levels from 0.010 and 0.061 microg kg(-1) ww, with an error <30%. The applicability of the method was tested experimentally in five mothers' milk samples, where only BDE 47 was detected at a maximum concentration of 10.45 microg kg(-1) lipid weight (lw). PLE succeeded in extracting all PBDEs from the sample with good reproducibility although the clean-up step had to be severely performed to eliminate sample interferences such as milk lipids and proteins.

Molecular tools for the identification of Tuber melanosporum in agroindustry.

PubMed

Séjalon-Delmas, N; Roux, C; Martins, M; Kulifaj, M; Bécard, G; Dargent, R

2000-06-01

Tuber melanosporum Vitt., Tuber magnatum Pico, and Tuber uncinatum Chat. can be differentiated by their morphological characters. Fraud problems have arisen recently with the importation to Europe of truffles from China. T. melanosporum is morphologically very close, but distinct from the Chinese species [Tuber indicum (Cooke and Massee) and T. himalayense BC (Zhang and Winter)]. We have optimized molecular tools to unequivocally identify T. melanosporum. DNA extraction from ascocarps of black truffles is not straightforward. Problems to obtain pure DNA are due to high contents of phenolic compounds, melanine, and various polymers (proteins, polysaccharides, etc). These compounds coprecipitate with the DNA during extraction and strongly inhibit the PCR reaction. We have developed an efficient and reliable protocol for DNA extraction from truffle ascocarps. It was used successfully for DNA extraction from mycorrhizal root tips as well as from canned preparations of T. melanosporum. Several approaches to identify T. melanosporum by PCR were developed. Two specific primers for T. melanosporum were designed after comparison of the ITS region of this species with those of three Chinese fungi. They proved to be efficient to specifically detect the presence of T. melanosporum by PCR. The mycorrhizal status of trees inoculated with T. melanosporum but unable to produce truffles was confirmed in a single-step PCR reaction. A multiplex PCR approach was also developed with three sets of primers (including a specific one for Chinese truffles) to detect, in one PCR reaction, the presence of any other Tuber species mixed with T. melanosporum ascocarps. This optimized protocol, in association with the specific primers we designed, is applicable to quality control in the truffle industry from the production stages to final commercial products.

Comparative study of two protocols for quantitative image-analysis of serotonin transporter clustering in lymphocytes, a putative biomarker of therapeutic efficacy in major depression.

PubMed

Romay-Tallon, Raquel; Rivera-Baltanas, Tania; Allen, Josh; Olivares, Jose M; Kalynchuk, Lisa E; Caruncho, Hector J

2017-01-01

The pattern of serotonin transporter clustering on the plasma membrane of lymphocytes extracted from human whole blood samples has been identified as a putative biomarker of therapeutic efficacy in major depression. Here we evaluated the possibility of performing a similar analysis using blood smears obtained from rats, and from control human subjects and depression patients. We hypothesized that we could optimize a protocol to make the analysis of serotonin protein clustering in blood smears comparable to the analysis of serotonin protein clustering using isolated lymphocytes. Our data indicate that blood smears require a longer fixation time and longer times of incubation with primary and secondary antibodies. In addition, one needs to optimize the image analysis settings for the analysis of smears. When these steps are followed, the quantitative analysis of both the number and size of serotonin transporter clusters on the plasma membrane of lymphocytes is similar using both blood smears and isolated lymphocytes. The development of this novel protocol will greatly facilitate the collection of appropriate samples by eliminating the necessity and cost of specialized personnel for drawing blood samples, and by being a less invasive procedure. Therefore, this protocol will help us advance the validation of membrane protein clustering in lymphocytes as a biomarker of therapeutic efficacy in major depression, and bring it closer to its clinical application.

Optimization of parameters for coverage of low molecular weight proteins

PubMed Central

Müller, Stephan A.; Kohajda, Tibor; Findeiß, Sven; Stadler, Peter F.; Washietl, Stefan; Kellis, Manolis; von Bergen, Martin

2010-01-01

Proteins with molecular weights of <25 kDa are involved in major biological processes such as ribosome formation, stress adaption (e.g., temperature reduction) and cell cycle control. Despite their importance, the coverage of smaller proteins in standard proteome studies is rather sparse. Here we investigated biochemical and mass spectrometric parameters that influence coverage and validity of identification. The underrepresentation of low molecular weight (LMW) proteins may be attributed to the low numbers of proteolytic peptides formed by tryptic digestion as well as their tendency to be lost in protein separation and concentration/desalting procedures. In a systematic investigation of the LMW proteome of Escherichia coli, a total of 455 LMW proteins (27% of the 1672 listed in the SwissProt protein database) were identified, corresponding to a coverage of 62% of the known cytosolic LMW proteins. Of these proteins, 93 had not yet been functionally classified, and five had not previously been confirmed at the protein level. In this study, the influences of protein extraction (either urea or TFA), proteolytic digestion (solely, and the combined usage of trypsin and AspN as endoproteases) and protein separation (gel- or non-gel-based) were investigated. Compared to the standard procedure based solely on the use of urea lysis buffer, in-gel separation and tryptic digestion, the complementary use of TFA for extraction or endoprotease AspN for proteolysis permits the identification of an extra 72 (32%) and 51 proteins (23%), respectively. Regarding mass spectrometry analysis with an LTQ Orbitrap mass spectrometer, collision-induced fragmentation (CID and HCD) and electron transfer dissociation using the linear ion trap (IT) or the Orbitrap as the analyzer were compared. IT-CID was found to yield the best identification rate, whereas IT-ETD provided almost comparable results in terms of LMW proteome coverage. The high overlap between the proteins identified with IT-CID and IT-ETD allowed the validation of 75% of the identified proteins using this orthogonal fragmentation technique. Furthermore, a new approach to evaluating and improving the completeness of protein databases that utilizes the program RNAcode was introduced and examined. Electronic supplementary material The online version of this article (doi:10.1007/s00216-010-4093-x) contains supplementary material, which is available to authorized users. PMID:20803007

[Extraction Optimization of Rhizome of Curcuma longa by Response Surface Methodology and Support Vector Regression].

PubMed

Zhou, Pei-pei; Shan, Jin-feng; Jiang, Jian-lan

2015-12-01

To optimize the optimal microwave-assisted extraction method of curcuminoids from Curcuma longa. On the base of single factor experiment, the ethanol concentration, the ratio of liquid to solid and the microwave time were selected for further optimization. Support Vector Regression (SVR) and Central Composite Design-Response Surface Methodology (CCD) algorithm were utilized to design and establish models respectively, while Particle Swarm Optimization (PSO) was introduced to optimize the parameters of SVR models and to search optimal points of models. The evaluation indicator, the sum of curcumin, demethoxycurcumin and bisdemethoxycurcumin by HPLC, were used. The optimal parameters of microwave-assisted extraction were as follows: ethanol concentration of 69%, ratio of liquid to solid of 21 : 1, microwave time of 55 s. On those conditions, the sum of three curcuminoids was 28.97 mg/g (per gram of rhizomes powder). Both the CCD model and the SVR model were credible, for they have predicted the similar process condition and the deviation of yield were less than 1.2%.

Essential motions and energetic contributions of individual residues in a peptide bound to an SH3 domain.

PubMed Central

Kolafa, J; Perram, J W; Bywater, R P

2000-01-01

We have studied protein-ligand interactions by molecular dynamics simulations using software designed to exploit parallel computing architectures. The trajectories were analyzed to extract the essential motions and to estimate the individual contributions of fragments of the ligand to overall binding enthalpy. Two forms of the bound ligand are compared, one with the termini blocked by covalent derivatization, and one in the underivatized, zwitterionic form. The ends of the peptide tend to bind more loosely in the capped form. We can observe significant motions in the bound ligand and distinguish between motions of the peptide backbone and of the side chains. This could be useful in designing ligands, which fit optimally to the binding protein. We show that it is possible to determine the different contributions of each residue in a peptide to the enthalpy of binding. Proline is a major net contributor to binding enthalpy, in keeping with the known propensity for this family of proteins to bind proline-rich peptides. PMID:10919999

Preparation of inulin-type fructooligosaccharides using fast protein liquid chromatography coupled with refractive index detection.

PubMed

Li, J; Cheong, K L; Zhao, J; Hu, D J; Chen, X Q; Qiao, C F; Zhang, Q W; Chen, Y W; Li, S P

2013-09-20

A fast protein liquid chromatography coupled with refractive index detection (FPLC-RID) method was firstly developed for preparation and purification of fructooligosaccharides with different degree of polymerization from burdock, Arctium lappa. After extraction with 60% ethanol and decolorization with MCI gel CHP20P, total fructooligosaccharides were purified on Bio-Gel P-2 column eluted with water at the flow rate of 0.3 ml/min, which was the optimized conditions. The obtained fructooligosaccharides with degree of polymerization of 3-9 were identified based on their methylation analysis, MS and NMR data. This method has the advantages of high automation, good recovery and easy performance, which could be used for preparation of FOS from other sources, as well as other targeted compounds without UV absorbance. Copyright © 2013 Elsevier B.V. All rights reserved.

Mitogenicity of M5 protein extracted from Streptococcus pyogenes cells is due to streptococcal pyrogenic exotoxin C and mitogenic factor MF.

PubMed Central

Schmidt, K H; Gerlach, D; Wollweber, L; Reichardt, W; Mann, K; Ozegowski, J H; Fleischer, B

1995-01-01

M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V beta 2, V beta 4, and V beta 8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cell-stimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V beta 2 and V beta 8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein. PMID:7591107

[Optimizing the extracting technique of ampelopsin from Ampelopsis cantoniensis Planch by a uniform design method].

PubMed

He, Zhi-feng; Zeng, Sa; Hou, Juan-juan; Liu, De-yu

2006-07-01

To optimize the preparation of ampelopsin from Ampelopsis Cantoniensis Planch. The extraction and purification process was studied by the uniform design with the extract of ampelopsin content and purity as markers. The facters which influence the extraction and the purification of ampelopsin content were studied by uniform design. The optimum extraction and purification process: the concentration for alcohol was 90%, and refluxing quartic, 1.5 h each time; extraction by petroleum ether quintic, the mount of active carbon was 1 g/100 g of the medicine material, and recrystaling thrice. This extraction process has higher yield of ampelopsin and is available for production.

Optimization study of Chromalaena odorata essential oil extracted using solventless extraction technique

NASA Astrophysics Data System (ADS)

Nasshorudin, Dalila; Ahmad, Muhammad Syarhabil; Mamat, Awang Soh; Rosli, Suraya

2015-05-01

Solventless extraction process of Chromalaena odorata using reduced pressure and temperature has been investigated. The percentage yield of essential oil produce was calculated for every experiment with different experimental condition. The effect of different parameters, such as temperature and extraction time on the yield was investigated using the Response Surface Methodology (RSM) through Central Composite Design (CCD). The temperature and extraction time were found to have significant effect on the yield of extract. A final essential oil yield was 0.095% could be extracted under the following optimized conditions; a temperature of 80 °C and a time of 8 hours.

Extraction of intracellular protein from Glaciozyma antarctica for proteomics analysis

NASA Astrophysics Data System (ADS)

Faizura, S. Nor; Farahayu, K.; Faizal, A. B. Mohd; Asmahani, A. A. S.; Amir, R.; Nazalan, N.; Diba, A. B. Farah; Muhammad, M. Nor; Munir, A. M. Abdul

2013-11-01

Two preparation methods of crude extracts of psychrophilic yeast Glaciozyma antarctica were compared in order to obtain a good recovery of intracellular proteins. Extraction with mechanical procedures using sonication was found to be more effective for obtaining good yield compare to alkaline treatment method. The procedure is simple, rapid, and produce better yield. A total of 52 proteins were identified by combining both extraction methods. Most of the proteins identified in this study involves in the metabolic process including glycolysis pathway, pentose phosphate pathway, pyruyate decarboxylation and also urea cyle. Several chaperons were identified including probable cpr1-cyclophilin (peptidylprolyl isomerase), macrolide-binding protein fkbp12 and heat shock proteins which were postulate to accelerate proper protein folding. Characteristic of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.

Ultrasound assisted extraction of pectin from waste Artocarpus heterophyllus fruit peel.

PubMed

Moorthy, I Ganesh; Maran, J Prakash; Ilakya, S; Anitha, S L; Sabarima, S Pooja; Priya, B

2017-01-01

Four factors three level face centered central composite response surface design was employed in this study to investigate and optimize the effect of process variables (liquid-solid (LS) ratio (10:1-20:1ml/g), pH (1-2), sonication time (15-30min) and extraction temperature (50-70°C)) on the maximum extraction yield of pectin from waste Artocarpus heterophyllus (Jackfruit) peel by ultrasound assisted extraction method. Numerical optimization method was adapted in this study and the following optimal condition was obtained as follows: Liquid-solid ratio of 15:1ml/g, pH of 1.6, sonication time of 24min and temperature of 60°C. The optimal condition was validated through experiments and the observed value was interrelated with predicted value. Copyright © 2016 Elsevier B.V. All rights reserved.

Intensification of marrubiin concentration by optimization of microwave-assisted (low CO2 yielding) extraction process for Marrubium vulgare using central composite design and antioxidant evaluation.

PubMed

Mittal, Vineet; Nanda, Arun

2017-12-01

Marrubium vulgare Linn (Lamiaceae) was generally extracted by conventional methods with low yield of marrubiin; these processes were not considered environment friendly. This study extracts the whole plant of M. vulgare by microwave assisted extraction (MAE) and optimizes the effect of various extraction parameters on the marrubiin yield by using Central Composite Design (CCD). The selected medicinal plant was extracted using ethanol: water (1:1) as solvent by MAE. The plant material was also extracted using a Soxhlet and the various extracts were analyzed by HPTLC to quantify the marrubiin concentration. The optimized conditions for the microwave-assisted extraction of selected medicinal plant was microwave power of 539 W, irradiation time of 373 s and solvent to drug ratio, 32 mL per g of the drug. The marrubiin concentration in MAE almost doubled relative to the traditional method (0.69 ± 0.08 to 1.35 ± 0.04%). The IC 50 for DPPH was reduced to 66.28 ± 0.6 μg/mL as compared to conventional extract (84.14 ± 0.7 μg/mL). The scanning electron micrographs of the treated and untreated drug samples further support the results. The CCD can be successfully applied to optimize the extraction parameters (MAE) for M. vulgare. Moreover, in terms of environmental impact, the MAE technique could be assumed as a 'Green approach' because the MAE approach for extraction of plant released only 92.3 g of CO 2 as compared to 3207.6 g CO 2 using the Soxhlet method of extraction.

Study on Process Parameters of Extraction of γ-aminobutyric Acid Instant Moringa oleifera Powder

NASA Astrophysics Data System (ADS)

Tiaokun, Fu; Suhui, Zhang; Neng, Liu; Jihua, Li; Shaodan, Peng; Changqing, Guo; Wei, Zhou

2017-12-01

To preliminary optimize the extraction of γ-aminobutyric acid instant tea powder from Moringa oleifera leaves,taking γ-aminobutyric acid Moringa oleifera leaves as raw material and pure water as extraction solvent and GABA content as the main evaluation index,the effects of extraction temperature, ratio of liquid to material and extraction time on biochemical components and sensory qualities of Moringa oleifera leaves extractor was studied by using one-factor-at-a-time experiment.The results showed shat the optimal extraction conditions for γ-aminobutyric acid instant tea powder were extraction temperature of 60 °C, material-to-water ration of 1:10 and extraction time of 40 min.

Natural rubber latex skin testing reagents: safety and diagnostic accuracy of nonammoniated latex, ammoniated latex, and latex rubber glove extracts.

PubMed

Hamilton, R G; Adkinson, N F

1996-11-01

Nonammoniated latex, ammoniated latex, and rubber glove extracts are the only sources of natural rubber (Hevea brasiliensis) latex that have potential for use as skin testing reagents in the diagnosis of latex allergy. Their diagnostic sensitivity and specificity as skin test reagents are unknown. We conducted a phase 1/2 clinical study to examine the safety and diagnostic accuracy (sensitivity and specificity) of nonammoniated latex, ammoniated latex, and rubber glove extracts as skin test extracts to identify the most efficacious source material for future skin test reagent development. Twenty-four adults not allergic to latex, 19 adults with hand dermatitis or pruritus, and 59 adults with a latex allergy were identified by clinical history. All provided blood and then received puncture skin tests and intradermal skin tests with nonammoniated latex, ammoniated latex, and rubber glove extracts from Malaysian H. brasiliensis latex by use of sequential titration. A glove provocation test and IgE anti-latex RAST were used to clarify positive history-negative skin test response and negative history-positive skin test response mismatches. All three extracts were biologically safe and sterile. After normalization to 1 mg/ml of total protein, all three extracts produced equivalent diagnostic sensitivity and specificity in puncture skin tests and intradermal skin tests at various extract concentrations. Optimal diagnostic accuracy was safely achieved at 100 micrograms/ml for intradermal skin tests (e.g., nonammoniated latex: puncture skin test sensitivity 96%, specificity 100%; intradermal skin test sensitivity 93%, specificity 96%). The presence of IgE antibody in skin was highly correlated with IgE anti-latex in serum (nonammoniated latex: r = 0.98, p < 0.001; ammoniated latex: r = 0.94, p < 0.001; rubber glove extract: r = 0.96, p < 0.001). All five available subjects with a positive history, negative skin test response, and absence of IgE antibody in serum had a negative glove provocation test response, indicating no clinical evidence of latex allergy. No systemic or large local allergic reactions were observed with puncture skin tests or intradermal skin tests. Equivalent diagnostic sensitivity and specificity were observed with the nonammoniated latex, ammoniated latex, and rubber glove extract skin test reagents after normalization for total protein; nonammoniated latex may be considered the reagent of choice on the basis of practical quality control and reproducibility considerations.

Authenticity determination of honeys with non-extractable proteins by means of elemental analyzer (EA) and liquid chromatography (LC) coupled to isotope ratio mass spectroscopy (IRMS).

PubMed

Dong, Hao; Xiao, Kaijun; Xian, Yanping; Wu, Yuluan

2018-02-01

The present work aims to systematically demonstrate the authenticity of honeys with non-extractable proteins for the first time, by means of EA-IRMS and LC-IRMS. Fifty-three pure honeys of various botanical and geographical origins were studied and a criterion on the basis of the stable carbon isotope ratio characterization of total honey and the main sugars was established for pure honeys. Parameters such as δ 13 C values of total honey and the main sugars were well utilized to identify honeys with non-extractable proteins. Thirty-five honeys from which protein could not be extracted were all identified as adulterated with C-4 sugars or C-3 sugars. The use of isotopic compositions and some systematic differences permit the honeys with non-extractable proteins to be reliably identified. The findings obtained in this work could supplement the AOAC 998.12 C-4 sugar method, with regard to honeys from which protein cannot be extracted. Copyright © 2017 Elsevier Ltd. All rights reserved.

Optimized aqueous extraction of saponins from bitter melon for production of a saponin-enriched bitter melon powder.

PubMed

Tan, Sing P; Vuong, Quan V; Stathopoulos, Costas E; Parks, Sophie E; Roach, Paul D

2014-07-01

Bitter melon, Momordica charantia L. (Cucurbitaceae), aqueous extracts are proposed to have health-promoting properties due to their content of saponins and their antioxidant activity. However, the optimal conditions for the aqueous extraction of saponins from bitter melon and the effects of spray drying have not been established. Therefore, this study aimed to optimize the aqueous extraction of the saponins from bitter melon, using response surface methodology, prepare a powder using spray drying, and compare the powder's physical properties, components, and antioxidant capacity with aqueous and ethanol freeze-dried bitter melon powders and a commercial powder. The optimal aqueous extraction conditions were determined to be 40 °C for 15 min and the water-to-sample ratio was chosen to be 20:1 mL/g. For many of its physical properties, components, and antioxidant capacity, the aqueous spray-dried powder was comparable to the aqueous and ethanol freeze-dried bitter melon powders and the commercial powder. The optimal conditions for the aqueous extraction of saponins from bitter melon followed by spray drying gave a high quality powder in terms of saponins and antioxidant activity. This study highlights that bitter melon is a rich source of saponin compounds and their associated antioxidant activities, which may provide health benefits. The findings of the current study will help with the development of extraction and drying technologies for the preparation of a saponin-enriched powdered extract from bitter melon. The powdered extract may have potential as a nutraceutical supplement or as a value-added ingredient for incorporation into functional foods. © 2014 Institute of Food Technologists®

Optimisation of phenolics recovery from Vitex agnus-castus Linn. leaves by high-pressure and temperature extraction.

PubMed

Lataoui, Mohammed; Seffen, Mongi; Aliakbarian, Bahar; Casazza, Alessandro Alberto; Converti, Attilio; Perego, Patrizia

2014-01-01

To optimise recovery of phenolics from Vitex agnus-castus Linn., a non-conventional high-pressure (2-24 bar) and temperature (100-180°C) extraction method was used under nitrogen atmosphere with methanol as a solvent. Optimal temperature was between 100 and 140°C, and optimal extraction time was about one half that of conventional solid/liquid extraction at room temperature. Final yields of total polyphenols, total flavonoids, o-diphenols and anthocyanins extraction were 2.0, 3.0, 2.5 and 11-fold those obtained by conventional extraction.

Optimization of headspace solid phase micro-extraction of volatile compounds from papaya fruit assisted by GC-olfactometry.

PubMed

da Rocha, Renier Felinto Julião; da Silva Araújo, Ídila Maria; de Freitas, Sílvia Maria; Dos Santos Garruti, Deborah

2017-11-01

Optimization of the extraction conditions to investigate the volatile composition of papaya fruit involving headspace solid phase micro-extraction was carried out using multivariate strategies such as factorial design and response surface methodology. The performance of different combinations of time for reaching the equilibrium in the headspace and time for maximum extraction of volatiles was evaluated by GC-olfactometry of the extract (intensity of papaya characteristic aroma), number of peaks and total area in the chromatogram. Thirty-two compounds were identified by GC-MS under the optimized extraction conditions, the majority of which were aldehydes, both in number of compounds and area. Major compounds were δ-octalactone, β-citral, benzaldehyde, heptanal, benzyl isothiocyanate, isoamyl acetate, γ-octalactone, (E)-linalool oxide and benzyl alcohol. Seven aldehydes and two other compounds are reported for the first time in papaya's volatile profile.

Simultaneous Microwave Extraction and Separation of Volatile and Non-Volatile Organic Compounds of Boldo Leaves. From Lab to Industrial Scale

PubMed Central

Petigny, Loïc; Périno, Sandrine; Minuti, Matteo; Visinoni, Francesco; Wajsman, Joël; Chemat, Farid

2014-01-01

Microwave extraction and separation has been used to increase the concentration of the extract compared to the conventional method with the same solid/liquid ratio, reducing extraction time and separate at the same time Volatile Organic Compounds (VOC) from non-Volatile Organic Compounds (NVOC) of boldo leaves. As preliminary study, a response surface method has been used to optimize the extraction of soluble material and the separation of VOC from the plant in laboratory scale. The results from the statistical analysis revealed that the optimized conditions were: microwave power 200 W, extraction time 56 min and solid liquid ratio of 7.5% of plants in water. Lab scale optimized microwave method is compared to conventional distillation, and requires a power/mass ratio of 0.4 W/g of water engaged. This power/mass ratio is kept in order to upscale from lab to pilot plant. PMID:24776762

Optimization of PEG-based extraction of polysaccharides from Dendrobium nobile Lindl. and bioactivity study.

PubMed

Zhang, Yi; Wang, Hongxin; Wang, Peng; Ma, ChaoYang; He, GuoHua; Rahman, Md Ramim Tanver

2016-11-01

Polyethylene glycol (PEG) as a green solvent was employed to extract polysaccharide. The optimal conditions for PEG-based ultrasonic extraction of Dendrobium nobile Lindl. polysaccharide (JCP) were determined by response surface methodology. Under the optimal conditions: extraction temperature of 58.5°C; ultrasound power of 193W, and the concentration of polyethylene glycol-200 (PEG-200) solution of 45%, the highest JCP yield was obtained as 15.23±0.57%, which was close to the predicted yield, 15.57%. UV and FT-IR analysis revealed the general characteristic absorption peaks of both JCP with water extraction (JCP w ) and PEG-200 solvent extraction (JCP p ). Thermal analysis of both JCPs was performed with Thermal Gravimetric Analyzer (TGA) and Differential Scanning Calorimeter (DSC). Antioxidant activities of two polysaccharides were also compared and no significant difference in vitro was obtained. Copyright © 2016 Elsevier B.V. All rights reserved.

Predicting nucleic acid binding interfaces from structural models of proteins

PubMed Central

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2011-01-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared to patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. PMID:22086767

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device.

PubMed

Battle, Katrina N; Jackson, Joshua M; Witek, Małgorzata A; Hupert, Mateusz L; Hunsucker, Sally A; Armistead, Paul M; Soper, Steven A

2014-03-21

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.

[Optimization of supercritical fluid extraction of bioactive components in Ligusticum chuanxiong by orthogonal array design].

PubMed

Hu, Li-Cui; Wu, Xun; Yang, Xue-Dong

2013-10-01

With the yields of ferulic acid, coniferylferulate, Z-ligustilide, senkyunolide A, butylidenephthalide, butylphthalide, senkyunolide I, senkyunolide H, riligustilide, levistolide A, and total pharmacologically active ingredient as evaluation indexes, the extraction of Ligusticum chuanxiong by supercritical fluid technology was investigated through an orthogonal experiment L9 (3(4)). Four factors, namely temperature, pressure, flow rate of carbon dioxide, co-solvent concentration of the supercritical fluid, were investigated and optimized. Under the optimized conditions, namely 65 degrees C of temperature, 35 MPa of pressure, 1 L x min(-1) of CO2 flow rate, 8% of co-solvent concetration, supercritical fluid extraction could achieve a better yield than the conventional reflux extraction using methanol. And the supercritical fluid extraction process was validated to be stable and reliable.

[Biochemistry and functional characterization of squid mantle meat (Dosidicus gigas)].

PubMed

Abugoch, L; Guarda, A; María Pérez, L; Isabel Donghi, M

2000-12-01

A study for the characterization of frozen giant squid mantle (meat) protein stored at -25 degrees C for 8 month was started. In the present research, the following functional properties were investigate: emulsifying, water holding and gel forming capacities. Optimal conditions for the separation and differentiation of miofibrillar and sarcoplasmatic proteins were also studied. It was found that the unfrozen giant squid mantle meat es capable of emulifying 2.817,4 g of oil/g of protein and holding capacity was 3.64 g of water/g of protein. Related to the gel forming capacity, it was not obtain, probably due to excessive storage of the meat. With regard to miofibrilar protein obtention of the squid mantle meat, it was found that two low ionic strength washings (I = 0.05), the sarcoplasmic proteins were practically eliminated from the protein matrix. The differentiation of miofibrilar and sarcoplasmatic proteins was obtained by PAGE-SDS of the squid mantle meat extracted at two different ionic strength (I = 0.05 and I = 0.5). This work demonstrates that the giant squid mantle protein has a high emulsifying and water holding capacity, and it can be used, as a raw material, for the improvement of sausage products. About the gelling products, more studies will be necessary with fresh squid mantle meat to conclude about this functional property.

Extraction of DNA from human embryos after long-term preservation in formalin and Bouin's solutions.

PubMed

Nagai, Momoko; Minegishi, Katsura; Komada, Munekazu; Tsuchiya, Maiko; Kameda, Tomomi; Yamada, Shigehito

2016-05-01

The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies. © 2015 Japanese Teratology Society.

Application of cation-exchange solid-phase extraction for the analysis of amino alcohols from water and human plasma for verification of Chemical Weapons Convention.

PubMed

Kanaujia, Pankaj K; Tak, Vijay; Pardasani, Deepak; Gupta, A K; Dubey, D K

2008-03-28

The analysis of nitrogen containing amino alcohols, which are the precursors and degradation products of nitrogen mustards and nerve agent VX, constitutes an important aspect for verifying the compliance to the CWC (Chemical Weapons Convention). This work devotes on the development of solid-phase extraction method using silica- and polymer-based SCX (strong cation-exchange) and MCX (mixed-mode strong cation-exchange) cartridges for N,N-dialkylaminoethane-2-ols and alkyl N,N-diethanolamines, from water. The extracted analytes were analyzed by GC-MS (gas chromatography-mass spectrometry) in the full scan and selected ion monitoring modes. The extraction efficiencies of SCX and MCX cartridges were compared, and results revealed that SCX performed better. Extraction parameters, such as loading capacity, extraction solvent, its volume, and washing solvent were optimized. Best recoveries were obtained using 2 mL methanol containing 10% NH(4)OH and limits of detection could be achieved up to 5 x 10(-3) microg mL(-1) in the selected ion monitoring mode and 0.01 microg mL(-1) in full scan mode. The method was successfully employed for the detection and identification of amino alcohol present in water sample sent by Organization for Prohibition of Chemical Weapons (OPCW) in the official proficiency tests. The method was also applied to extract the analytes from human plasma. The SCX cartridge showed good recoveries of amino alcohols from human plasma after protein precipitation.

Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling

PubMed Central

2014-01-01

Background Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses. Results Using combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent. Conclusions This study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS. PMID:24980254

Development of a simple and valid method for the trace determination of phthalate esters in human plasma using dispersive liquid-liquid microextraction coupled with gas chromatography-mass spectrometry.

PubMed

Ebrahim, Karim; Poursafa, Parinaz; Amin, Mohammad Mehdi

2017-11-01

A new method was developed for the trace determination of phthalic acid esters in plasma using dispersive liquid-liquid microextraction and gas chromatography with mass spectrometry analysis. Plasma proteins were efficiently precipitated by trichloroacetic acid and then a mixture of chlorobenzene (as extraction solvent) and acetonitrile (as dispersive solvent) rapidly injected to clear supernatant using a syringe. After centrifuging, chlorobenzene sedimented at the bottom of the test tube. 1 μL of this sedimented phase was injected into the gas chromatograph for phthalic acid esters analysis. Different factors affecting the extraction performance, such as the type of extraction and dispersive solvent, their volume, extraction time, and the effects of salt addition were investigated and optimized. Under the optimum conditions, the enrichment factors and extraction recoveries were satisfactory and ranged between 820-1020 and 91-97%, respectively. The linear range was wide (50-1000 ng/mL) and limit of detection was very low (1.5-2.5 ng/mL for all analytes). The relative standard deviations for analysis of 1 μg/mL of the analytes were between 3.2-6.1%. Salt addition showed no significant effect on extraction recovery. Finally, the proposed method was successfully utilized for the extraction and determination of the phthalic acid esters in human plasma samples and satisfactory results were obtained. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Orthogonal array design in optimizing the extraction efficiency of active constituents from roots of Panax notoginseng.

PubMed

Dong, T T X; Zhao, K J; Huang, W Z; Leung, K W; Tsim, K W K

2005-08-01

The root of Panax notoginseng (Radix Notoginseng, Sanqi) is a commonly used traditional Chinese medicine, which is mainly cultivated in Wenshan of Yunnan China. The identified active constituents in Radix Notoginseng include saponin, ssavonoid and polysaccharide; however, the levels of these active constituents vary greatly with different extraction processes. This variation causes a serious problem in standardizing the herbal extract. By using HPLC and spectrophotometry, the contents of notoginsenoside R(1), ginsenoside R(g1), R(b1), R(d), and ssavonoids were determined in the extracts of Radix Notoginseng that were derived from different processes of extraction according to an orthogonal array experimental design having three variable parameters: nature of extraction solvent, extraction volume and extraction time. The nature of extraction solvent and extraction volume were two distinct factors in obtaining those active constituents, while the time of extraction was a subordinate factor. The optimized condition of extraction therefore is considered to be 20 volumes of water and extracted for 24 h. In good agreement with the amount of active constituents, the activity of anti-platelet aggregation was found to be the highest in the extract that contained a better yield of the active constituents. The current results provide an optimized extraction method for the quality control of Radix Notoginseng. Copyright (c) 2005 John Wiley & Sons, Ltd.

Extraction optimization and UHPLC method development for determination of the 20-hydroxyecdysone in Sida tuberculata leaves.

PubMed

da Rosa, Hemerson S; Koetz, Mariana; Santos, Marí Castro; Jandrey, Elisa Helena Farias; Folmer, Vanderlei; Henriques, Amélia Teresinha; Mendez, Andreas Sebastian Loureiro

2018-04-01

Sida tuberculata (ST) is a Malvaceae species widely distributed in Southern Brazil. In traditional medicine, ST has been employed as hypoglycemic, hypocholesterolemic, anti-inflammatory and antimicrobial. Additionally, this species is chemically characterized by flavonoids, alkaloids and phytoecdysteroids mainly. The present work aimed to optimize the extractive technique and to validate an UHPLC method for the determination of 20-hydroxyecdsone (20HE) in the ST leaves. Box-Behnken Design (BBD) was used in method optimization. The extractive methods tested were: static and dynamic maceration, ultrasound, ultra-turrax and reflux. In the Box-Behnken three parameters were evaluated in three levels (-1, 0, +1), particle size, time and plant:solvent ratio. In validation method, the parameters of selectivity, specificity, linearity, limits of detection and quantification (LOD, LOQ), precision, accuracy and robustness were evaluated. The results indicate static maceration as better technique to obtain 20HE peak area in ST extract. The optimal extraction from surface response methodology was achieved with the parameters granulometry of 710 nm, 9 days of maceration and plant:solvent ratio 1:54 (w/v). The UHPLC-PDA analytical developed method showed full viability of performance, proving to be selective, linear, precise, accurate and robust for 20HE detection in ST leaves. The average content of 20HE was 0.56% per dry extract. Thus, the optimization of extractive method in ST leaves increased the concentration of 20HE in crude extract, and a reliable method was successfully developed according to validation requirements and in agreement with current legislation. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparative analyses of universal extraction buffers for assay of stress related biochemical and physiological parameters.

PubMed

Han, Chunyu; Chan, Zhulong; Yang, Fan

2015-01-01

Comparative efficiency of three extraction solutions, including the universal sodium phosphate buffer (USPB), the Tris-HCl buffer (UTHB), and the specific buffers, were compared for assays of soluble protein, free proline, superoxide radical (O2∙-), hydrogen peroxide (H2O2), and the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR) in Populus deltoide. Significant differences for protein extraction were detected via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). Between the two universal extraction buffers, the USPB showed higher efficiency for extraction of soluble protein, CAT, GR, O2∙-, GPX, SOD, and free proline, while the UTHB had higher efficiency for extraction of APX, POD, and H2O2. When compared with the specific buffers, the USPB showed higher extraction efficiency for measurement of soluble protein, CAT, GR, and O2∙-, parallel extraction efficiency for GPX, SOD, free proline, and H2O2, and lower extraction efficiency for APX and POD, whereas the UTHB had higher extraction efficiency for measurement of POD and H2O2. Further comparisons proved that 100 mM USPB buffer showed the highest extraction efficiencies. These results indicated that USPB would be suitable and efficient for extraction of soluble protein, CAT, GR, GPX, SOD, H2O2, O2∙-, and free proline.

Design of distributed systems of hydrolithospere processes management. Selection of optimal number of extracting wells

NASA Astrophysics Data System (ADS)

Pershin, I. M.; Pervukhin, D. A.; Ilyushin, Y. V.; Afanaseva, O. V.

2017-10-01

The article considers the important issue of designing the distributed systems of hydrolithospere processes management. Control effects on the hydrolithospere processes are implemented by a set of extractive wells. The article shows how to determine the optimal number of extractive wells that provide a distributed control impact on the management object.

The extraction of liquid, protein molecules and yeast cells from paper through surface acoustic wave atomization.

PubMed

Qi, Aisha; Yeo, Leslie; Friend, James; Ho, Jenny

2010-02-21

Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells via SAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics.

In vitro and in vivo characterization of hazelnut skin prick test extracts.

PubMed

Akkerdaas, Jaap H; Wensing, Marjolein; Knulst, André C; Aalberse, Rob C; Hefle, Susan L; van Ree, Ronald

2003-01-01

Hazelnut allergy ranks among the most frequently observed food allergies. Clinical symptoms range from the oral allergy syndrome to life threatening anaphylaxis. Diagnosis of hazelnut allergy partially relies on in vivo testing by means of skin prick testing (SPT). The aim of this study was to characterize hazelnut SPT extracts both in vitro and in vivo. Hazelnut SPT extracts were investigated for protein concentration and composition. The major hazelnut allergen Cor a 1, lipid transfer protein (LTP) and thaumatin-like-protein (TLP) were monitored by competitive RIA and immunoblotting. SPT extracts (n = 6) were analyzed for skin reactivity and the correlation between the SPT extract protein concentration and the mean skin reactivity (HEIC) was determined in a group of hazelnut-allergic patients (n = 30). For one SPT extract, the threshold level for Cor a 1 was determined in Cor a 1-monosensitized patients (n = 5). Protein concentrations ranged from 0.2-14 mg/ml. Although some proteins were present in most extracts (bands at 10, 22-28, 32 and around 48 kDa), clear differences in composition were observed (both intra- and inter-variability). The concentration of the major hazelnut allergen Cor a 1 differed up to a factor 50 (0.6-32 micrograms/ml). LTP was virtually absent in 3/9 SPT extracts and variable quantities of TLP were detected by immunoblotting. Some patients (6/30) had a false-negative SPT with 3/6 SPT extracts. There was a clear correlation between the protein concentration and the mean HEIC (RPearson = 0.87). The threshold level for Cor a 1 was +/- 3.2 ng/ml as assessed with one of the products investigated. Heterogeneous protein concentration/composition of SPT extracts results in variable skin test responses. The absence of potentially severe allergens like LTP may lead to false-negative SPT results that jeopardize a patient's safety. From these results it can be concluded that there is a strong need for standardization of products for SPT.

Response surface modeling and optimization of ultrasound-assisted extraction of three flavonoids from tartary buckwheat (Fagopyrum tataricum)

PubMed Central

Peng, Lian-Xin; Zou, Liang; Zhao, Jiang-Lin; Xiang, Da-Bing; Zhu, Peng; Zhao, Gang

2013-01-01

Background: Buckwheat (Fagopyrum spp., Polygonaceae) is a widely planted food crop. Flavonoids, including quercetin, rutin, and kaempferol, are the main bioactive components in tartary buckwheat (Fagopyrum tataricum (L.) Gaertn). From the nutriological and pharmacological perspectives, flavonoids have great value in controlling blood glucose and blood pressure levels, and they also have antioxidant properties. Objective: To optimize the conditions for extraction of quercetin, rutin, and kaempferol from F. tataricum. Materials and Methods: A combination of ultrasound-assisted extraction (UAE) and response surface methodology (RSM) was used for flavonoid extraction and yield assessment. The RSM was based on a three-level, three-variable Box-Behnken design. Results: Flavonoids were optimally extracted from F. tataricum by using 72% methanol, at 60°C, for 21 minutes. Under these conditions, the obtained extraction yield of the total flavonoids was 3.94%. Conclusion: The results indicated that the UAE method was effective for extraction of flavonoids from tartary buckwheat. PMID:23930003

Extraction of Oil from Flaxseed (Linum usitatissimum L.) Using Enzyme-Assisted Three-Phase Partitioning.

PubMed

Tan, Zhi-Jian; Yang, Zi-Zhen; Yi, Yong-Jian; Wang, Hong-Ying; Zhou, Wan-Lai; Li, Fen-Fang; Wang, Chao-Yun

2016-08-01

In this study, enzyme-assisted three-phase partitioning (EATPP) was used to extract oil from flaxseed. The whole procedure is composed of two parts: the enzymolysis procedure in which the flaxseed was hydrolyzed using an enzyme solution (the influencing parameters such as the type and concentration of enzyme, temperature, and pH were optimized) and three-phase partitioning (TPP), which was conducted by adding salt and t-butanol to the crude flaxseed slurry, resulting in the extraction of flaxseed oil into alcohol-rich upper phase. The concentration of t-butanol, concentration of salt, and the temperature were optimized to maximize the extraction yield. Under optimized conditions of a 49.29 % t-butanol concentration, 30.43 % ammonium sulfate concentration, and 35 °C extraction temperature, a maximum extraction yield of 71.68 % was obtained. This simple and effective EATPP can be used to achieve high extraction yields and oil quality, and thus, it is potential for large-scale oil production.

[Study on the extraction of the total alkaloids from Caulopyhllum robustum].

PubMed

Li, Yi-ping; Yang, Guang-de; He, Lang-chong

2007-02-01

To study the technological parameters of the extraction process of the total alkaloids from Caulopyhllum robstum. Taspine, whiVh is main component of the total alkaloids from Caulopyhllum robustum, was selected as an evaluating marker and determined by HPLC. The orthogonal test was used to optimize extracting conditions in the process of acid water extraction. Then the optimized conditions for purification using cation exchange resin were investigated. The optimized conditions in the process of acid water extraction were 1% hydrochloric acid as much as seven times of the medicine amount for 24hs and three times. Then the extraction of acid water was purified with a column of macroporous cation exchange resin LSD001 at 2 ml/min of flow rate, then eluted with 10BV of 4% aqueous ammonia ethanol. The extraction ratio of the total alkaloids was 1. 35% and the content of taspine of the total alkaloids was 6. 80%. This technology is simply, cheap effective and feasible for manufacture in great scale.

Optimization by response surface methodology of lutein recovery from paprika leaves using accelerated solvent extraction.

PubMed

Kang, Jae-Hyun; Kim, Suna; Moon, BoKyung

2016-08-15

In this study, we used response surface methodology (RSM) to optimize the extraction conditions for recovering lutein from paprika leaves using accelerated solvent extraction (ASE). The lutein content was quantitatively analyzed using a UPLC equipped with a BEH C18 column. A central composite design (CCD) was employed for experimental design to obtain the optimized combination of extraction temperature (°C), static time (min), and solvent (EtOH, %). The experimental data obtained from a twenty sample set were fitted to a second-order polynomial equation using multiple regression analysis. The adjusted coefficient of determination (R(2)) for the lutein extraction model was 0.9518, and the probability value (p=0.0000) demonstrated a high significance for the regression model. The optimum extraction conditions for lutein were temperature: 93.26°C, static time: 5 min, and solvent: 79.63% EtOH. Under these conditions, the predicted extraction yield of lutein was 232.60 μg/g. Copyright © 2016 Elsevier Ltd. All rights reserved.

Algorithms and semantic infrastructure for mutation impact extraction and grounding.

PubMed

Laurila, Jonas B; Naderi, Nona; Witte, René; Riazanov, Alexandre; Kouznetsov, Alexandre; Baker, Christopher J O

2010-12-02

Mutation impact extraction is a hitherto unaccomplished task in state of the art mutation extraction systems. Protein mutations and their impacts on protein properties are hidden in scientific literature, making them poorly accessible for protein engineers and inaccessible for phenotype-prediction systems that currently depend on manually curated genomic variation databases. We present the first rule-based approach for the extraction of mutation impacts on protein properties, categorizing their directionality as positive, negative or neutral. Furthermore protein and mutation mentions are grounded to their respective UniProtKB IDs and selected protein properties, namely protein functions to concepts found in the Gene Ontology. The extracted entities are populated to an OWL-DL Mutation Impact ontology facilitating complex querying for mutation impacts using SPARQL. We illustrate retrieval of proteins and mutant sequences for a given direction of impact on specific protein properties. Moreover we provide programmatic access to the data through semantic web services using the SADI (Semantic Automated Discovery and Integration) framework. We address the problem of access to legacy mutation data in unstructured form through the creation of novel mutation impact extraction methods which are evaluated on a corpus of full-text articles on haloalkane dehalogenases, tagged by domain experts. Our approaches show state of the art levels of precision and recall for Mutation Grounding and respectable level of precision but lower recall for the task of Mutant-Impact relation extraction. The system is deployed using text mining and semantic web technologies with the goal of publishing to a broad spectrum of consumers.

Optimization of clean extraction methods to isolate carotenoids from the microalga Neochloris oleoabundans and subsequent chemical characterization using liquid chromatography tandem mass spectrometry.

PubMed

Castro-Puyana, María; Herrero, Miguel; Urreta, Iratxe; Mendiola, Jose A; Cifuentes, Alejandro; Ibáñez, Elena; Suárez-Alvarez, Sonia

2013-05-01

A novel experimental design was used to optimize the extraction of carotenoids from Neochloris oleoabundans using pressurized liquid extraction with food-grade solvents such as ethanol and limonene. Experimental factors, including the extraction temperature and the solvent composition, were optimized using a three-level factorial design. The response variables extraction yield and total amount of carotenoids were assessed. The statistical analysis of the results provided mathematical models to predict the behavior of the responses as a function of the factors involved in the process. The optimum conditions predicted by the model developed in this study were 112 °C as the extraction temperature and 100% ethanol as the extraction solvent. Chemical characterization of the extracts obtained was performed by means of high-performance liquid chromatography-tandem mass spectrometry. The results obtained demonstrated that, under certain growth conditions (photoautotrophically cultured in a medium supplemented with 0.3 g L(-1) KNO3), N. oleoabundans accumulated significant total amounts of the carotenoids (from 57.4 to 120.2 mg carotenoids per gram of extract depending on the extraction conditions), mainly lutein, cantaxanthin, zeaxanthin, and astaxanthin monoesters and diesters.

Ionic liquid-based microwave-assisted extraction of flavonoids from Bauhinia championii (Benth.) Benth.

PubMed

Xu, Wei; Chu, Kedan; Li, Huang; Zhang, Yuqin; Zheng, Haiyin; Chen, Ruilan; Chen, Lidian

2012-12-03

An ionic liquids (IL)-based microwave-assisted approach for extraction and determination of flavonoids from Bauhinia championii (Benth.) Benth. was proposed for the first time. Several ILs with different cations and anions and the microwave-assisted extraction (MAE) conditions, including sample particle size, extraction time and liquid-solid ratio, were investigated. Two M 1-butyl-3-methylimidazolium bromide ([bmim] Br) solution with 0.80 M HCl was selected as the optimal solvent. Meanwhile the optimized conditions a ratio of liquid to material of 30:1, and the extraction for 10 min at 70 °C. Compared with conventional heat-reflux extraction (CHRE) and the regular MAE, IL-MAE exhibited a higher extraction yield and shorter extraction time (from 1.5 h to 10 min). The optimized extraction samples were analysed by LC-MS/MS. IL extracts of Bauhinia championii (Benth.)Benth consisted mainly of flavonoids, among which myricetin, quercetin and kaempferol, β-sitosterol, triacontane and hexacontane were identified. The study indicated that IL-MAE was an efficient and rapid method with simple sample preparation. LC-MS/MS was also used to determine the chemical composition of the ethyl acetate/MAE extract of Bauhinia championii (Benth.) Benth, and it maybe become a rapid method to determine the composition of new plant extracts.