Gut microbiota remodeling drived by dietary millet protein prevents the metabolic syndrome

Metabolic syndrome(Met S)is a chronic disease associated with the disturbance of gut microbiota homeostasis.Metabolites derived from gut microbes play essential roles in Met S prevention and therapy.Here,we focused on the inhibitory effect of the extract of millet bran protein(EMBP)on a high-fat diet(HFD)-induced Met S,aiming to identify gut microbiota and their metabolites that involve in the anti-Met S activity of EMBP.The obesity,chronic inflammation,insulin resistance in Met S mouse models were abolished after EMBP treatment.The protective mechanism of EMBP against HFD-induced Met S may depend on improved gut barrier function.Using microbiome analysis,we found that EMBP supplementation improved gut microbiome dysbiosis in Met S mice,specifically upregulating Bacteroides acidifaciens.The fecal microbiota transplantation(FMT)also demonstrated this phenomenon.In addition,metabolomic analysis showed that EMBP mediates metabolic profiling reprogramming in Met S mice.Notably,a microbiota-derived metabolite,gamma-aminobutyric acid(GABA),is enriched by EMBP.In addition,exogenous GABA treatment produced a similar protective effect to EMBP by improving NRF2-dependent gut barrier function to protect HFDinduced Met S.The results suggest that EMBP suppress host Met S by remodeling of gut microbiota as an effective candidate for next-generation medicine food dual purpose dietary supplement to intervene in MetS.

Optimization of Two-Dimensional Gel Electrophoresis for Kenaf Leaf Proteins

To establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis （2-DE） analysis in kenaf leaf tissues, three extraction methods （trichloroacetic acid/acetone, urea/thiourea, and phenol extraction methods） were applied to the extraction of kenaf leaf protein. The results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and 2-DE gels. Furthermore, the 2-DE system was optimized for four aspects： the pH range of IPG （immobilized pH gradient） stripes, sampling methods, sample volumes, and concentration of polyacrylamide gels. The data presented showed that the phenol extraction method is the best method to perform 2-DE analysis of kenaf leaf protein. The protein extracted from phenol extraction method reached the purity of （26.40±0.859）%, showed （25.67±1.53） protein bands in one dimension SDS-PAGE gels, and （1 374±54.44） protein spots on 2-DE gels. The research also indicates that kenaf leaf protein spots were distributed mainly within the pH range of 4-8. More clear background with a better distribution effect and many protein spots could be obtained on 2-DE gels under the conditions of active rehydration loading, 24 cm IPG strips （linear pH gradient of 4-7）, 1.4 mg samples, and 12% SDS-PAGE gels.

Deep eutectic solvents and alkaline extraction of protein from seabuckthorn seed meal: a comparison study

Seabuckthorn seed meal(SSM) is a waste of oil extraction industry that rich in protein. In order to seek suitable protein extraction method, three different deep eutectic solvents(DESs)(including choline chlorideglycerol, choline chloride-oxalic acid and choline chloride-urea) were developed for extracting protein from SSM and compared with alkaline. Result indicated that alkaline could effectively extract 56.9% protein from SSM and its protein content was 73.1%, higher than DES at 31.0%-41.4% and 64.3%-67.5%, respectively. However, compared to alkali, DES led to a product with less β-sheet, more β-turn, more essential amino acids, higher total amino acid content, especially choline chloride-urea which extracted protein showing an integrated and similar protein weight distribution compared to SSM. Also, this protein extracted chloride-urea showed a highest digestibility in vitro(by pepsin)(54.2%). These results indicated that choline chloride-urea extraction is better than alkaline extraction for SSM.

Protein Extraction Methods for Two-Dimensional Electrophoresis from Baphicacanthus cusia(Nees)Bremek Leaves-A Medicinal Plant with High Contents of Interfering Compounds

Protein extraction is a critical step for two-dimensional electrophoresis （2-DE）. Different plant samples require different and adaptive protein extraction protocols. The leaves of medicinal plant, Baphicacanthus cusia （Nees） Bremek are notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds （especially the secondary metabolites and pigments）. This study was aimed to establish a routine procedure for the proteomic analysis ofB. cusia leaves, and a new protocol for the protein extraction was developed by optimizing trichloroacetic acid （TCA）/ acetone extraction method. The efficiency of this protocol was demonstrated by comparison with 3 published protein extraction methods （chloroform/acetone, Mg/NP-40, Tris-base/acetone）. The results showed that the optimized TCA/ acetone precipitation extraction method gave a relatively high protein yield （9.263 mg g^-1 fresh weight）, high-resolution separation, clear protein profiles, the highest proteins spots （1 31 t protein spots）, and displayed less contamination in 2- DE gels. Therefore, the results suggested that the optimized TCA/acetone method was the most effective among the 4 methods for B. cusia leaves.

Methodological approach to the isolation of functionally active proteins from the tissues of marine hydrobionts: an example of Adamussium colbecki

Enzymes from cold-adapted organisms have significant application potential. Because of their unique properties they have been found to be useful in various industries. Despite indisputable practical interest, cold active enzymes also represent a valuable model for fundamental research into protein folding and catalysis. Many investigators have focused their attention on marine hydrobionts, which are growing in importance as a promising source of enzymes. The nature of the source not only determines the availability and the cost of biomolecules of interest but also determines the choice of method for their extraction. A simple and convenient methodological approach of two-stage extraction of proteins has been tested on the Antarctic marine hydrobiont--Adamussium colbecki. This method extracts enough effective protein directly from primary raw materials, as well as when using leftover crude precipitates. The electrophoretic pattern of proteins showed the presence of molecules in a wide range of molecular weights in the samples of A. colbecki after the first and the second stage of extraction. The general proteolytic activity in the first and the second extracts were examined using a zymogram technique. Our experiments revealed that the second extract of A. colbecki contained thermo stable protease exhibiting a molecular weight of 95 kDa in a gelatin zymogram. Further biochemical assays, using different substrates, were conducted to partially identify the types of hydrolases present in the first and the second extracts. Our results revealed the presence of enzymes with collagenolytic and some amylolytic activities preserved in the second extracts. But no esterase or amidase trypsin-like activities were found in the second extract, in contrast to the first extract where this type of activity was significant.

A Simple Procedure for Extraction of Surface Protein of Salmonella Serotypes and Escherichia coli Strains Isolated from Poultry and Pigs

Salmonella and E.coli possess different surface protein structures that can induce protective immune responses.Identification of these proteins capacitates development of diverse applications in prevention and diagnosis that contribute to effectively control disease-causing enterobacteria pathogens such as Salmonella and E.coli.A simple procedure for obtaining protein complexes of Salmonella serotypes and E.coli is performed in this study.A sonication process with heat treatment of whole bacteria induced the release of protein complexes.Concentration of the protein extract was quantified using protein quantification Kits-Rapid,and protein complex profile was obtained by SDS-PAGE(Sodium dodecyl sulfate polyacrylamide gel electrophoresis)and silver staining.The concentrations of protein ranged from 29.45 to 45.35μg/mL in the Salmonella protein extracts,and from 25.35 to 36.72μg/mL in the E.coli protein extracts.Six major groups of proteins from E.coli(YfiO,NipB,OmpF,YfgL,Talc,YaeT)and four major groups of proteins from Salmonella(Flagellin,OmpA,Porin,SEF21)were preliminarily determined by a simple procedure of extraction based on the molecular weight.

Overview on pulse proteins for future foods:ingredient development and novel applications

We are facing the challenge of climate change and food insecurity for a growing population.The current mode of animal protein production via animal agriculture is resource-intensive and unsustainable.Therefore,there is a need to find alternative sources of food protein that are environmentally sustainable.Plant-based proteins,specifically pulse-based proteins,provide a promising solution to the problem.This review aims to provide an overview and perspective on extraction,functionality,digestibility,sensory,and new food applications of pulse proteins.Two main methods,namely wet fractionation and dry fractionation are used to extract pulse-based proteins.As compared to dry fractionation,wet fractionation yields high purity protein,but the process alters the structure and function of the proteins.Various biochemical and physical techniques can be used to assist wet extraction process to increase protein yield and/or reduce extraction time.The main techno-functional properties of plant-based proteins determining their practical applicability are solubility,water/oil holding capacity(WHC/OHC),gelation,emulsification,foaming,and rheological properties.Nutritionally,pulse proteins are deficient in one or more essential amino acids.Strategies to overcome the deficiency are discussed.Volatile and non-volatile compounds inherent to pulses or developed during processing are mostly responsible for the off flavors in the extracted protein.Approaches to improve the pulse protein flavor and remove or modify off-flavors are discussed.Pulse-based protein ingredients have applications in bakery products,pasta,meat analogues,dairy alternatives,and beverages.Beyond these applications,there is a need to explore novel applications of pulse proteins in infant and children’s formula,beverages,breakfast cereals,flavor development,and extruded snack products,develop new applications such as personalized and precision nutrition and bioactive peptides,and employ innovative technologies such as 3D printing,extrusion,and artificial intelligence for pulse protein research.This review presents the current status,limitations,and future perspectives for developing pulse protein ingredients as future foods.This review aims to foster thinking and generate novel ideas for future research.

Recent Advances in Protein Extraction and Chiral Separation of Biomolecules

Reverse micelles create unique environment in organic media. They are capable of solubilizing hydrophilic biomolecules （e.g., proteins, peptides, amino acids, and DNAs） in their aqueous interior. This feature brings about the practical use of biomaterials in organic media because reverse micelles solubilize them with the intrinsic activity. In this paper, we focus on recent two topics concerning protein extraction and chiral separation of biomolecules using liquid membranes. In the first topic, we present recent attempts to extract proteins from an aqueous solution into isooctane using reverse micelles, and some important operational parameters to achieve an efficient protein transfer are discussed. Furthermore, novel function of reverse micelles as a protein activation medium is introduced. In the reverse micellar phase, denatured proteins were completely reactivated in the reverse micellar solution. The reverse micellar technique is found to be a useful tool not only for protein separation but also for protein refolding. Furthermore, we found that a cyclic ligand carixarene has an extraction ability to set up optimum conditions for protein transfer. In the second topic, we have found that a supported liquid membrane （SLM） encapsulating enzymes shows high enantioselectivity （enantioselective excess value is over 96%） in the transport of racemic pharmaceutical compound ibuprofen. A different experiment also suggests that the α-chymotrypsin-catalyzed reactions droved the enantioselective transport of L-phenylalanine based on the enantioselectivity of the enzyme. The SLM encapsulating the surfactant-enzyme complex enabled the highly enantioselective separation of racemic mixtures. It can be envisioned that arrangement of appropriate enzymes in the SLM system will allow enantioselective separation of various useful organic compounds.

Time-sequential changes of differentially expressed miRNAs during the process of anterior lumbar interbody fusion using equine bone protein extract, rhBMP-2 and autograft

The precise mechanism of bone regeneration in different bone graft substitutes has been well studied in recent researches. However, miRNAs regulation of the bone formation has been always mysterious. We developed the anterior lumbar interbody fusion （ALIF） model in pigs using equine bone protein extract （BPE）, recombinant human bone morphogenetic protein-2 （rhBMP-2） on an absorbable collagen sponge （ACS）, and autograft as bone graft substitute, respectively. The miRNA and gene expression profiles of different bone graft materials were examined using microarray technology and data analysis, including self-organizing maps, KEGG pathway and Biological process GO analyses. We then jointly analyzed miRNA and mRNA profiles of the bone fusion tissue at different time points respectively. Results showed that miRNAs, including let-7, miR-129, m iR-21, miR-133, miR-140, miR-146, miR-184, and miR-224, were involved in the regulation of the immune and inflammation response, which provided suitable inflammatory microenvironment for bone formation. At late stage, several miRNAs directly regulate SMAD4, Estrogen receptor 1 and 5-hydroxytryptamine （serotonin） receptor 2C for bone formation. It can be concluded that miRNAs play important roles in balancing the inflammation and bone formation.

A survey of current trends in computational predictions of protein-protein interactions

Proteomics become an important research area of interests in life science after the completion of the human genome project.This scientific is to study the characteristics of proteins at the large-scale data level,and then gain a holistic and comprehensive understanding of the process of disease occurrence and cell metabolism at the protein level.A key issue in proteomics is how to efficiently analyze the massive amounts of protein data produced by high-throughput technologies.Computational technologies with low-cost and short-cycle are becoming the preferred methods for solving some important problems in post-genome era,such as protein-protein interactions(PPIs).In this review,we focus on computational methods for PPIs detection and show recent advancements in this critical area from multiple aspects.First,we analyze in detail the several challenges for computational methods for predicting PPIs and summarize the available PPIs data sources.Second,we describe the state-of-the-art computational methods recently proposed on this topic.Finally,we discuss some important technologies that can promote the prediction of PPI and the development of computational proteomics.

PRELIMINARY STUDY OF EXTRACTABLE PROTEIN BINDING USING MALEIC ANHYDRIDE COPOLYMER

A preliminary study of using maleic anhydride copolymer for protein binding has been carried out. The polymeric films were prepared by compression of the purified resin and annealing the film to induce efficient back formation of the anhydride groups. The properties of the film surface were analyzed by attenuated total reflection Fourier transforms infrared spectroscopy and water contact angle measurements. The protein content was determined by Bradford assay. To obtain optimum conditions, immersion time for protein binding was examined. Results revealed that proteins can be successfully immobilized onto the film surface via covalent linkage. The efficiency of the covalent binding of the extractable protein to maleic anhydride-polyethylene film was estimated at 69.87 ug/cm2, although the film had low anhydride content （3%） on the surface.

Proteomic Analysis of Stichopus japonicus and Evaluation of the Antiaging Properties of Its Peptide

The sea cucumber Stichopus japonicus is one of the"Eight Treasures of Seafood"and contains a number of bioactive components involved in multiple physiological and pharmacological functions.Proteins and peptides are generally considered to be responsible for these beneficial properties.In this study,a total of 3478 proteins and 17390 peptides were identified in Stichopus japonicus by proteomics methods.Among them,4 proteins were involved in 8 metabolic pathways,especially oxidative phosphorylation and cell senescence.Subsequently,lifespan assay and oxidative stress test were performed to investigate the peptides prepared from sea cucumber protein hydrolyzate using the aging model of Caenorhabditis elegans.The results of the anti-aging experiment demonstrated that high-dose peptides significantly prolonged the lifespan of nematodes(30.50%),and improved their capacity to inhibit oxidative stress.The results provide evidence supporting the development of bioactive proteins and peptides derived from Stichopus japonicus as functional foods and lay the foundation for the research of an anti-aging drug.

Effect of Huannao Yicong Prescription (还脑益聪方) Extract onβ-Amyloid Precursor Protein Metabolic Signal Transduction-Related Protein in Brain Tissue of Dementia Model Transgenic Mouse

Objective： To observe the effect of Huannao Yicong Prescription （还脑益聪方, HNYC, a Chinese medical compound） extract on β-amyloid precursor protein （APP） metabolic signal transductionrelated protein kinase C （PKC）, tyrosine amyloid protein kinase （TrKA）, and glycogen synthase kinase-3 （GSK-3） in brain tissue of transgenic mouse dementia model induced by APP. Methods： Sixty dementia model transgenic 3-month-old mice induced by APP695V7171 were randomly allocated in four groups： the model group （A）, the Donepezil （0.65×10^-3 g.kg-1.d-1）-treated group （B）, and the two HNYC-treated groups （C and D） with high dosage （2.8 g.kg^-1.d^-1） and low dosage （1.4 g.kg^-1.d^-1） of HNYC extract, respectively, 15 mice in each group. Besides, a normal control group was set up with 15 C57BL/6J mice with the same age and genetic background as the model mice. The drugs for treatment were administered once a day by dissolving in equal-volume distilled water through gastric infusion, continued for 6 months, to mice in group A and to normal control group equal-volume distilled water was administered instead. Spatial learning and memory capacity of mice were observed by Morris water maze; their one-time escape response memory capacity was tested by diving platform; and changes of PKC, TrkA, and GSK-3 levels in hippocampus and cortex of brain were detected by Western blotting. Results： HNYC extract showed significant effects on increasing the time of model mice for swimming through the flat roof and the swimming time and path in the fourth quadrant （P〈0.05 or P〈0.01）. Diving platform test showed that the latent times in Groups B and C were longer than that in Group A significantly （P〈0.05 and P〈0.01）. Compared with the normal control group, PKC and TrkA protein expression levels in hippocampus and cortex of model mice＇s brain lowered significantly （P〈0.01）, while GSK-3 protein expression increased significantly （P〈0.01）; compared with Group A （the model group）, hippocampal and cortical levels of PKC protein expression in the intervened groups （B-D） as well as those of TrkA in Group C were higher （P〈0.01 or P〈0.05）, while hippocampal levels of GSK-3 in intervened groups were lower （P〈0.01）. Conclusion： HNYC extract could obviously increase the protein expressions of PKC and TrkA and decrease the expression of GSK-3 protein in brain tissue of transgenetic mice model of dementia, and regulate APP metabolic signal transduction path, and thus to suppress the production of A β, which is one of the dominant mechanisms for improving learning/memory capacity of dementia model animals.

Chemical characteristics changes associated with extracted protein and salt addition and effect of different additives in the curing process on sensory acceptance of liquid fermented fish from tilapia frame

Liquid fermented fish(Nam pla ra)is a traditional local condiment and popular consumption in Thailand.According to previous research,tilapia frame could be used as a raw material for the production,but the amount of protein received is still below the standard.In addition,there is no information of additives in curing process on consumer preferences.In this research,response surface methodology was used to study the influence of extracted protein from soybean meal(5-15%)and salt(15-20%)contents on chemical characteristics of Nam pla ra after 4 months of fermentation.The amount of roasted rice and roasted rice bran addition in curing process on sensory preferences were also studied.The results showed the linear effect of extracted protein and salt contents on NaCl content(P<0.05).When extracted protein increased,NaCl content of sample decreased,while increased salt concentration,NaCl content increased.Extracted protein showed linear relationship with autolytic degradation products and formaldehyde nitrogen of sample(P<0.05).Increase in extracted protein concentration caused an increment to both autolytic degradation products and formaldehyde nitrogen.In addition,salt content showed linear relationship with protein,total nitrogen,ammonia nitrogen and amino nitrogen(P<0.05).When increased salt concentration,protein and all kind of nitrogen tested in this study decreased.The statistical models for protein and NaCl contents(as an index of product quality)and our experimental validation,indicated that the models were appropriate to predict values.For acceptability test,there was no significant different in all attributes scores of cured samples with 10%roasted rice,roasted rice bran and mixture of both.In addition,their showed a significant different in all attributes scores lower than the commercial sample.In conclusion,the highest protein content was found in a treatment prepared by added salt content 15%by weight of digested material.Addition of proteins extracted from soybean meal did not lead to increase in protein content of finished product.However,chemical properties of all treatments could meet Thai community product standard.

Effect of Early Intervention with Extract of Huannao Yicong Decoction(还脑益聪方) on the Pathologic Picture of Hippocampus and Neurocyte Apoptosis in APP Transgenic Mice Model of Dementia

Objective：To observe the effect of early intervention using extract of Huannao Yicong Decoction （还脑益聪方,HYD） on the pathological picture of hippocampus,neurocyte apoptosis,and associated regulatory genes inβ-amyloid precursor protein（APP） transgenic mice model of dementia.Methods：Sixty APP695^（V7171） transgenic mice were randomly divided into four groups of 15.The model group was treated with distilled water, the positive control group was treated with donepezil（0.65 mg/kg）,and the two HYD groups were treated with high dose（2.8 g/kg） and low dose（1.4 g/kg） HYD,respectively.All testing drugs were administered through gastrogavage by dissolving in equal volume of distilled water,once a day for six successive months.In addition, a normal control group with 15 healthy C57BL/6J mice of the same age and genetic background was set up with distilled water treatment.The pathologic picture of brain tissue was observed by microscopy with HE stain;the amount of apoptosis cells in the hippocampal CA1 area was detected by TUNEL;and expressions of associated genes,Bcl-2,and Bax were determined by immunohistochemical method.Results：Pathologic pictures of hippocampus showed that in the model group,cells messily arranged,neurons markedly decreased, and the surrounding tissue of some cells was loosened with edema,necrosis,and widened gap with glia cells proliferation.Compared with those in the normal group,the amount of apoptosis cells in the CA1 area was increased,Bcl-2 expression decreased,and Bax expression increased significantly,with markedly reduced Bcl-2/Bax ratio in the model group.Compared to the model group,the pathological changes were significantly milder in the HYD-treated groups,showing rather regularly arranged cells,significantly increased neurons, only few denatured necrotic cells with milder edema,less proliferation of glia cells,and obviously reduced cell apoptosis in CA1 area（P0.05 or P0.01）.Besides,Bcl-2 expression was up-regulated and Bax expression down-regulated,and Bcl-2/Bax ratio significantly increased in the two HYD groups（P0.05 or P0.01）. Conclusion：Early intervention with HYD could improve the abnormal pathologic picture of hippocampus and regulate the expressions of associated genes to suppress cell apoptosis,which might be its mechanism of action in alleviating cognitive functional disorder.