Differential Analysis Of Buffalo Whey Proteins And Screening,validation Of Colostrum Antimicrobial Peptides

Buffalo(Bubalus bubalis)is an important livestock breed adapted to the southern climate and has good economic development value.In recent years,milk buffalo breeding has developed into a characteristic industry in Guangxi agricultural economy,and has established the leading technology system of milk buffalo breeding and improvement in China.Buffalo milk is the world’s second largest milk source,which has great development potential,its rich nutritional value,high added value of products,known as the "king of milk".In addition to its high nutritional value,buffalo whey is also rich in proteins and endogenous peptides for antioxidant,blood pressure lowering,blood lipid lowering,immune regulation and other functions.The content of these bioactive substances varies with the lactation stage.Therefore,understanding the temporal expression and functional characteristics of buffalo whey proteins is the basis of product development.Isobaric Tags for Relative and Absolute Quantitation combined with Liquid Chromatography-tandem Mass Spectrometry technology to develop the dynamic changes of proteins and peptides from buffalo colostrum and mature milk,and analyze the immune mechanism of buffalo colostrum.On this basis,the antimicrobial peptides from colostrum were screened and verified,which provided a theoretical basis for the further development of buffalo colostrum products and contributed to the development of milk buffalo industry.The main contents of this study are as follows:(I)Differential anylysis of protein expression between buffalo colostrum and mature milk.i TRAQ technique was used to investigate the dynamic changes of buffalo whey protein in different lactation stages and to analyze its function.A total of 585 proteins were identified in buffalo colostrum and mature milk,and 107 differentially expressed proteins were found in buffalo mature milk as control group.Gene Ontology analysis showed that differentially expressed proteins were mainly located in organelles and cytoplasm,and were mainly involved in biological processes such as immune regulation.Dynamic changes of whey proteins in colostrum and mature milk were mainly related to immune regulation and energy metabolism.Analysis of the KEGG pathway showed that most of the proteins involved in the Interleukin(IL)and interferon signaling pathways jump-start the anti-inflammatory cycle.Differential protein interaction network analysis revealed five nodal proteins related to immunity,anti-inflammatory and antibacterial.Cytokine induction protein-Sh2(CISH)interacts with receptor tyrosine kinase(RTK)to regulate the transduction of IL-2,IL-4 and IL-13,and plays a role in the regulation of immune response from regulating the proliferation and differentiation of T cells and B cells.Meanwhile,functional analysis of endogenous peptides showed that buffalo colostrum contained more anti-inflammatory,antibacterial and immunomodulatory functional peptides.The specific expression of these proteins and endogenous peptides may be one of the physiological bases of the acquired immunity provided by buffalo colostrum.(Ⅱ)Screening and verification of antimicrobial peptides in buffalo colostrum.The enzyme digestion products were obtained by pancreatic enzyme treatment of buffalo colostrum whey protein.According to the physicochemical properties of the peptides,the enzyme digestion peptides and endogenous free peptides were purified by ultrafiltration and liquid phase separation.The bacteriostatic efficiency of each isolated component against Escherichia coli and Staphyloccocus aureus was tested.It was confirmed that buffalo colostrum contained more bacteriostatic peptides,and the short peptides below 10 k Da in colostrum had higher bacteriostatic efficiency.The components with the strongest antibacterial effect were identified by LC-MS/MS mass spectrometry.The amino acid sequence and other information of the candidate antibacterial peptides were obtained through database retrieval and prediction,and then the candidate peptides with antibacterial effect were obtained by synthetic method.Four peptides with antibacterial activity were obtained,among which,the antibacterial efficiency of N1 against four kinds of pathogens was greater than 80%,showing a broad spectrum of antibacterial potential.Stability tests showed that N1 had certain tolerance under different p H values,temperature,osmotic pressure and other conditions,which provided the basis for the development and application of buffalo milk antibacterial peptides.In conclusion,proteomics technology was used in this study to analyze the changes of bovine colostrum and mature milk whey proteins.Antibacterial,immune-related proteins and free peptides in colostrum may be the physiological basis for colostrum to provide acquired immunity.Antibacterial peptides were screened from buffalo colostrum and their functions were verified,which laid a theoretical foundation for the development of antibacterial peptides derived from buffalo milk.