Proteomic Analysis Of Anchovy And Cow Milk

Fish and cow milk are important protein-rich food sources in the sea and on land,respectively,with high nutritional and functional value.In this paper,we optimized the conditions of degreasing and enzymatic hydrolysis during the preparation of anchovy antimicrobial peptides.Furthermore,mass spectrometric-based proteomics analysis was used to analyze the peptide composition in the active peptide mixture,and anchovy antimicrobial peptides were synthesized and verified,which provided theoretical support and reliable technical scheme for the development of Marine animal antimicrobial peptides,and provided direction for the future research on the development of potential bioactive peptides.A filter-aided sample preparation method was used to separate proteins and endogenous peptides in cow milk,and an LC-MS/MS method was established to study the composition of proteins and endogenous peptides in cow milk,that provides useful clues to the development of food that is closer to people’s needs.The main research contents,methods and results of this paper are as follows:(1)With anchovy as the research object,the antimicrobial peptides of anchovy were prepared by degreasing and enzymolysis of extracts from anchovy.The inhibition zone diameter of Escherichia coli and Staphylococcus aureus was used as the index to optimize the preparation conditions of anchovy antimicrobial peptides.The results showed that the protein content of anchovy,anchovy after degreasing and anchovy after degreasing and enzymolysis were 16.84 g/100 g,37.41 g/100 g and 42.88 g/100 g,respectively.The inhibitory activity of anchovy enzymolysis mixture against Staphylococcus aureus showed a concentration dependence.The higher the concentration,the stronger the inhibitory effect.However,the mixture had no inhibitory activity against Escherichia coli.The minimum inhibitory concentration and minimum bactericidal concentration of anchovy antimicrobial peptides against Staphylococcus aureus were 12.5 mg/m L and 100 mg/m L,respectively.Furthermore,response surface method was used to optimize the preparation process of anchovy antimicrobial peptides.The optimal reaction conditions were substrate concentration: 4 %;the amount of enzyme: 2750 u/g;and reaction time: 6 h.The inhibition zone diameters of 25mg/m L and 50 mg/m L of anchovy enzymolysis mixture against Staphylococcus aureus at 12 hours were 5.95 mm and 8.15 mm,respectively.(2)Further proteomic analysis of anchovy antimicrobial peptides was performed.The peptides identified by LC-MS/MS were compared with the antimicrobial peptide database,and4 antimicrobial peptides with potential activity were synthesized.The antibacterial activity of the synthetic peptides was verified by plate drilling method.87 peptides were identified in the anchovy antimicrobial peptide mixture,corresponding to 68 proteins.When 2 mg antimicrobial peptides were added,the antimicrobial peptide GTEDELDKY had inhibitory activity against Escherichia coli,and the antimicrobial zone diameter was 7.0 mm.Antimicrobial peptides DREGESGPS,YIGRPIPQ and YEDEINKR showed inhibitory activity against Staphylococcus aureus,and the antimicrobial zone diameters were 5.2 mm,6.7 mm and 11.0 mm,respectively.(3)The upper layer proteins of milk were obtained by the method of filter-aided sample preparation after fresh cow milk was de-caseinated.The endogenous peptides of milk were collected by fresh cow milk solution treated directly with filter-aided sample preparation method.The protein and endogenous peptide separated by filter-aided sample preparation method were fractionated by liquid phase and Stage Tip at high p H,respectively,and the solution were collected.The LC-MS/MS technology was used for analysis,Max Quant software was used to search the original data collected by LC-MS/MS.The results showed that the number of milk proteins identified from unfractionated samples was 183,corresponding to 1608 peptides.956 endogenous peptides were identified from unfractionated samples,corresponding to 56 source proteins.After fractionating milk proteins and endogenous peptides,580 proteins were identified,and the corresponding peptides were 4368.After fractionation,1694 endogenous peptides were identified,corresponding to 126 source proteins.The main proteins identified after fractionating were β-lactoglobulin,α-lactoalbumin and albumin.It can be seen that the amount of proteins and endogenous peptides in cow milk after fractionating was 2.2 times and 0.8 times higher than that in unfractionated cow milk.This work could not only provide data support for the screening of bioactive peptides,but also provide sensitive and accurate methods for the analysis and detection of bioactive peptides,thus providing technical support for the development of protein(polypeptide)products in China.