The Studies On Endogenous Peptide Composition Of Human Milk And Its Correlation With Lactation

There are a variety of protease-specific cleavage peptides in the human mammary gland.These endogenous peptides can better satisfy the weaker gastrointestinal digestive effects and may also be active in infants.The analysis of endogenous peptides in different human milk has been a global active research field,which not only helps to provide a theoretical basis for the promotion of gastrointestinal digestive capacity and the establishment of immune system,but also provides a useful exploration for the development of infant formulas that are closer to human milk.In this thesis,the endogenous peptides in human milk were separated and purified by ultrafiltration.A method for studying the composition of endogenous peptides in human milk by LC-MS/MS was established.The human milk endogenous peptides of different individuals in different lactation periods were established.Peptide composition characteristics were analyzed for screening potentially active antimicrobial peptides.The main parts of this paper were divided in three sections:,The main results were as follows:(1)Protein concentration and peptide concentration.The protein concentration of colostrum was 12.423 mg/mL-44.424 mg/mL and the peptide concentration was 0.98 mg/mL-5.40 mg/mL;the protein concentration of mature milk was 9.366 mg/mL-22.254 mg/mL and peptide concentration was 0.52 mg/mL-6.12 mg/mL.Correlation analysis showed no significant significance between the two variables of endogenous peptide concentration and protein concentration.The data of all samples showed that protein concentration and endogenous peptide concentration were higher in colostrum than in mature milk.(2)The profile of endogenous peptides.The number of endogenous peptides identified by LC-MS/MS of the mixed samples was 771-1691,and the number of proteins releasing the endogenous peptide was 24-55.The number of endogenous peptides identified by LC-MS/MS for independent samples was 380-2536,and the number of proteins releasing endogenous peptides was 21-137.Statistical analysis showed no significant differences between the number of peptides in different lactation periods,while the number of protein precursors was significantly greater for colostrum amples than mature milk samples.The top ten proteins in different samples have high similarity.The majority of milk peptides in both colostrum samples and mature milk samples were derived from β-casein(>50%).Other major protein precursors included immunoglobulin receptor,αs1-casein,osteopontin and κ-casein,etc.There were 950 peptides in the fully mixed colostrum and mature milk samples,which accounted for 40% of the total peptides.There were 14 active peptides that were matched in the MBPDB database,involving immunological activity,antioxidant activity,ACE inhibitory activity,antibacterial activity,and stimulating proliferation activity.(3)High-intensity peptide.A total of 70 peptides of the top 20 endogenous peptides with the highest intensity among the four groups mixed samples.These peptides were derived from αs1-casein,lactoferrin,immunoglobulin receptor,osteopontin and β-casein.Among the 70 peptides,66 peptides were derived from β-casein with the highest intensity,and these peptides were mainly concentrated in the amino acid range of 180-226 of the β-casein sequence.(4)Escherichia coli and Staphylococcus aureus were selected as representative strains to verify whether the selected peptide fragments had broad-spectrum antibacterial ability.The results of inhibition zone experiments showed that the six peptides obtained by screening had obvious inhibitory effects on Escherichia coli and Staphylococcus aureus.When the sample loading was 3.5 mg,the inhibition zone diameters for the two bacteria were 0.85-0.50 cm and 0.85-0.40 cm,respectively.