Preparation And Proteomic Analysis Of Antibacterial Peptides From Milk

As an important food source for protein intake,milk has high nutritional value and edible value.Milk protein can not only promote the absorption of trace elements and vitamins,but also produce a variety of biologically active components with physiological activities.In this paper,casein was taken as the research object,the preparation process of casein antimicrobial peptides was set up and optimized,and the types and contents of casein antimicrobial peptides were systematically analyzed by proteomics technology.In addition,some characteristics of milk derived antimicrobial peptides were studied.The antimicrobial peptides were screened by combining the peptide spectrum data identified by high performance liquid chromatography-mass spectrometry with the structure-activity relationship of antimicrobial peptides,and their activities were verified.This study provides theoretical support and a technical scheme for the development of antibacterial peptides derived from milk,and provides a direction for the future research of peptides with potential biological activity.The paper is divided into three parts,and the main research methods and results are as follows:(1)Casein was used as raw material to prepare casein antimicrobial peptides according to the preset preparation process.Using casein antimicrobial peptide as raw material,the inhibition rate of streptococcus mutans and porphyromonas gingivalis as index,the factors were screened by single factor method.The central combination test was designed by Box Behnken,the preparation process of casein antimicrobial peptide was further optimized,regression simulation equation was established.The three optimum reaction conditions were as follows: substrate concentration 5.5%,enzyme dosage 11000 U /g,reaction time 5 h;substrate concentration 4%,enzyme dosage10000 U /g,reaction time 3.2 h;substrate concentration 5.5%,enzyme dosage 11000 U/g,reaction time 4 h.The antibacterial rates of the obtained products against Streptococcus mutans were 92.43%,95.73% and 87.88%,respectively.The inhibitory rates against porphyromonas gingivalis were 93.61%,92.67% and 89.76%,respectively.The minimum inhibitory concentrations of casein antimicrobial peptides against streptococcus mutans and porphyromonas gingivalis in the range of 3.125-12.500mg/mL.(2)The proteomic analysis of casein antimicrobial peptides was further carried out,and the peptide compositions of casein antimicrobial peptides from different processes were compared.The results showed that 1239,1291,1151 peptides were identified in the three samples respectively,and 991 peptides were identified jointly,accounting for68% of the total number of peptides.The identified peptides were matched with active peptides in the MBPDB database.57,63 and 56 active peptides were matched from samples 1# 2# and 3#respectively.There were 10 antimicrobial peptides from k-casein P02668 F(63-70),?-casein P02666 in all three samples F(113-120)F(121-128)and F(208-224),?-s2-casein P02663 F(163-176)and F(213-222),?-s1-casein P02662 F(16-39)F(30-37)and F(110-119)and ?-lactoglobulin P02754 F(41-56)and F(141-151).Thereinto,the antimicrobial peptides derived from ?-casein were located in the range of ?-casein sequence 181-217,and the peptides produced in this region had the deepest coverage and the highest intensity.The active peptides were identified to the peptides with 80% homology.The active peptides in MBPDB database were matched with 314 potential active peptides,and the potential active peptides increased the activity of promoting cell proliferation Among them,113 had potential antibacterial activity.(3)The emulsification stability and potential of the optimized casein antimicrobial peptide were analyzed,and the morphological changes of bacteria treated by casein antimicrobial peptides were characterized by scanning electron microscopy.The results showed that the emulsifying ability increased first and then decreased with the increase of the standing time of casein antibacterial peptides,and the emulsifying stability decreased as a whole.After treatment with casein antimicrobial peptide or chlorhexidine,the morphology of the bacteria changed,and the surface of the bacteria appeared incomplete depression,and the optimized casein antimicrobial peptide had good antibacterial activity.The nephelometry was used to verify the bacteriostatic effect of the screened synthetic peptides.The experimental results showed that peptide 2 had the best inhibitory effect on gram-positive bacteria Streptococcus mutans and gramnegative bacteria porphyromonas gingivalis,proving its broad-spectrum antibacterial ability.