Study On The Cell Membrane Chromatographic Preparation Method And Antibacterial Properties Of Antimicrobial Peptide From Casein

Recently the dairy industry has been developing rapidly in our country. However, thesingle product structure and the low added value objectively request the deep processing ofmilk. Casein is a crucial component of milk, and utilizing casein is one way to improve theeconomic value of milk. The antimicrobial peptide from casein is expected to be a naturalantibacterial peptide and used widely. However, the complicated purification steps ofantimicrobial peptide limits its applications. Therefore, establishing an efficient method forpurification is urgent.Casein was hydrolyzed with protease in this paper. The method of solid-phase extractioncombined with HPLC fingerprint (CMC), was developed to purificate antibacterial peptideaccurately. The antimicrobial and stabilization of the antibacterial peptide, and effect on cellgrowth curve and cell wall components were stuided, which provides the basis theory for thedevelopment and utilization of casein.Firstly，casein was hydrolyzed by alcalase, pepsin, neutral protease and alkaline protease.Using the antibacterial activity against S. aureus as an index, pepsin was selected for the bestenzyme. Results showed that the optimal conditions for preparing antibacterial peptide: caseinconcentration10mg/mL, the ratio of enzyme to substrate (E/S)1:15, pH2.0, temperature37℃, reaction time3h.Secondly，combined treatment with repeated freezing and thawing, ultrasound was usedto break the Saccharomyces cerevisiae cell walls, and then prepare cell membrane withgradient centrifugation technique. The Saccharomyces cerevisiae cell membrane stationaryphase was prepared with the silica gel as a carrier, which properties was characterized bySEM. The experiment was carried out with repeated freezing and thawing (water content25%,freezing and thawing4times) followed by ultrasound (400W, duration of ultrasound10s,15min, Saccharomyces cerevisiae concentration20mg/mL). The results showed that the brokenratio of Saccharomyces cerevisiae cell walls reached89.31%, which was2.4times only therepeated freezing and thawing, and1.5times only by ultrasound. SEM showed that thebumpy surface of SEMSP had been encircled. Csmax, the maximum adsorption capacity ofSCM protein on the silica surface, was2.38mg/g; K*, the Langmuir constant, was325.61mL/mg.Thirdly, CMC was developed to screen antibacterial peptide from casein hydrolysatessuccessfully. With MALDI-TOF-MS/MS technology, the amino acid sequence of antibacterialpeptide was Leu-Ala-Leu-Lys-Lys-Tyr-Lys-Val-Pro-Gln-Leu. Antibacterial experiment showed that the separation of casein antibacterial peptide with cell membranechromatography was feasible. The antimicrobial peptide had antibacterial activity againsgram-positive and gram-negative and had good thermal stability. It could resist the changes ofthe ionic strength, and was stable at room temperature.Lastly, the effect of antibacterial peptide on growth curve, the damage of innermembrane of S. aureus in the living cell environment was measured by optical densitymethod. Results showed that the antibacterial peptide from casein could destroy the wallmembrane structure and lead to the death of S. aureus.