Preparation Of Lactoferrin Hydrolysates And Antimicrobial Effect Of Its Modifiers

Lactoferrin is an important source of antimicrobial peptides. Hydrolyzation of lactoferrin was performed by pepsin. Its modifiers were prepared by plastein reaction, plastein reaction introducing amino acid, or deamidation modification. Antibacterial activity of the modified products was evaluated. The results of the study were as followed:(1) Preparation of lactoferrin hydrolysates by pepsin hydrolysis of lactoferrin The concentration of lactoferrin was fixed in5%, reaction time4h, reaction temperature37℃, E/S (680U·g-1,1000U·g-1) and pH (2,2.5,3) were optimized, and antibacterial activity of hydrolysates was detected. Hydrolysis conditions was determined:substrate concentration of5%, pH with2.5, E/S for1000U·g-1, the reaction time of4h, the reaction temperature of37℃.(2) Selection of enzymes in the plastein reaction modification Antibacterial activity of plastein reaction modifiers by papain or pepsin was investigated. The free amino contents of modification products of papainwas increased significantly, antibacterial activity decreased at the same time; the free amino content of modification products of pepsin also decreased significantly, but antibacterial activity increased. Thus pepsin was selected as the catalytic enzyme of plastein reaction.(3) Optimization of reaction conditions in the plastein reaction modification Plastein reaction was catalyzed by pepsin using the single factor experiment. The substrate concentration, E/S, reaction pH, reaction temperature, and reaction time were studied in the modification reaction. Antibacterial activity was investigated. Optimization of reaction conditions were:substrate concentration40%, E/S2kU·g-1, reaction pH5, reaction temperature37℃, reaction time12h. Antibacterial activity for5strains (Escherichia coli, Bacillus subtilis, Bacillus cereus, Micrococcus luteus,&Staphylococcus aureus) was increased by41%-65%.(4) Introducing of extrinsic amino acids in the plastein reaction modification Optimization of reaction conditions in the plastein reaction modification were used, and tryptophan or arginine was added to the reaction system. The addition ratios of exogenous amino acid (ratio of amino acids of total free amino content to lactoferrin hydrolysate) were optimized. For the modifiers introducing tryptophan in the plastein reaction, the addition ratio of was0.7, and antibacterial activity increased by55%-79%; for the modifiers introducing arginine, the addition ratio of was0.3, and antibacterial activity increased by54%-76%.(5) Deamidation modification of lactoferrin hydrolysates The effect of antibacterial activities in different deamidation degrees was investigated. Along with the increasing of the reaction time, lactoferrin hydrolysate deamidation degrees were increased, and the degrees of hydrolysis were increased correspondingly, while the antibacterial activities were decreased. The results showed that the deamidation modification was very important to the antibacterial activity of lactoferrin hydrolysate.(6)It was found that lactoferrin can promote the growth of Escherichia coli in some way in our study of the inhibitory effect of it on the growth of Escherichia coli. The results showed that if antimicrobial protein was developed as a food preservative,"select the appropriate concentration,""strictly limited shelf-life", and "controlling the initial contamination of raw food materials" should be emphasized.(7) The hydrolysization of LF by cell lysis liquid of Escherichia coli was also tested. The antibacterial activity and free amino content of test products were investigated. Results showed that lactoferrin was not hydrolyzed, and the antimicrobial activity appeared no significant difference. It might be because that the types and doses of proteases in Escherichia coli were different or because of the structure change of LF affter the inhibitory reaction. Direct evidence needs further research.