Study On Enzymatic Preparation And Isolation Technology Of Antimicrobial Peptide From Whey Protein

In this study, whey protein was used as the raw material, and trypsinase was used as thehydrolysis enzyme. The parameters of the enzymatic hydrolysis were optimized to determine thebest hydrolysis process. And the ultrafiltration were used for the isolution of the polypeptidewhich were finally found have obviously antibacterial activity. The research were included as thetwo following parts:1. Study on the parameter optimization of whey protein enzymatic hydrolysates. Withantibacterial activity as the evaluation index, different degree of hydrolysis of whey proteolyticdigestion liquid was studied and analyzed Oxford cup plate, turbid metric, filter paper methodwere used respectively to dectect the antibacterial activity. The results showed that the mostantimicrobial activity was obtained when the degree of hydrolysis in enzyme solution was18%.Oxford cup plate was found to be the most convinent method to perform effective andintuitive.The diameter of inhibition zone measured by Oxford cup plate method reaches1.32cmwhen the degree of hydrolysis in enzyme solution achieves18%.With antibacterial activity as theevaluation Index, single factor test (substrate concentration, pH, temperature andenzyme-substrate ratio, etc) and response surface regression analysis were carried out on the basisof degree of hydrolysis. The optimum enzyme hydrolysis conditions of trypsin were identified as:substrate concentration8.3%, pH8.5, temperature46.0℃, enzyme-substrate ratio5000U/g, andtime3h. The attained degree of drolylsis was about18.91%,and bacteriostatic circle diameter was1.58cm, which provided strong antibacterial activity for staphylococcus aureus.Studying on theenzymatic hydrolysis product of the molecular weight distribution,found that molecular mass oflactoferrin antimicrobial peptide distributed mainly in2000-8000Da.2. Study on the conditions of whey protein peptide ultrafiltration. With membrane flux as theevaluation index, influence of membrane pressure, initial concentration and temperature ofmaterial liquid, and time on ultrafiltration process were researched by the single factor test andresponse surface regression analysis. The results showed that the optimum operating conditionsare as follows: a TMP of0.5bar, an initial material liquid concentration of16.4mg/mL, a materialliquid initial temperature of8.5℃and a ultrafiltration time of60min. Under the conditions,whey polypeptides achieve the most antibacterial activity. The bacteriostatic circle diameter andmembrane flux are about1.67cm and70.25L/m2·h, The contents of amino acids in whey proteinpeptides were performed, in which the highest amount of free amino acids was leucine andsecondly was lysine. Their contents were19.26%and18.46%respectively.In this paper, the trypsin hydrolysis progress of whey protein were optimized for theproduction of antimicrobial peptides The ultrafiltration and gel filtration chromatographytechnology were used to sepearte and purify the protein peptide The results show that wheyprotein peptides with strong antibacterial activity for staphylococcus aureus can be obtained basedon and the hydrolysis process and the separation technology proposed in the paper.