The Influence Of Hydrolytic Proceeding On The Antibacterial Activity Of The Lactoprotein Hydrolysate

Bioactive peptides are the peptides that can regulate the metabolism, and exist in the protein in the form of polypeptide chains'sequences. The hydrolysis of natural protein by protease is the most feasible approach to attain diverse bioactive peptides. The hydrolysis method includes two types, namely batch-type hydrolysis and continuous hydrolysis utilizing the enzymatic membranes bioreactors (EMB). The former is easy and simple to control and has low automaticity; while the latter can be operated continuously and has efficient output.Antibacterial peptide which is one of the important categories of bioactive peptides offers vital protection to organism to resist microbial attack. The antibacterial peptide restrains the microorganism through various mechanisms, such as destroying the structure of cell membrane, inducing the cell apoptosis and inhibiting the cell respiration and DNA synthesis. Lactoprotein, as one kind of the natural protein, is an important material in the production of antibacterial peptide. The hydrolysates of the casein, whey protein and lactoferrin which are the ingredients of lactoprotein have different influences on the microorganism.This study compasses three parts. Firstly, the study optimized the separation technique of lactoprotein, and suggested new detection methods for some of the proteins. Secondly, using the batch-type reaction, different kinds of lactoprotein were hydrolyzed for the purposes of the optimization of hydrolysis parameters and the analysis of hydrolysates'antibacterial activities. Thirdly, antibacterial peptide was continuously produced by a designed enzymatic membrane bioreactor.The casein was separated from defatted milk powder and reach 7.8% yield through the isoelectric point precipitation. The whey was processed with the ultrafiltration membrane (10K) in the neutral pH condition, in order to erase the lactose and salinity and produce high purified whey protein isolate.In this study, a method to determine the lactoferrin in milk products by HPLC was identified. In a bid to obtain a configuration of lactoferrin that can be precipitated easily, the lactoferrin in milk was saturated by additional ferrous ion. Subsequently, the milk was precipitated by 60% thanol to eliminate the sugar, and dissolved in acetic acid buffer to remove casein protein and extract lactoferrin. The remaining product was then measured by HPLC, using LiChrosorb RP-C18 as separation column, linear gradient elution by methanol and water with the assistance of 0.1%TFA. The linear range of the lactoferrin detection is between 0.4 to 2mg/mL (R2= 0.9980), and the average recovery rate was 95.4%. This method is useful in detecting the lactoferrin in the milk products.In addition, an alternative method to determine the lactoferrin in milk powder, based on iron binding function of lactoferrin, was proposed and compared with the HPLC method. First, ferrous ion was added to lactoferrin to achieve a saturated iron ion binding. And then, the protein was precipitated by the ethanol and dissolved in water repeatedly in order to eliminate the excessive iron ion. The amount of iron in lactoferrin was measured by atomic absorption spectrophotometer. Through the correlation between the amounts of iron and protein, the amount of lactoferrin can be calculated. This method has advantages in terms of accuracy and speediness. The lactoferrin recovery rate is above 96%. The lactoferrin detection ranges from 3.5 to 100 mg/g. This method can be useful in the detection of lactoferrin in the infantile milk powder.The colostrums were preprocessed with the isoelectric point precipitation and pressure filtration to remove the caseins. In the condition of pH 7.5, the ethanol was mixed into the whey up to 50% of the mixture. The addition of the ethanol assists the precipitation of lactoferrin in the whey and to be harvested as crude product. The crude product was further purified with low pressure liquid chromatography and desalted with tangential flow ultra filter to yield high purity lactoferrin. The HPLC and HPEC analyses of lactoferrin indicated that the impurity content of lactoferrin is below the range of detection.The parallel test was employed to design the experiments for the optimization of lactoferrin hydrolysis. The calculation of response surface regression performed with software Mathematic 7.01 showed that the optimum parameters of lactoferrin hydrolysis by the pepsin are substrate concentration 2%, temperature 37℃, pH 2.2-2.3, and dosage of enzyme 7U/g, when the aim of experiment is to maximize the degree of proteolysis.The orthometric experiments aimed at optimization of whey protein hydrolysis were designed by the software SPSS 18.0. The optimized parameters attained from the orthometric experiments were further used to formulate a univariate general linear model. The data of parameters estimated by the univariate general linear model were compared with the real data using the variance analysis. The results revealed that the optimum parameters for the whey protein hydrolysis by the alcalase include the ratio of enzyme to substrate 5%, pH 8.0 and temperature 45℃. The hydrolysate does not inhibit the pathogenic microorganism, but facilitate the growth of probiotics.The casein was hydrolyzed by the mixture of trypsin and neutral proteinase (ratio 1:1). The response surface analysis in the Box-Behken function was used to design the experiments, employing the software Design Expert. The results of experiments were then analyzed with regression analysis, which revealed that the degree of hydrolysis can reach 15%, with pH 7.4, substrate density 32g/L, ratio of enzyme to substrate 2.5%, and 90 minutes of hydrolysis. When the degree of hydrolysis reaches 15%, the antibacterial activity of polypeptide product is strongest. The polypeptide can also boost the growth of probiotics.In this study, lactoferrin was continuously hydrolyzed to generate lactoferrcin by a designed EMB apparatus. The membrane used in the experiment was the polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 10k. The study found that the EMB can have stable output, if the supplement rate of substrate is well controlled and the enzyme was added on line. The production of lactoferrcin and the conversion rate of lactoferrin were higher in the EMB reactor than in the batch reactor.