Research On Preparative Technique Of Antibacteial Peptides From Casein

Antibacterial peptides are called antimicrobial peptides or peptide antibiotics, usually the amino acids number of it less than 100, have positive charge.Besides discovered the antibacterial peptides in bacteria, plants and animals, researchers showed that food proteins wound release antibacterial peptides after proper enzymatic hydrolysis.Casein is one kind of complete protein (has 8 kinds of amino acid essential for organism), its content in milk proteins is 78.8%.In recent years, the researchers show that casein has not only nutritional function, but also has physiological function.It is the important source of bioactivity peptides which includes antibacterial peptides, casomorphin, antihypertensive peptides, antithrombotic tetrapeptide, the peptides promoting mineral ions absorption, and so on. The studies show that these bioactivity peptides can be obtained from the enzymatic hydrolysis of casein with suitable enzyme. Antibacterial peptides have widely antibacterial function, which is being attentioned by society increasingly because of its advantages of well stability and water solubility and unique antibacterial mechanism and harmless to normal cells of higher animals. At present, the researches about antibacterial peptides obtained from food protein are less. The antibacterial peptides obtained from enzymatic hydrolysis of casein with pepsin was researched in this paper. The following are the research contents:The first part: study of hydrolysis condition. The effect of substrate concentration ([S]),enzyme concentration ([E]),pH and temperature CO on hydrolysis degree(DH)which meaured by colorimetric method were studied. Orthogonal design of four factors three levels was taken to optimize the hydrolysis condition of casein with pepsin. The results show that the optimum hydrolysis condition was substrate concentration 1%, enzyme concentration 2%, pH1.5, temperature 40℃.The second part: based on the results of the first part, showed different hydrolysis time (0h,0.5h,lh,1.5h,2h,2.5h,3h,3.5h,4h,4.5h,5h,5.5h,6h)to study using rotary evaporator to concentrating the hydrolysate of different hydrolysis time , concentration multiple was 1:2,1:4 and 1:6.and make bacteriostatic experiment(filter paper method)of these concentration hydrolysate using staphylococcus aureus, bacillus cereus, aspergillus niger, gas production bacillus and escherichia coli. The results showed that: the antibacterial effectall to aspergillus niger of the hydrolysate of different concentration multiple and hydrolysis time were not obvious. When the hydrolysis time was 3.5h and 5h, the hydrolysate of different concentration multiple have better antibacterial effectall to the other bacteria , and the hydrolysis time of 3.5h was better than 5h, the inhibition spectrum of staphylococcus, aureusbacillus and cereusescherichia coli were most obvious.The third part: based on the results of the second part, studied on the antibacterial substance in casein. Adopt Superdex 75 10/300 column to separate casein hydrolysate of hydrolysis time 3.5h. Absorbed the standard on the column under optimal conditions, using 0.5ml/min buffer solution to elute. Based on the eluted volume, draw calibration curve, separated the hydrolysis sample of hydrolysis time 3.5h.Make bacteriostatic experiment of solution samples of different peak .The result was peak 4 has strongest inhibition spectrum.Then in order to purfied peptides, The bud compatriot bacillus choosing a sample of wax is a peak as instructing bacterium to sift the peak having antibiosis activity 4—1. And, according to the K_av,=-0.3853 lg M+2.0126standard, the curve judgement washes the molecular weight(that is the antibacterial substance obtained from the casein hydrolysis ) taking off a peak being 3386 Da. These studies will help the study on foods sourced- antibacterial peptides.