Preparation And Study Of The Antimicrobial Peptides Of Casein

The milk proteins have rich nutrition,of which the casein accounts about 76%～86% includingαs1-CN,αs2-CN,β-CN andκ-CN.As an important nutrient,they have an wide applications.For example,they can be used for the production of the active peptides derived from the casein,for the strengthening of the baked protein and for the thickening of the margarine.Because of the widespread use of the antibiotics for a long time,the drug resistance of the pathogen has been greatly improved.People urgently need to develop new type of antibiotics among which antibacterial peptides derived from the casein have become one of the hot topics of the studies.The research used the casein as raw materials and hydrolyzed them with trypsin.Then,the separation and purification of antibacterial peptides of enzymatic hydrolysate was done.Finally,substances with antibacterial activities were obtained and their molecular weight was determined.The research is composed of 4 parts.The first part optimized the conditions of enzymolysis process of casein by trypsin. Using the degree of hydrolysis as an index,single factor experiment was done to study the influence of substrate concentration, enzyme-substrate ratio,pH and temperature on hydrolysis, and further,the single factor optimal conditions were determined.On this basis of using Box-Behnken central combination design,we optimized hydrolysis conditions by response surface analysis.The experimental result of the single factor experiment showed:the optimal hydrolysis conditions were substrate concentration 50mg/mL,enzyme-substrate ratio 1.5%,pH 8,temperature 55℃.Further result of response surface analysis showed the optimal hydrolysis conditions were:substrate concentration 47.2mg/mL,enzyme-substrate ratio 1.92%,pH8.31, temperature 54.8℃.The second part determined the optimal time of hydrolysis on which antibacterial peptides derived from the casein were released.According to the reported molecular weight of antibacterial peptides derived from the casein,we selected the polyethersulfone membrane with molecular weight cut off 10000Da or 20000Da for the ultrafiltration separation of the hydrolysate of the casein with different hydrolysis time and used the filter paper diffusion method or the agar hole diffusion method for the determination of the antibacterial effect of the liquid.The result showed that the optimal conditions of ultrafiltration was inlet pressure0.01MPa,outlet pressure 0.03MPa,the flow rate of penetration 20mL/min and the optimal time of the hydrolisis of casein by trypsin to release antimicrobial peptides was 4.5hr.The third part studied the stability of antibacterial peptides of the casein hydrolysate and their antibacterial performance.Using the Escherichia coli as the indicator bacteria,we studied the stability of antibacterial peptides of the casein hydrolysate under the high temperature,different pH and different storage time and used the Escherichia coli, Staphylococcus aureus,Bacillus subtilis and Bacillus cereus as indicator bacterias to study the antibacterial performance of antibacterial peptides of the casein hydrolysate.The result showed,antibacterial peptides of the casein hydrolysate can inhibit the Escherichia coli, Staphylococcus aureus,Bacillus subtilis and Bacillus cereus with broad spectrum antibacterial properties.The antibacterial peptides of the casein hydrolysate had the stability toward high temperature and also had a good stability of antibacterial performance toward Escherichia coli under the alkaline condition.The antibacterial performance was enhanced under the acid condition.Their powder form had good storability at the room temperature.The fourth part used the ion exchange chromatography to separate antibacterial peptides of the casein hydrolysate whose molecular weights were also determined.By selecting Q-Sepharose Fast Flow as the chromatography medium,the separation conditions were studied and we also determined the elution volume of antibacterial peptides of the casein under the optimal separation conditions through the antibacterial performance,and the molecular weight of the antibacterial peptides.The result showed,the ion exchange chromatography could separate antibacterial peptides of the casein hydrolysates well.The optimal step elution conditions were as follows:pH10.5, elution flow rate0.5mL/min,0.07M NaCl,50mL;0.13MNaCl,45mL;0.25MNaCl,40mL;1MNaCl,50mL.By the further separation, the elution volume of antibacterial peptides of the casein is 18.52mL and its molecular weight was measured to be 3162Da.