| 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. Antibacterial peptides released from casein during enzyme proteolysis belong to food-borne bioactive peptides with high security. Therefore it can be expected as a new antimicrobial agent that could be widely used.This research used the casein as raw materials and hydrolyzed with pepsin. Then the separation and purification of antibacterial peptides of enzymatic hydrolysate was done. Finally, substances with antibacterial activities were obtained. Their molecular weight and amino acid composition were determined and analyzed by RP-HPLC-MS-MS. After this, the stability of antibacterial peptides of the casein hydrolysate and their antibacterial performance were studied in vitro. The main results are as follows:(1) Isoelectric point precipitation method was used for extraction of casein from milk. Single factor experiment was done to study the influence of pH, temperature and stirring intensity on the yield of casein. Based on one-factor experimental design and L9(34) orthogonal design, the optimal condition was pH4.8, temperature 55℃, agitating speed 60 rpm.(2) Casein was hydrolyzed by different protease for antibacterial peptides preparation. Among them, the pepsin hydrolyzate showed the strongest antibacterial activity to Staphylococcus aureus. Using Box-Behnken response surface model, hydrolysis conditions were optimized. The result showed the optimal hydrolysis conditions were:substrate concentration 2.6%, enzyme-substrate ratio 3%, pH2.6, temperature 37.7℃and enzymolysis time 4h.(3) Ultrafiltration and two-step gel filtration were used on the enzymatic hydrolysis treatment, with molecular weight of ultrafiltration membrane 10KDa, and operating conditions for ultrafiltration was:the operating pressure 0.2MPa, initial concentration 30mg/mL, feed temperature 25℃, operating time 120 minutes. Antibacterial peptides components G-25-3 was obtained with the antibacterial effect against Staphylococcus aureus 97.4%.(4) Amino acid analysis showed that the proline content of G-25-3 component was significantly higher than other types of amino acids, reaching 22.1%. In addition, the proportion of basic amino acids was 17.4%, the proportion of hydrophobic amino acids was 53% and the hydrophilic amino acids accounted for 45.1% of the total. The component molecular weight was estimated to be about 2416.8Da by gel filtration using Sephadex G 25. RP-HPLC-MS-MS analysis showed that G-25-3 contains a variety of peptide components.(5) The stability of G-25-3 component and its antibacterial performance were researched. S aureus was used as the indicator bacteria to determine the minimum inhibitory concentration of the components and to study the effects of high temperature, different pH, different storage time and repeated freezing and thawing on the antibacterial activity. The results showed that the MIC was 125μg/mL, and it had high thermal stability. Under acidic conditions, the component displayed significant antibacterial effect, but declined in neutral conditions, whereas in alkaline conditions, the inhibitory effect had a sharp decline.Repeated freezing and thawing 3 times had no significant effect on the antimicrobial activity and its powder form had good storability at the room temperature.
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