Selection Of Milk-Source Lactic Acid Bacteria Producing Antimicrobial Peptides And Cultivation For Antimicrobial Activity

Antibacterial peptides have the merits of small molecular weight, good thermal stability, broad spectrum and unique mechanism of antibacterial, moreover, they have high performance, no residue and drug resistance for antibiotics. The antibacterial peptides have become focus of international research; Antibacterial peptides from milk-source lactic acid bacteria are inartificial antibiotic molecules with broad spectrum, high performance, stable and safety produced. Now antibacterial peptides has been used to prevent mastitis of dairy cow, treatment diarrhea of piglets, promote growth of chickens, feed additive and food preservatives. In this experiment, according to mechanism of action by antibacterial peptide, we determined antibacterial activities of different milk-source lactic acid bacteria; then we established the training techniques of the selected bacteria, then, a new method of high expression of antibacterial peptide of lactic acid bacteria was constructed. We found the milk-source lactic acid bacteria played a role of bacteriostasis to raw milk. Thereby our research will lay the foundation for theoretical and application of antibacterial peptides.In this experiment, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus casei, Leuconostoc mesenteroides, Lactococcus lactis lactate subsp. seven kinds of lactic acid bacteria were isolated and identified and cultured for antimicrobial activities. Lactobacillus casei and Lactobacillus acidophilus were selected and identified, by16S rDNA, blast analysis, and species-specific primers PCR. Inhibitory effect was used as the evaluation index, By changing the medium of nutrients and environmental conditions, we established the target training of producing antimicrobial peptides of milk-source lactic acid bacteria. The optimum culture medium nutrients and environmental conditions of target training methods for milk-source lactic acid bacteria producing antimicrobial peptides were established by the orthogonal experiment. Finally, the antimicrobial peptides were used in milk preservation experiments, observed to evaluate the effect of fresh milk observation.The results of this experiment were as follows:①Lactobacillus acidophilus and Lactobacillus casei strain were selected from seven kinds of lactic acid bacteria showing a strong antibacterial activities;②Protease activity assay showed that the antibacterial molecules were of proteins;③Identification has been made by16S rDNA, blast analysis, and species-specific primers PCR.The results showed that these strains were Lactobacillus acidophilus and Lactobacillus casei;④The orthogonal test showed that the best nutritional environment of Lactobacillus acidophilus was sucrose3%, beef extract4%, NaCl0.5%, Tween-801.5%, initial pH5.0; the best nutrition environment of Lactobacillus casei was sucrose1%, soybean peptone3%, NaCl0.7%, Tween-801.0%, initial pH6.0;⑤Lactobacillus acidophilus and Lactobacillus casei were individual co-cultured with E.coli and Staphylococcus aureus. After24h,48h,72h,96h,120h, the antimicrobial activities significantly rised;⑥After adding of metabolic precursors, adjusting the initial pH, temperature and antagonism of target cultivation, the antibacterial diameter of Lactobacillus acidophilus got to23.53±0.06mm, without the target cultivation Lactobacillus acidophilus antibacterial diameter was11.63±0.15mm, the antibacterial activity rised by about102%. After target cultivation, antibacterial diameter of Lactobacillus casei was21.27±0.25mm, without target cultivation the antibacterial diameter of Lactobacillus casei was12.50±0.10mm, antimicrobial activity rised by70.2%;⑦Adding antimicrobial peptides of milk-source lactic acid bacteria to the fresh milk could effectively control postacidification under condition of4℃and25℃, moreover, the organization and the sense organ quality of milk were good.This experment indicates that the target training can significantily increase the ability of the milk-soure lactic acid bacteria producing antimicrobial peptides. Antimicrobial peptides used in milk preservation can help to extend the shelf life of milk. We have screened out two strains of strong ability of producing antimicrobial peptides:Lactobacillus acidophilus and Lactobacillus casei. Target training method for milk-soure lactic acid bacteria producing antimicrobial peptides was first established. The detailed mechanism needs to be further explored to improve the production of antimicrobial peptides. It has broad application prospects in the husbandry and food industry.