Study On The Optimization Of Fermentation Conditions Of Antimicrobial Substances Produced By Lactobacillus Casei And Functional Evaluation On Its Probiotics

The pollution of drug residues in the products of livestock and poultry and drug-resistant harmful bacteria had seriously affected the quality of livestock product. As an ideal substitute of antibiotics, antimicrobial peptides were the focus of attention for researchers at home and abroad, which has a broad-spectrum antibacterial activity, not easy to produce drug resistance to bacteria and any other characters. As a new green feed additives, the probiotics contained antimicrobial peptides has broad application prospects, which not only has the antibacterial effect of antimicrobial peptides, but also prebiotic effect of probiotics.For the development of a new feed probiotics contained antimicrobial peptides, in this study, three strains of Lactobacillus spp. producing antimicrobial peptides were chosen as the research objects, fermentation conditions for antimicrobial peptides production by Lactobacillus casei and its biological characteristics were investigated. And the composite microorganisms agent was composed of the Lactobacillus casei and the recombinant yeasts expressing AvBD9. Functional evaluation of the composite probiotics was preliminarily analyzed by average daily gain, immune organ index and the changes of intestinal microflora. The specific contents and results were showed as follows:1. Screening of Lactobacillus producing antimicrobial substancesIn order to select out Lactobacillus of high-level production of antimicrobial peptides, the antimicrobial activity of the cell-free supernatant (CFS) of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum was investigated by the agar diffusion assay with Escherichia coli and Staphylococcus aureus as indicator bacteria. The fermentation supernatant can inhibit indicator strains strongly excluding inhibitive effect of H2O2 and organic acid, but the inhibitory activity decreased sharply after treatment with trypsin. The results showed that the CFS of Lactobacillus casei had a stronger antimicrobial activity than the other two strains and its inhibitory material was a kind of protein.2. Optimization of fermentation conditions for antimicrobial peptide production by Lactobacillus caseiDifferent factors such as fermentation time, temperature, inoculum dose, liquid volume on the production of antimicrobial peptides were investigated in order to optimize the fermentation conditions by single factor and orthogonal tests. The results showed that when Lactobacillus casei was cultured for 42h at 30℃ in the conditions of liquid volume of 20% and inoculum dose of 2%, the antimicrobial activity of the CFS came to the highest.3. Study on biological characteristics of antimicrobial peptide produced by Lactobacillus caseiCharacteristics of antimicrobial peptides by Lactobacillus casei were analyzed. The protease hydrolysis tests showed that it was sensitive to proteolytic enzymes such as trypsin, neutral protease and papain but not pepsin, and partially digested by proteinase K. The stability test results of the temperature and pH of the CFS showed that it was heat-stable and could maintain good antimicrobial activity at low pH conditions. The action mode of the CFS was confirmed as bactericidal. The antimicrobial assays showed that the CFS had inhibitory activities against common pathogens including Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Pasteurella, Pseudomonas aeruginosa, Citrobacter, Salmonella, but had no significant inhibitory activity towards Candida albicans, Pichia.4. The functional evaluation of the composite probiotics composed of Lactobacillus casei producing antimicrobial peptide and Pichia pastoris expressing AvBD9The function of the composite probiotics composed of Lactobacillus casei and Pichia pastoris expressing AvBD9 at 1:1 ratio was evaluated by average daily gain, immune organ index, the dynamic changes of intestinal flora. With one-day-old chicks as object, they were divided into four groups, group Ⅰ was control group, group Ⅱ was Lactobacillus casei, group Ⅲ was recombinant Pichia pastoris, group IV was the composite probiotics and the test lasted for 42d. The results showed that, compared to the control group, group Ⅱ, group Ⅲ and group Ⅳ could improve the average daily weight gain of chicks and immune organ index, while group Ⅳ improved more outstandingly(P<0.01). And group Ⅱ, group Ⅲ, group Ⅳ had an impact on the advantage flora and intestinal flora structure, while group Ⅳ affected more outstandingly and could improve the diversity of intestinal microflora and the advantage flora could be in steady state earlier in the cecum and ileum.In summary, an antimicrobial peptide-producing strain of Lactobacillus casei was screened out. The antimicrobial activity of the CFS was increased by optimizing the fermentation conditions.The antimicrobial peptide had a good biological characteristic. The antimicrobial assays showed the CFS could have antimicrobial activity against several pathogenic microbes. The animal tests showed that the composite probiotics could improve the performance, immune organ index and the intestinal microbial environment of chicks to some extent, which laid a basis for animal feed additives in the research and development of the use of it.