Screening Of Lactic Acid Bacteria From Piglet And It’s Agent Preparation

In this study,lactic acid bacteria with strong acid-producing ability were screened from the faeces of healthy piglets.The growth characteristics of the strains,tolerance to the animal digestive tract,the inhibition of pathogens and the adhesion to intestinal epithelial cells were studied.The main research results are as follows:(1)Screening and identification of lactic acid bacteria.26 suspected strains were obtained by MRS supplemented with bromocresol violet.Seven strains with tolerance to low pH and high concentration bile were obtained after secondary screening.The NCU156 and NCU301 were determined as Lactobacillus plantarum and Pediococcus pentosaceus respectively through physiological and biochemical and molecular biology means.(2)Growth characteristics of NCU156.NCU156 can produce large amounts of lactic acid resulting reduce of culture medium p H to about 3.6.0-2 h is the delay period,0-12 h is the logarithmic phase,12-32 h is the stability period,and enter the decay period after 32 h.(3)Tolerance to the animal gastrointestinal environment.The survival rate of NCU156 in MRS at pH 2.5 for 4 h was 87.31% and in MRS with 3 g/L bile salt 4h was 41.88%.The number of viable cells maintained above 108 CFU/mL after 14 h in the artificial gastric juice,indicating that NCU156 could pass through the animal digestive tract.(4)Adhesion properties.The adherence of NCU156 was bacterial and Ca2+concentration dependence in a certain range,balanced at 2 h,most stable in the middle of stationary phase.In the inhibition experiment of pathogens adhesion to Caco-2,the exclusion inhibition effect was the strongest,followed by competitive inhibition and replacement inhibition finally.(5)Antibacterial properties.The antibacterial effect of NCU156 on the seven common pathogens showed that the inhibition to Pseudomonas aeruginosa was the strongest,the diameter of the inhibition zone was more than 23 mm,the NCU156 cells didn’t have bacteriostasis.The bacteriostasis of the fermentation broth was not affected by the treatment of high temperature,peroxidase and protease.So the antibacterial activity may be mainly derived from the produce of lactic acid.The antibiotic sensitivity results show that NCU156 was sensitive to tetracycline and penicillin,resistance to cephalosporins,glycopeptides and quinolones.(6)Optimization of the process of NCU156 agent preparation.The number of viable cells was used as an indicator.The production process was optimized with one-factor-at-a-time combined with response surface methodology.Results showed that NCU156 achieved the highest biomass after 46.40 h incubation at 30.6 ℃,with substrate to water ratio of 1:0.61 and inoculum concentration of 4.45%.The number of NCU156 could be achieved 9.88 lg(CFU/g)under the best drying condition of 50 ℃ hot air drying 4 h.