Isolation And Screening Of Lactobacillus From Chickens And Study On Their Biological Characteristics

In this study, we collected healthy chicken's intestinal tract and feces samples from different places in Sichuan province. After culturing and screening,42 strains were isolated such as Lactobacillu salivarius, Lactobacillus feumentum, Lactobacillus desidiosus, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus intestinalis, Lactococcus sp et al.A series of tests about the isolated strains were taken, including the determination of acidogenicity, the tolerance of low pH value, high bile salt concentration and high protease.4 strains were screened, which were able to descend the pH value of the liquid nutrient medium from original 6.0 to 3.78,3.55,3.62 and 3.76 respectively, and can survive more than 80% in the condition of pH 2.5,0.3% bile salt and 1% trypsin. They were named LR-1, LR-5, LR-6 and LR-15.The viable count of strain LR-5 and LR-6 reached to 1.79×1010 cfu/ml and 1.15×1010cfu/ml respectively within 16h. So their growth velocity were found to be the fastest in these 4 strains of Lactobacillus after cultured in 37℃for 48h.18-20g Kumming mice were feeded with 109 cfu/ml of these 4 Lactobacillus strains per os, once a day,0.5ml a time, for 15 days. Not a single mouse was found ill or dead.The bacteriostatic effect of the 4 Lactobacillus strains to Staphylococcus aureus, Escherichia Coli and Salmonella were obvious observed by the inhibition zones between 17.51-22.68 mm,12.74-21.29 mm and 11.32-16.46mm respectively. Among them, the strain LR-5's ability of bacteriostasis was extremely significant stronger than the other 3 strains (p<0.01). In the mixed culture experiment, the viable count of the Staphylococcus aureus, Escherichia Coli and Salmonella reduced to 0×100cfu/ml from 2.01×108 cfu/ml, 4.92*10 cfu/ml and 3.15x10 cfu/ml respectively, while cocultured with the 4 Lactobacillus strains for 24h, which showed the bacteriostatic effect of the 4 Lactobacillus strains. The strain LR-5 needed only 18h to resist the pathogenic bacteria completely, while other 3 needed 24h, which was the most outstanding in the 4 Lactobacillus strains.The adhesion test revealed that the adherent ability of strain LR-5 to human colonadenocarcinoma cell (Caco-2 cell) was highly performanced. The orthogonal tests' result showed that the optimal combination for adhesion of strain LR-5 was on condition of pH 6, spent culture supernatant (SCS),109cfu/ml,37℃for 3h. The mean count of adhesion was 59.23±1.40 per cell, while the strain LR-1, LR-6 and LR-15 were only 30.03±1.14.36.63±0.31 and 30.57±0.91 per cell(p<0.01).Comprehensive analysing each factor of the 4 Lactobacillus strains, such as the acidogenicity, the tolerance of low pH value, high bile salt concentration and high protease, growth characteristic, bacteriostatic effect and adherent ability to Caco-2 cell, we finally screened an excellent chicken source Lactobacillus strain which was strain LR-5. The strain LR-5 was eventually identified as Lactobacillus reuteri with GenBank accession number JF745115 via conventional bacteriology identification and 16S r RNA.