The Isolation, Identification And Genetic Diversity Analysis Of Lactobacilli Of Two Sources

Lactobacillus is the biggest genus in the lactic acid bacteria,it's so important that it's widely used in food industry.Be probiotics,lactobacilli have received many attentions because of its probiotic function.As a traditional lactic acid fermentation vegetables production,sauerkraut is rich in living lactic acid bacteria in its juice;especially the Lactobacillus brevis and Lactobacillus plantarum are predominant.Besides,lactic acid bacteria are the dominance flora in the digestive tract of pig.The lactobacilli present normally in the digestive tract of pig contain:Lactobacillus acidophilus,Lactobacillus reuteri,Lactobacillus brevis,Lactobacillus fermentum and Lactobacillus salivarius,in which the Lactobacillus acidophilus and Lactobacillus reuteri are dominance.The traditional identify method of the lactobacilli is depends on the phenotypic characteristics of the lactobacilli,although it's still the main methods now,more research indicate that the accurate classification of lactobacilli needs the identification on gene level.Test on phenotypic characteristics only can easily cause the inaccuracy of the identify result,so combining with the molecular biology identification,the result can be trust.The main contents of this study are isolation lactobacilli of animal origin and plant origin,identification of them,and analysis the genetic polymorphism of them.This research mainly included the following five parts:1.The isolation,purification and preservation of lactobacilliTake the wild fermentation sauerkraut and pig digestive tract contents as the simple,the modified MRS medium as the isolation medium,cultivate under anaerobic conditions,lactobacilli of these two source were isolated,purification and preservation.According to the morphological characteristics and catalase test,39 strains and 29 strains were identified as lactobacilli.2.The physiological and biochemical identification of lactobacilliAccording to the gelatin liquefaction test,indole test,H₂S creation and nitrate reduction test, we discovered that 34 and 25 strains isolated from these two sources belong to Lactobacillus.And confirm the species according to the fermentation experiment about carbohydrate.3.Identify of lactobacilli by 16S rDNA sequence cluster analysisAmplify the 16S rDNA sequence of the isolation strains L1～L20 using a pair of primer which amplify lactic acid bacteria only,obtaining a fragment length 1500bp around,and then the 16S rDNA sequence was alignments with type strains blast on the GengBank.The result can be obtained according to the homology.Finally,1 Lactobacillus casei strain,4 Lactobacillus brevis strains,1 Lactobacillus coryniformis strain,3 Lactobacillus plantarum strains,1 Lactobacillus sakei strain;4 Lactobacillus reuteri strains,2 Lactobacillus johnsonii strains,2 Lactobacillus acidophilus strains,1 Lactobacillus fermentum strain,1 Lactobacillus salivarius strain were identified.4.Genetic polymorphism analysis of the lactobacilli by the PCR-RFLP techniqueUsing three restriction enzyme:Xbaâ… ,Mspâ… and Aluâ… digest the 16S rDNA of isolation strains L1～L20,the Mspâ… and Aluâ… produce an abundant enzyme map,divided L1～L20 into 10 groups,present the polymorphism,reflect the genetic polymorphism of isolation strains.5.Discriminate lactobacilli of different species by PCR-DGGEAmplify the V7～V8 hypervariable region of the isolation strains L1～L20 using a pair of primer which amplify lactic acid bacteria only,the result of the DGGE showed that these isolation strains can be discriminated by this method;the result is matched with the traditional method and 16S rDNA sequence cluster analysis.59 strains were isolated from two source,part of them were analysis and identified,these laid the foundation for them to be used in different domain;besides,the role of some molecular biology methods in identification and discrimination of lactobacilli was analysis,which provides basis for the systematized identification of lactobacilli.