Isolation,Identification,Diversity And Application Of Lactic Acid Bacteria From Traditional Fermented Dairy Products In Inner Mongolia

Traditional natural fermentation methods have long been used in Inner Mongolia to make dairy products with ethnic characteristics,mainly milk tofu,sour horse milk,sour cream(uzhimo)and milk skin,etc,which have better preserved the rich lactic acid bacteria resources in the local natural environment.The unique climate,environment and characteristic processing methods of food products in the region have led to diverse populations of lactic acid bacteria in traditional fermented dairy products.In this study,we isolated and purified lactic acid bacteria from homemade milk tofu and sour cream(Urimo)samples collected in the agricultural and pastoral areas of Inner Mongolia,identified lactic acid bacteria species relationships using conserved sequence 16 S rRNA,and then analyzed the phylogeny,genetic differentiation and population structure of lactic acid bacteria;based on the characteristic indexes of acid tolerance,salt tolerance,acid production ability and drug resistance of lactic acid bacteria in the culture medium;and acid production ability,viscosity production,self-reliance in skim milk.Based on the characteristic indexes of acid tolerance,salt tolerance,acid production ability and drug resistance in the culture medium,as well as the acid production,viscosity production,autolysis,water holding property,curd time and curd performance in skim milk,we screened the excellent strains for the production of fresh cheese;through analyzing the sensory quality,physical and chemical indexes and microbial composition of cheese,we determined the compound combination of lactic acid bacteria,and made the lyophilized bacteriological agent according to the compound ratio.The results showed that a total of 81 strains of lactic acid bacteria were isolated and purified from the two traditional fermented dairy products collected,mainly distributed in Pediococcus acidilacticii,Pediococcus pentosaceus,Lactobacillus brevis,Lactobacillus helveticu,Lactobacillus paracasei,Lactobacillus diolivorans,Lactobacillus otaqui,Lactobacillus plantarum,Lactobacillus kefir,Lactococcus lactis,Enterococcus faecalis,Enterococcus faecium and Enterococcus durans,among which E.faecium and Lac.lactis were the dominant species.The phylogenetic status of lactic acid bacteria from different sampling sites was cross-distributed,indicating that the species relationship of lactic acid bacteria was not significantly correlated with the geographical distribution pattern.Most of the lactic acid bacteria had a relatively homogeneous colony structure,a long genetic distance(Dxy),a low gene exchange index(Nm),and a low nucleotide polymorphism(Pi),indicating that the frequency of genetic recombination and gene exchange in the evolutionary history of these lactic acid bacteria was relatively rare and showed a strong genetic stability.The analysis of the probiotic characteristics revealed that the fermentation performance of Lactobacillus in MRS medium showed a large variability,with Lac.lactis showing the strongest acid production,acid tolerance and bile salt tolerance,IMUNJL0215 and IMUNJL0273 the strongest acid production,IMUNJL0221 and IMUNJL0018 the best acid tolerance,IMUNJL0272 and IMUNJL0221 the highest bile salt tolerance.The 81 strains of Lactobacillus showed complete resistance to nalidixic acid;high resistance rate to streptomycin,ciprofloxacin and vancomycin;low resistance rate to erythromycin and chloramphenicol;95.06% of Lactobacillus had multi-drug resistance.Based on the above characteristics,13 excellent strains were initially screened,namely L.plantarum IMUNJL0018,L.otaqui IMUNJL0024,L.plantarum IMUNJL0088,Lac.lactis IMUNJL0092,E.durans IMUNJL0096,L.brevis IMUNJL0105,P.acidilacticii IMUNJL0115,Lac.lactis IMUNJL0156,P.pentosaceus IMUNJL0215,L.diolivorans IMUNJL0221,L.paracasei IMUNJL0235,E.faecium IMUNJL0244,L.kefir IMUNJL0249.In the experiment of skim milk fermentation by lactic acid bacteria,it was found that strains IMUNJL0235 and IMUNJL0092 fermented yogurt had lower acidity and showed stronger acid production ability;IMUNJL0244 and IMUNJL0215 fermented yogurt had higher viscosity production characteristics;IMUNJL0235 and IMUNJL0215 fermented yogurt had better water holding ability and no whey precipitation;the autolysis of IMUNJL0105 and IMUNJL0115 fermented yogurt was moderate and the taste was good;the coagulation time of IMUNJL0088 and IMUNJL0115 fermented yogurt was short;the coagulation performance of IMUNJL0088,IMUNJL0092,IMUNJL0115 and IMUNJL0235 fermented yogurt was the best,and then 7 groups of lactic acid bacteria compound fermenters were identified.The results of fresh cheese making experiments showed that the sensory evaluation of cheese made by compound fermenters GLFP01 and GLFP02 was the highest,and the physicochemical indexes such as protein,fat,ash and p H were excellent and significantly different(P<0.05),and the dominant flora were Lactobacillus and Enterococcus in the compound fermenters,which had a simple structure and low abundance and effectively inhibited the growth and reproduction of miscellaneous bacteria.20% skim milk powder,10% alginate and 3% glycerol were selected as protective agents for lyophilized GLFP01 and GLFP02 bacteriophages,and the survival rates reached 85.37±3.55% and 93.63±1.56%.It laid the foundation for the development of lactic acid bacteria resources in traditional fermented dairy products in Inner Mongolia and their utilization in food,and provided excellent compound freeze-dried bacterial agents for the production of fresh cheese.