Isolation, Diversity Analysis And Characteristics Of Strains From Inner Mongolia Original Cheeses

Genetic diversity and phylogeny of50strains isolated from5different traditionalcheeses in different areas of Inner Mongolia were determined by analysis of phynotype,physiological and biochemical characteristics, and molecular biology methods. At the sametime, A series of functional properties such as acid-producing ability, survival in simulatedgastric juice. the ability to produce exocellular polysaccharide were performed on6representative stains. The work provides useful data on microbial diversity in conventionaldairy products, and has very important significance on screening and improvement of higheffective Lactobacillus.The results from physiological property testing showed as follows:(1) Isolates fromcheese Ch1growed well on salt-tolerance, while isolates from Ch4and Ch5cheeses growedworse.(2) All the isolates grew well under the condition of weak acidity, and could not growat a strong acid condition (pH<3.24).(3) Most isolates from cheeses Ch2, Ch4and Ch5hadstronger adaptive capacity to temperature, and could grow at a low temperature of15℃andat a high temperature of45℃.(4) Most isolates from the cheeses could not use citric acid asthe sole carbon source. Isolates from cheeses Ch2and Ch5could utilize all the carbohydratestested except for citric acid, and had strong ability of carbon source utilization.(5) All theisolated were evaluated for resistance against7antibiotics, andshowed various levels of antibiotic resistance. The isolates from cheeses Ch1and Ch3showedhigh rates of resistance to certain antibiotic such as acheomycin, and the isolates from cheeseCh2showed multi-antibiotic resistance. All the isolates from cheese Ch4could grow in themedium with a higher concentration of kanamycin (20μg/mL) or chloramphenicol (50μg/mL).Most isolates from cheese Ch5was resistant to all seven antibiotics except for gentamycin.(6)Except the isolates from cheese Ch1, all the isolates showed the ability of yogurt cheese. Theisolates from cheese Ch5produced yogurt cheese, and most of them had a shorter gelationtime. Most isolates produced lactic acid, but the milk flavors were diffused from strains4-Eand4-F.(7) All the isolates from the five cheeses showed no antimicrobial activity against thethree indicator strains Escherichia coli、Staphylococcus aureus and Bacillus subtilis.Genetic diversity of the50isolates were determined by analysis of16S rRNA RFLP andSDS-PAGE analysis of cell wall proteins. Results of16S rRNA PCR-RFLP showed that in thesimilarity of65%, all the isolates were clustered into2groups. Group II included only3isolates from ch3cheese3-A,3-F and3-J. Howerer, Gruop I inclued most of the isolated andwere further divided into2groups IIa and IIb in the similarity of70%. IIa includes all theisolates from cheeses ch1and ch4, and IIb includes all the isolates from cheeses ch2and ch5 and the other isolates from cheese ch3. Results of SDS-PAGE analysis of cell wall proteinsshowed that in the similarity of70%，the isolates were divided into10subgroups and sixsingle strains of1-N,2-C,2-I,2-K,4-A and5-N. Compared of two means, the UPGMA of cellwall proteins was more refining and detailing.Six isolates were selected on the basis of diversity characteristics. Their genes of16S rDNAwere amplified and sequenced. Then a phylogenetic dendrogram was constructed bycomparing them to the sequences retrieved from GenBank database. Results indicated strain1-D belonged to Micrococcus, Strains2-A and4-A were determined as Pediococcus andLeuconostoc, respectively, Stains3-D,3-J, and5-k were identified as Lactobacillus. All of thesix strains were Lactobacillaceae except strain1-D.Functional properties of the6representative stains were further studied. The results showedthat:(1) The variation in acidity was similar to that in pH. In the fermentation process, strain1-D produced little lactic acid. Other strains produced less lactic acid in the first12hours.Howerer after the lag phase, the lactic acid concentration increased exponentially withtime. And it was found that the maximum production was obtained after48hours growth.(2)In order to evaluate the survival of the strains in acidic conditions, we compared the survivalof six strains in simulated gastric juice, pH3.0. Strain4-A survived and grew in gastric juiceand it showed strong tolerance. Strains2-A,3-J and5-K had a high tolerance to the simulatedgastric juice, while strains1-D and3-D exhibited a poor survivor.(3)Among the strains, strain1-D produced highest concentration of extracellular polysaccharides (EPS)，while the lowestconcentrations were strains3-D and5-K.(4) As a non-lactic acid bacterium, strain1-Dexhibited little activation of β-galactosidase.Among the lactic acid bacteria, strain3-J hadhighest enzyme activity, while the lowest was strain5-K.A variation curve of pH wasperformed during β-galactosidase production, and the results showed that there was a positivecorrelation between activation of β-galactosidase and acid-producing ability.