Study On Ultrastructure And Fermentation Properties And Establishment Of PCR-SSCP Analysis Of Microorganisms Diversity In Kefir

Kefir is an acidic and mildly alcoholic fermented dairy product that is fermented by kefir grains based on milk and so on.In this article,first of all, the ultrastructure of kefir grains was observed by scanning electron microscope (SEM).Secondly, the changes of pH, acidity, microbial counts and utilization of lactose during kefir fermentation were studied. Third, traditional methods were used to isolate and identify the microbes in the kefir. At last, we evaluated three DNA extraction methods (i.e.lysozyme, sonication and CTAB methods) and established a reliable extraction method which was used for the microbial diversity analysis of kefir by PCR-SSCP.Using scanning electron microscope to observe the ultrastructure of kefir grains,there were a great amount of lactococcus, lactobacillus and yeast. However, there were few microbes in the kefiran, and these microbes did not have the integrity cell.Therefore,microorganisms nearest to the surface of kefir grains were much more important.In the fermentation of kefir, pH of kefir was decreased sharply, and its value dropped to 4.102 from 6.798 and the acidity rose to 96.9°T from 16.1°T after fermentation of 24h. In addition, microbial counts were evaluated.The colony counts of lactococcus were higher than lactobacillus in the initial fermentation stage.At the end of fermentation, the colony counts of lactococcus,lactobacillus and yeast reached 1.6×107 cfu/mL,7.7×107 cfu/mL,1.1×105 cfu/mL respectively. The fermentation of kefir resulted that the content of lactose was decreased significantly, and its value declined to 1.90mg/mL from 4.05mg/mL in the fermentative period.Three strains were isolated from kefir grains,named Y1,Y2 and R1.Y1, Y2 and R1 strains were identified to Kluyveromyces sp.,Candida sp.and L.lactis subsp.lactis respectively.Experiments were performed to determine the influence of three DNA extraction methods (i.e.lysozyme, sonication and CTAB methods) from kefir on the microbial diversity analysis by PCR-single strand conformation polymorphism (PCR-SSCP).The results showed that the band of DNA extracts using CTAB was clearer than that using other methods.In addition, the quantity and purity of DNA extracted using CTAB were the highest and reached respectively 915μg/ml and 1.694.The results from the experiments indicated that the CTAB-based DNA extraction method was the most efficient method for DNA extraction from kefir. The heterogeneity of PCR products,amplified from community DNA with universal primers spanning the V3 region of 16S rRNA genes,was analyzed by using SSCP.The results showed that the SSCP profile based on the sonication method gave the highest microbial diversity of kefir. One conclusion from these results was that the DNA extraction method was an important factor affecting the SSCP-based microbial diversity analysis of kefir.