Investigation of Microbial Ecology of Kefir Grains and Viili Starters and Studying the Mechanisms of Their Biofilm Formation
Journal
臺灣大學動物科學技術學研究所學位論文
Date Issued
2011
Date
2011
Author(s)
王聖耀
Wang, Sheng-Yao
Advisor
陳明汝
Abstract
Kefir and viili are traditional fermented milk and characterized as health-promoting properties. Specificities of kefir and viili are mainly attributed to the presence of a complex microbial population. Interestingly, microorganisms in the kefir can form the grains as the starter but no grain was found in viili. The mechanism of grain formation was unavailable. To investigate the major microorganisms associated with grain formation and to assess the microbial stability of subculture in different fermentation materials, it is necessary to understand the microbial population and their interaction among different bacteria and yeast in kefir grains and viili starters. Thus, the objectives of this study were to (1) identify the complex microbial compositions present in Taiwanese kefir grains and viili starters, (2) compare the microbial distribution of both starters made from different types of milk, and (3) study the possible mechanisms of microbial aggregation and biofilm formation.
A combination of conventional microbiological cultivation with polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) successfully identified 4 yeast species from both starters. Kluyveromyces marxianus, Saccharomyces turicensis, and Pichia fermentans were found in Taiwanese kefir grains with 81, 17, and 2% distribution, respectively, while Klu. marxianus, S. unisporus, and P. fermentans were identified in viili starters with 68, 9, and 23% distribution, respectively. For lactic acid bacteria (LAB), Lactococcus lactis subsp. cremoris was the major LAB (95%), followed by Leuconostoc mensenteroides subsp. mensenteroides in Taiwanese viili starters, whereas, Lactobacillus kefiranofaciens was 55% of the total isolates, constituting the most dominant LAB found in kefir grains followed by Lb. kefiri (36%), Leu. mensenteroides (5%) and Lc. lactis (5%).
The type of milk demonstrated greatly influence on the microbial ecology, which might further affect the product characteristics. For bovine viili, Lactococcus lactis subsp. cremoris TL1 (EPS forming strain) and Klu. marxianus were the most dominant species found in samples. On the contrary, Lc. lactis subsp. cremoris TL4 (non-EPS forming strain) was the most prevalent LAB in caprine samples. The physical and chemical properties of the kefir and viili samples were also significantly influenced by the type of milk. The caprine sample showed a lower viscosity than the other samples. Fermentation stage was another important factor affecting the microbial dynamics in kefir. At the beginning of kefir fermentation, 4 LAB and 2 yeasts were detected. The situation dramatically changed at the end of fermentation. Only Lc. lactis and Klu. marxianus were detected. In contrast, the microbial dynamics in viili were unchanged during the fermentation.
After study the microbial ecology in kefir and viili samples, we further investigated the possible mechanisms of grain formation and aggregation by studying cell surface properties and interaction among LAB and yeasts. Results indicated that kefir grain LAB, Lb. kefiranofaciens, possessed the strong self-aggregation ability and Lb. kefiri showed a significant biofilm formation. The SEM revealed that short-chain lactobacilli, Lb. kefiri, were occupied on the surface and the long-chain lactobacilli, Lb. kefiranofaciens, were aggregated in the center of kefir grains. Besides kefir LAB, the S. turicensis demonstrated a good flocculation property. A significant co-aggregation among S. turicensis and kefir LAB (Lb. kefiranofaciens and Lb. kefiri) was observed when co-cultured kefir yeast with LAB. Conversely, LAB and yeasts in viili did not discover a significant self-aggregation and biofilm formation.
For investigation of formation and aggregation of kefir grains, 8 LAB and 6 yeast strains were successfully identified from kefir grains and viili starters in this study. Then, we assessed the surface properties and interaction among LAB and yeasts. Lb. kefiranofaciens, Lb. kefiri, and S. turicensis isolated from kefir grains might involve in grain formation. According to above results, we make hypotheses for grain formation. At the beginning, Lb. kefiranofaciens and S. turicensis started to self-aggregate and form a small granule. The biofilm producer, Lb. kefiri, then attached to the surface of granules and co-aggregated with other strains and components in milk to form the grains. After sub-culturing, more strains were attached on the grains resulting in the grain growth. The SEM observation was parallel to our hypothesis.
Subjects
kefir grain
microbial ecology
biofilm
aggregation
Type
thesis
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