探討克弗爾粒與viili菌元之菌群分布並研究其分離菌株之生物膜形成機制

Abstract

本研究以台灣地區克弗爾粒(kefir grain)與viili菌元(starter)為試驗對象，針對菌元顆粒形成之可能機制進行三部分之探討，首先進行克弗爾粒與viili菌元之菌種鑑定與分離，並比較克弗爾與viili之菌群分布變化，最後評估分離菌株之表面特性與微生物間之交互作用，期能釐清克弗爾粒形成之可能機制。 在克弗爾粒與viili菌元之菌種鑑定部分，本試驗結合傳統微生物培養方式與聚合酶連鎖反應-變性梯度膠體電泳(PCR-DGGE)成功自兩菌元中鑑定出4種酵母菌，其中台灣地區克弗爾粒具3種酵母菌Kluyveromyces marxianus、Saccharomyces turicensis及Pichia fermentans分別佔81、17及2 %，而台灣地區viili則被鑑定出Klu. marxianus、S. unisporus及P. fermentans分別佔68、9及23 %，至於乳酸菌方面，台灣地區viili菌元中主要以Lactococcus lactis subsp. cremoris為主要優勢乳酸菌(95 %)，其次為Leuconostoc mensenteroides subsp. mensenteroides，而克弗爾粒佔最大比例之乳酸菌為Lactobacillus kefiranofaciens (55 %)，其次為Lb. kefiri (36 %)、Leu. mensenteroides (5 %)及Lc. lactis (5 %)。 在第二部分之試驗發現不同種類乳原料對克弗爾與viili之菌群分布有顯著影響效果，因而造成後續發酵製品之理化特性發生改變。在牛乳viili之菌相分析顯示胞外多醣產生菌株Lc. lactis subsp. cremoris TL1與Klu. marxianus兩者為優勢菌種，相反地不具胞外多醣產生能力之Lc. lactis subsp. cremoris TL4則為羊乳viili之優勢乳酸菌，至於牛乳與羊乳克弗爾最大菌群分布差異在於優勢酵母菌不同，除了菌群分布受到影響外，不同乳原料之發酵製品理化特性亦有顯著地差異，羊乳克弗爾或viili之黏度均低於其他組別，但保水性則有較佳表現。而不同發酵階段亦會顯著影響克弗爾之菌群分布變化，在克弗爾發酵初期存在4種乳酸菌及2種酵母菌，但發酵終了僅存Lc. lactis及Klu. marxianus，而viili之菌群分布在發酵階段並無明顯改變。 在研究克弗爾與viili之菌相分布情況之後，針對分離自克弗爾粒與viili菌元之8株乳酸菌株與6株酵母菌株進行試驗，分析菌體表面特性與乳酸菌及酵母菌間交互作用，以進一步探討菌元顆粒形成與聚集之可能機制。結果顯示克弗爾粒分離之乳酸菌Lb. kefiranofaciens具有較強之自聚集能力，而Lb. kefiri能夠顯著地形成生物膜，若透過掃瞄式電子顯微鏡發現短桿狀之Lb. kefiri主要分布並貼附於克弗爾粒之表面，而長鏈狀Lb. kefiranofaciens則聚集於克弗爾粒之中心，除了克弗爾乳酸菌外，S. turicensis顯示具有非常強之絮凝能力，於共培養克弗爾酵母菌與乳酸菌試驗中亦發現S. turicensis與乳酸菌(Lb. kefiranofaciens、Lb. kefiri)具顯著共聚集能力；相反地，存在於viili之乳酸菌與酵母菌並無顯著自聚集及生物膜形成能力。 綜言之，由上述試驗結果證實Lb. kefiranofaciens、Lb. kefiri與S. turicensis可能參與克弗爾粒之形成，推測克弗爾粒形成初期由Lb. kefiranofaciens與S. turicensis開始共聚集並形成小形菌種顆粒，而生物膜形成菌株Lb. kefiri會進一步黏附於小顆粒表面，並與其他菌株或是乳成分共聚集形成克弗爾粒，而隨著不斷地繼代培養，更多的菌株(主要是Lb. kefiri)會黏附於此特殊生物膜，造成克弗爾粒變大變多，此推測與電子顯微鏡觀察混合克弗爾乳酸菌與酵母菌之混合菌群生物膜有相類似之處。

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.