糖液克弗爾粒於不同發酵基質中之菌相分布與其分離菌株抗腸炎機能性之研究

Abstract

本研究目的為探討糖液克弗爾粒於黑糖液、牛乳與羊乳中發酵後之微生物菌相分布，並測試從中分離的乳酸菌株其抗腸炎之機能性。實驗藉由聚合酶連鎖反應–變性梯度膠體電泳及DNA定序等方法鑑定乳酸菌與酵母菌之種類，並以掃描式電子顯微鏡觀察其結構及菌群分布之變化；從糖液克弗爾粒中分離之乳酸菌株，則透過Caco-2細胞單層膜完整性評估，與DSS誘發結腸炎小鼠動物模式探究其保護腸道之能力。 菌種鑑定結果顯示，從不同發酵基質中共分離出6種乳酸菌（Leuconostoc mesenteroides、Lactobacillus mali、Lactobacillus hordei、Lactococcus lactis、Enterococcus faecalis和Bifidobacterium psychraerophilum）與4種酵母菌（Zygosaccharomyces fermentat、Saccharomyces cerevisiae、Dekkera bruxellensis、Pichia fermentans）；而直接抽取細菌之DNA可另外發現到Pseudomonas spp.、Enterobacter spp.、Sporolactobacillus spp.和Zymomonas mobilis之存在，以掃描式電子顯微鏡觀察之結果亦發現，不同發酵基質對於糖液克弗爾之菌元結構與微生物菌相分布有顯著影響。 從糖液克弗爾粒中分離出來之Lb. mali可顯著提升Caco-2細胞單層膜之跨上皮電阻值，並增加趨化激素CCL20之分泌量；在DSS誘發結腸炎小鼠動物模式中，亦可減緩因潰瘍性結腸炎而導致之體重減輕、糞便潛血與結腸長度縮短等臨床症狀，組織切片結果同樣應證其對腸道上皮具有保護之功效。

Sugary kefir is a fermented beverage, made from sugar and water with or without fruit (usually figs and lemon) added, whose fermentation is induced by grains. Sugary kefir grains are small transparent mucilaginous masses, also consisting of a polysaccharide gel embedding lactic acid bacteria (LAB) and yeasts. In contrast to milk kefir grains made of complex heteropolysaccharides namely kefiran, the sugary kefir grains are mainly consist of dextran, an alpha 1-6 linked glucose polymer. Sugary kefir grains are likely to differ from the milk kefir grains because the materials for fermentation differ widely in their composition. In the present study, we fermented sugary kefir grain in brown sugar, bovine and caprine milk, and evaluated the effect of different fermentation materials on the microbial distribution in both grains and filtrates using culture-dependent and culture-independent methods. The identification results indicated that the strains in the grains were significantly affected by the fermentation materials. Three LAB species (Leuconostoc mesenteroides, Lactobacillus mali, Lactobacillus hordei) were found in the grains fermented with brown sugar. Whereas, four species including Leu. mesenteroides, Lactococcus lactis, Bifidobacterium psychraerophilum and Enterococcus faecalis were identified in the grains fermented with bovine and caprine milk. For PDA results, three yeasts (Zygosaccharomyces fermentati, Saccharomyces cerevisiae, Dekkera bruxellensis) were found in all three grains. Only Pichia fermentans was identified in the grains grown in the bovine and caprine milk. The profiles obtained by culture-independent denaturing gradient gel electrophoresis (DGGE) generally agreed with the results obtained by culture-dependent methods. However, two additional DGGE bands in the sugary kefir samples were found. Further identification by DNA sequencing revealed that they were Zymomonas mobilis and Sporolactobacillus spp. Additional three bands were also found in the milk samples, they were Pseudomonas spp. and Enterobacter spp. It is worth to notice that the strains and their distributions were different between the grains and filtrates. Additionally, we examined the anti-colitis effects of lactobacilli isolated from sugary kefir grains using in-vitro and on the in-vivo chemical-induced colitis model. Results indicated that Lb. mali could strengthen the epithelial barrier function in vitro by increasing the transepithelial electrical resistance (TEER) with significantly upregulated the level of CCL20 in both apical and basolateral sites. The in-vivo effects of Lb. hordei and Lb. mali on the regulation of intestinal physiology indicated that both strains could ameliorate DSS-induced colitis with a significant attenuation of bleeding score and colon length shortening. In the present study, we demonstrated that the fermentation materials presented remarkable differences in the microbial ecological profiles both in the grains and the filtrates. The strains were varied depending on the carbon and energy sources under grain fermentation. Furthermore, Lb. mali clearly demonstrated an anti-colitis effect.