傳統蒙古發酵乳製品中乳酸菌之分離及分子鑑定研究

Abstract

「益生菌」是一群在宿主體內達到一定數量時對宿主的健康有益處的微生物。乳酸菌一般被認為是安全的(Generally recognized as safe, GRAS)，和傳統發酵食品，尤其是傳統發酵乳製品，有密切的關係。數百年來，蒙古地區的遊牧民族持續的製作各種發酵乳製品，如Airag（發酵馬乳）和Tarag（發酵之牛、山羊、犛牛乳）等等。為了要從這些傳統發酵乳中篩選新的潛力益生菌株，乳酸菌株的收集就變得非常重要。本研究中使用塗盤培養以及分子生物學的技術篩選蒙古傳統發酵乳中具潛力益生菌能力的乳酸菌株。 依據Enterobacterial Reptitive Intergenic Consensus PCR (ERIC-PCR)的結果為基礎，從106個分離自菌落中歸類出40個獨立菌株。透過16s rRNA以及持家基因(housekeeping gene)中的pheS 和 rpoA兩種基因的鑑定，這些菌株分屬八個菌種：Lactobacillus crustorum、Lactobacillus diolivorans、Lactobacillus kefiri、Lactobacillus paracasei、Lactobacillus plantarum subsp. plantarum、Lactococcus. lactis、Leuconostoc lactis以及Leuconostoc pseudomensenteroides。其中Lactobacillus kefiri為最主要的菌種（24株），Lactobacillus crustorum為排第二的菌種（6株）。 接著，本研究針對低pH值、含膽鹽環境的生存耐受性試驗、胞外多醣（EPS）生產量進行測試並作為挑選潛力益生菌能力的項目。結果顯示，Lactobacillus crustorum、Lactobacillus diolivorans、Lactobacillus kefiri在低pH值（pH 3.0）、含膽鹽（0.3% bile）環境具有較佳的生存能力，且Lactobacillus crustorum和Lactobacillus kefiri同時也具有較好的胞外多醣（EPS）生產量。再經過其他條件的測試，兩株Lactobacillus crustorum的菌株（MCC0017及MCC0029）被預期為新的潛力益生菌株。

“Probiotics” are live microorganisms that provide health benefits on the host, when administered in adequate amounts. Lactic acid bacteria (LAB) are regarded as “Generally recognized as safe (GRAS)”, and are mainly involving in the fermentation of traditional fermented foods, especially in fermented milk products. For centuries, the nomadic peoples of Mongolia have been producing various kinds of traditional fermented milk products such as Airag (fermented mare’s milk) and Tarag (fermented milk of cows, goats and yaks). To explore the potential probiotic strains, it is very important to develop the culture collection at the first step. In this study, culture- and molecular-based methods were used to investigate the potential LAB probiotic strains in the traditional Mongolian fermented milk products. Based on Enterobacterial Reptitive Intergenic Consensus PCR (ERIC-PCR) profiles, a total of 106 isolates isolated from five Mongolian traditional fermented milk products were categorized into 40 different strains, and identified as belonging to 8 species (Lactobacillus crustorum, Lactobacillus diolivorans, Lactobacillus kefiri, Lactobacillus paracasei, Lactobacillus plantarum subsp. plantarum, Lactococcus. lactis, Leuconostoc lactis, and Leuconostoc pseudomensenteroides) by 16S rRNA and housekeeping gene (pheS and rpoA) sequencing. Lactobacillus kefiri was isolated as the predominant species (24 strains), followed by Lb. crustorum (6 strains). By the screening tests, survival in low pH and bile salt and production of exopolysaccharides (EPS) were characterized as the potential probiotic abilities. As a result, almost of the strains in Lb. crustorum, Lb. diolivorans, and Lb. kefiri showed high survival ability in artificial gastric (pH 3.0) and intestinal (0.3% bile) juices. And, almost of the strains in the Lb. crustorum and Lb. kefiri species showed high ability to produce EPS. Finally, features of survival abilities in low pH and bile salt, and EPS production demonstrated that the two Lb. crustorum strains (MCC0017, MCC0029) could be novel prospective probiotics.