- 學位論文
益生菌Lactobacillus kefiranofaciens HL1之亞種鑑定與最適化培養基之建立並應用共培養策略製做發酵乳
Subspecies identification and medium optimization of probiotic strain Lactobacillus kefiranofaciens HL1 and its co-culture strategy for manufacturing fermented milk
摘要
菌種Lactobacillus kefiranofaciens的分類可再劃分為兩種特性迥異的亞種──L. kefiranofaciens subsp. kefiranofaciens與L. kefiranofaciens subsp. kefirgranum，然而，兩個亞種之間高度相似的遺傳背景使得準確的鑑定與分類十分困難。本實驗室先前由台灣克弗爾粒中所分離的L. kefiranofaciens HL1 (HL1)，已被證實為具有抗氧化、抗老化功效之潛力益生菌，然而其亞種層級仍屬未知。在本研究中，為進一步確認HL1的分類地位至亞種層級，乃結合表現型與基因型特徵，加以綜合研判。隨後，基於後續可能的加工應用或商品化的目的，亦測定最佳培養條件並建立最適化培養基以將HL1的菌數產量進行最大化。最終，採取共培養的方式，利用微生物間的交互作用以進行發酵，開發出屬於HL1的發酵乳。 為了進行亞種的分類，利用了多種鑑菌技術，包含了形態觀察、基因序列分析、ERIC-PCR與RAPD指紋、醣類發酵試驗，以及全細胞蛋白質指紋圖譜分析，並透過與兩個亞種的標準菌株（L. kefiranofaciens subsp. kefiranofaciens BCRC 16059T與L. kefiranofaciens subsp. kefirgranum BCRC 80410T）進行比較以分類。結果顯示，在RAPD與全細胞蛋白質指紋圖譜的分析中，發現HL1的親緣關係最接近於亞種kefirgranum，然而其他技術則不具有足夠之亞種層級的鑑定能力。明確鑑定出HL1的亞種分類後，接著測定其最佳的各種培養條件，發現在溫度30°C、pH 6.0且不需添加還原劑的情況下，HL1有顯著較好的生長情形，另一方面，亦發現葡萄糖與酵母抽出物為分別為最適合之碳氮源，且額外補充脫脂乳粉與鈣離子有提升菌數的效果。接著應用反應曲面法進一步尋求最大化的HL1菌數產量，獲得之最適化培養基配方為：1%葡萄糖、4.91 g-nitrogen/L酵母抽出物與4%脫脂乳粉，並且此最適化培養基相較於一般MRS培養基提升約15.1倍的菌數產量。最終，評估HL1與數株候選菌株分別進行共培養之影響以開發新式益生菌發酵乳，基於菌落面積分析、MRS培養液與脫脂乳中的活菌數與產酸情形的結果，發現Lactococcus lactis subsp. cremoris APL 015 (APL 015) 最適合做為與HL1共培養的菌株以製作發酵乳，並透過觀察發酵乳中的發酵曲線，證實其可大幅改善發酵產酸能力，且對於HL1活菌數無負面影響。此外，共培養亦不會在發酵乳成品的理化及感官特性方面造成缺陷。 綜上所述，潛力益生菌HL1經過多種鑑定後方能歸類於L. kefiranofaciens subsp. kefirgranum，並且成功利用反應曲面法建立最適化培養基，將其菌數產量最大化。其後，透過一系列評估，最終將HL1與APL 015進行共培養，開發了一種具有特色的新式益生菌發酵乳製品。
並列摘要
Lactobacillus kefiranofaciens has two subspecies, kefiranofaciens and kefirgranum, with the distinct phenotypic features. However, high similarity of genetic background between these two subspecies makes accurate and definitive identification become a very challenging task. Our lab previously isolated a potential probiotic L. kefiranofaciens HL1 (strain HL1) from kefir grains. In the present study, we first classified strain HL1 to subspecies level. We then optimized the growth medium to improve propagating and fermenting performance of strain HL1 for the industrial application and possible commercialization. Furthermore, a fermented milk product was also developed by the utilization of microbial interaction in co-culture fermentation. For classification of subspecies, numerous approaches, including morphological characterization, ERIC-PCR and RAPD fingerprinting, 16S rRNA and housekeeping gene sequence analysis, carbohydrate fermentation tests, and whole cell protein profiling, were conducted by comparison with two type strains (L. kefiranofaciens subsp. kefiranofaciens BCRC 16059T and L. kefiranofaciens subsp. kefirgranum BCRC 80410T). Results indicated that strain HL1 was more phylogenetically related to subspecies kefirgranum based on the findings of the RAPD fingerprinting and whole cell protein profiling. Other technics did not possess enough discriminatory power to identify subspecies level. After taxonomy of subspecies, optimal culture conditions for strain HL1 were determined. We observed a significantly higher growth at the temperature of 30°C, initial pH of 6.0 without reducing agent. The best carbon and nitrogen sources for growth medium was glucose and yeast extract, respectively, with supplements of skim milk and calcium. Subsequently, response surface methodology (RSM) was carried out to further maximize the yield of biomass. Results showed that the biomass of this optimized medium with 1% glucose, 4.91 g-nitrogen/L yeast extract, and 4% skim milk, increased 15.1-fold than regular MRS medium after RSM optimization. Finally, the strain HL1 was co-cultured with each of 4 selected starter culture for development of novel probiotics product. Results of colony size test on MRS agar, microbial viability and acidification in both MRS broth and skim milk suggested that Lactococcus lactis subsp. cremoris APL 015 was suitable for co-culturing with strain HL1. This co-culture combination showed remarkably improved fermentation ability and no negative impact on viability of strain HL1 in the later fermentation kinetics experiment. Moreover, there were no deterioration in physicochemical properties and sensory evaluation of final products when co-culturing. In summary, strain HL1, classified as L. kefiranofaciens subsp. kefirgranum, was successfully maximized its biomass by RSM. Afterwards, we developed a novel fermented milk product through coculturing strain HL1 with Lc. lactis subsp. cremoris APL 015.