克弗爾微生物於甘蔗丁之固定化系統與其發酵應用

Abstract

克弗爾是含酒精氣泡風味之傳統發酵乳，近年來已被廣泛證實具健康機能性。因透過克弗爾粒的傳統製備過程緩慢且菌相無法標準化，商業上則使用種菌元 (mother culture) 或克弗爾分離菌株 (pure cultures) 進行生產，惟後者面臨風味不足和產氣特性之包裝疑慮。本研究以甘蔗丁作載體，與篩選純化自台灣新竹地區克弗爾粒之乳酸菌株進行培養，開發屬天然固定化且具重複利用性之發酵菌元體，期可改善上述情況並提升發酵穩定性。 甘蔗丁與克弗爾乳酸菌株Lactobacillus kefiranofaciens HL1、Lactobacillus kefiri HL2、Leuconostoc mesenteroides HL3和Lactococcus lactis HL4經過24小時培養，所形成固定化菌元主要由生長快速且可利用乳糖和蔗糖之Lc. lactis HL4 (82%) 所貼附，菌數約9 log CFU/g。重複28批次發酵期間，菌液 (free cells, F) 與固定化菌元 (immobilized cells, I) 發酵樣品之酸鹼值維持穩定pH 4.8； 而冷凍乾燥後之固定化菌元 (freeze-dried immobilized cells, FD) 可使樣品從pH 4.8逐漸下降至pH 4.4。I組產物之酸生成隨發酵批次逐漸上升並於後期顯著高於F組，I、F、FD組之平均酸度分別為0.55%、0.65% 和0.74%，而Lc. lactis HL4與 Leu. mesenteroides HL3 為當中主要產酸菌株。檢測表觀黏度、離水力和胞外多醣含量，I、F組產品之結果相似。 發酵期間，I組於甘蔗丁和發酵乳中微生物數維持10 log CFU/g (CFU/mL)，以random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) 進行菌相分析，發現各自優勢菌群分別為Leu. mesenteroides HL3 (44%)、Lc. lactis HL4 (38%) 和Lc. lactis HL4 (79%) 且展示高穩定性。FD組之甘蔗丁菌量與I組相仿，但可釋放至發酵乳中微生物數卻下降1 log值。透過掃描式電子顯微鏡，可觀察四株克弗爾乳酸菌株貼附於甘蔗丁之孔隙結構並和周圍發酵乳固形物黏合而鑲嵌其中，然發酵期間甘蔗丁維持其原有形態。在壓力耐受性測試，I組面對酸壓力 (pH 2.5, 3小時) 之存活率較F組高約0.04%，而在膽鹽壓力 (1% 膽鹽，1小時) 其存活優勢限於重複批次發酵前期。將FD菌元置4°C 和 -20°C進行保存試驗，為期28天中其微生物數可維持9 log CFU/g。 綜言之，以甘蔗丁作食品級載體，克弗爾乳酸菌株之固定化菌元所產發酵乳屬低酸性食品 (pH > 4.6)，其菌數和酸度可符合益生菌發酵乳規定。當中所展現之穩定菌相變化和冷凍乾燥保存之活菌維持，有利於商業生產之標準化 (standardization)。以此固定化菌元所開發之新式發酵乳 (new-type fermented milk) 可增加乳製品多樣性以供廣大消費群眾選擇。

Kefir is a traditional fermented milk with alcoholic and acidic flavor which well-known by its functionalities. However, artisanal preparation of kefir is not efficient for standardization while commercial production with mother or pure cultures has inferior tastes. The objective of this study is to develop a natural immobilized starter culture with sugar cane pieces as carries in ameliorating the issues above. Lactobacillus kefiranofaciens HL1, Lactobacillus kefiri HL2, Leuconostoc mesenteroides HL3 and Lactococcus lactis HL4 isolated from Taiwanese kefir grains were cultured with sugar cane pieces for 24 hours and immobilized with 9 log CFU/g. Lc. lactis HL4 (82%) which was relatively superior in growth and utilization of sugars including lactose and sucrose was dominated. During repeated-batch fermentation for 28 cycles, both immobilized (group I) and free (group F) cells culture performed stable pH 4.8 while freeze-dried immobilized cells (group FD) could further drop to pH 4.4. The average acidity of groups F, I and FD were 0.55%, 0.65% and 0.74% respectively with gradual increase tendency. In the mixed culture, Lc. lactis HL4 and Leu. mesenteroides HL3 were mainly contributed to acid production. Besides, the apparent viscosity, syneresis and EPS amount were comparable between groups F and I. During fermentation, viable cells on sugar cane pieces and fermented samples in group I were 10 log CFU/g (or CFU/mL). The analysis of RAPD-PCR revealed that Leu. mesenteroides HL3 (44%) and Lc. lactis HL4 (38%) were majorly distributed on sugar cane pieces while 79% of Lc. lactis HL4 (79%) was found in fermented samples. The microbial composition was preserved throughout the batches. In group FD, cells being released to the samples were decreased by 1 logarithm. Under the observation of SEM, each strains was embedded within porous structures of sugar cane pieces and believed to be strengthen in adherence with milk solid particles. In addition, group I showed a 0.04% increase in survival rate to group F under acid stress (pH 2.5, 3 hours) while its superiority under bile stress (1% bile, 1 hour) appeared in earlier batches of fermentation. For storage test in 28 days, viable cells of freeze-dried immobilized pieces were maintained (9 log CFU/g). Taken together, the product of immobilized cell starter culture with sugar cane pieces was low acidic (pH > 4.6) while performing adequate amount of live microorganism and acidic properties. Stable microbial composition shown was crucial for commercialization of mixed strains culture. This immobilized system involving kefir microorganism and sugar cane pieces could create a new-type fermented beverage for human consumptions on dairy products.