評估以克弗爾分離菌株製作克弗爾對其理化、微生物及感官性質的影響

Abstract

克弗爾是一種源自於高加索、東歐與巴爾幹等地區的傳統發酵乳，近年來，克弗爾的機能性使其逐漸在世界各地受到歡迎，但由於克弗爾粒在結構上與微生物組成上的特性，使得克弗爾在商業上的生產面臨許多難題。因此，本實驗將使用從克弗爾粒中分離出來的純菌株進行發酵乳製作，以期達到簡化克弗爾生產過程，以及保存傳統克弗爾的風味與質地等目的。 實驗將分為兩個部分。第一部分為克弗爾的製作，分為使用克弗爾粒的傳統組以及使用克弗爾粒分離之純菌株的純菌株組 (Lactobacillus kefiranofaciens M1、Lb. kefiri B15、Lactococcus. lactis MK4、Kluyveromyces marxianus E3)。冷凍的克弗爾粒會以10% (w/v) 的比例加入牛乳中，於20℃發酵20小時，並重複三次，使其恢復活性，待活性恢復後，每週繼代一次即可。四種純菌株會各自於培養基中活化三次，並接種於牛乳中使其適應牛乳的環境，之後根據它們在克弗爾粒中的數量加入牛乳中混合，於20℃發酵48小時。發酵後，樣品皆分裝並保存於4℃，待後續分析。第二部分為物理化性質分析。兩種發酵乳將會保存21天，並每7天取樣進行分析。分析項目包含，pH值、滴定酸度、質地、黏度、總菌數、qPCR (quantitative polymerase chain reaction)、有機酸含量與消費者喜好性試驗。實驗結果顯示，純菌株組的pH值 (4.21-4.39) 顯著高於傳統組 (4.04-4.14)，並且純菌株組在保存期間有逐漸下降的現象。滴定酸度於傳統組 (1.13%-1.16%) 顯著高於純菌株組 (0.86%-1.07%)，在兩組中皆會隨著保存期間增加而上升。質地分析的結果顯示，純菌株組在堅實度、稠度、內聚性與內聚功皆高於傳統組，而兩組間沒有顯著差異，四個項目也都會在保存期間下降。黏度在純菌株組中顯著較高，且會在保存期間下降，而傳統組則有上下起伏的現象。乳酸菌數量在純菌株組中顯著高於傳統組，其數量約維持在9 log CFU/mL，而傳統組的數量則在第7與14天之間減少了1.17 log CFU/mL，同時酵母菌增加了0.98 log CFU/mL。酵母菌突然的增加可能導致乳酸菌的減少。以qPCR定量四株菌的數量後發現，傳統組中以Lb. kefiranofaciens為優勢菌種，而純菌株組則為Lc. lactis。除了傳統組的K. marxianus之外，其他菌株皆會在保存期間內減少。有機酸分析的結果指出，傳統組除了檸檬酸之外，乳酸、甲酸、乙酸與丁酸含量皆高於純菌株組，其含量除了傳統組的檸檬酸之外，在保存期間皆無顯著變化。酒精含量在兩組間無顯著差異，而在保存期間皆有顯著的增加。消費者喜好性試驗指出，純菌株組第0天的分數最高，不過仍低於傳統組。其中影響接受度最主要的因素為口感與風味，有受試者指出純菌株組的第14與21天有明顯的酒味，而第14天的因為有細小的凝乳塊，使其口感得分最低。綜上所述，以克弗爾粒分離的四株純菌株確實可以進行發酵，並製成發酵乳，達到簡化生產過程、免去使用克弗爾粒的目的。純菌株組在微生物數量方面與傳統組不同，未來也可針對其機能性做更深入的探討，以評估其機能性是否相似於傳統克弗爾或更好。不過純菌株克弗爾的發酵時間較長，並且在保存期間會產生過於明顯的酒味，因此後續可針對菌株的數量做調整，以縮短發酵時間與提升風味。此外，不同批次間發酵乳的品質穩定性與一致性也須做更進一步的試驗，以使其更加適合用於商業生產。

Milk kefir, an ancient fermented beverage, originates from the Caucasus Mountains, Eastern Europe, and the Balkans, with a history dating back centuries. In recent years, kefir has gained global popularity due to its health-promoting properties, resulting in increased market availability. However, the unique biological structure and variability of kefir grains pose challenges for industrial-scale production. Therefore, this study aimed to simplify the production process and preserve the traditional sensory qualities of kefir by using a defined mixture of kefir-isolated microorganisms. This study consists of two main parts. The first part focused on kefir production. Traditional kefir was made with kefir grains, and pure culture kefir was made with four dominant kefir-derived strains (Lactobacillus kefiranofaciens M1, Lactobacillus kefiri B15, Lactococcus lactis MK4, and Kluyveromyces marxianus E3). Frozen kefir grains were activated by three successive fermentations (10% w/v in fresh milk at 20 °C for 20 hours), followed by weekly subculturing. For pure culture kefir, each strain was individually activated in broth, then combined based on their proportional abundance in kefir grains and inoculated into milk, followed by fermentation at 20 °C for 48 hours. All samples were stored at 4 °C for subsequent analysis. The second part evaluated the physicochemical and microbial characteristics of both kefir types every seven days over a 21-day storage period. Parameters assessed included pH, titratable acidity, total microbial counts, qPCR (quantitative polymerase chain reaction)-based microbial quantification, texture profile, viscosity, organic acid content, and consumer preference. Results showed that the pH of pure culture kefir (4.21-4.39) was significantly higher than that of traditional kefir (4.04-4.14), and declined gradually during storage. Titratable acidity was consistently higher in traditional kefir and increased slowly over time in both groups. Texture profile analysis revealed that firmness, consistency, cohesiveness, and work of cohesion were higher in pure culture kefir, though differences were not statistically significant. All texture attributes declined during storage. Viscosity was significantly higher in the pure culture group, but also decreased over time. LAB counts were significantly different between the groups; pure culture kefir maintained ~9 log CFU/mL, whereas traditional kefir experienced a ~1 log reduction from day 7 to 14. Yeast counts increased by ~1 log in both groups during storage, potentially contributing to the LAB decline in traditional kefir. Quantitative-PCR (qPCR) analysis indicated that Lb. kefiranofaciens dominated in traditional kefir, while Lc. lactis was most abundant in pure culture kefir. All strains, except K. marxianus, declined during storage in both groups. The organic acid assay indicated that except for citric acid, the contents of lactic acid, formic acid, acetic acid and butyric acid are higher in traditional group than pure culture group. The concentrations for organic acids had no significant difference during storage except for citric acid in traditional group. For ethanol content, there were no significant differences between two groups, but the concentration increased significantly during storage. The consumer preference test revealed that the pure culture group at day 0 had the highest score, but still lower than traditional group. According to the results, the main factors that affect the acceptance were texture and flavor. Some testers reported that there was distinct alcohol taste in pure culture group at day 14 and 21. Furthermore, there were small curds presence in pure culture group at day 14, hence it got the lowest score. In conclusion, kefir can be successfully produced using a simplified four-strain system. However, batch-to-batch consistency and stability remain challenges. Further optimization of microbial ratios and inoculation methods is needed for industrial scalability, as well as for reducing fermentation time and improving the flavor and texture during storage.