藉由共培養方法探討克弗爾微生物的交互作用與克弗爾粒之形成

Abstract

克弗爾是一種以克弗爾粒顆粒作為菌元之傳統發酵乳，其中含有乳酸菌 (LAB) 和酵母菌共生於多醣和蛋白基質中。許多研究指出傳統克弗爾具有對健康有益之好處。然而，克弗爾粒之形成機制仍然不清楚。本研究旨在藉由克弗爾優勢菌種共培養法來探討微生物的相互作用與克弗爾粒之形成。 透過三世代定序分析，臺灣克弗爾粒中含有五株優勢乳酸種與兩株優勢酵母菌種，其中有一株乳酸菌種，名為Lentilactobacillus parakefiri為之前未被分離與鑑定。因此，第一部分將臺灣乳清克弗爾粒中分離並以表觀型及基因型法進行鑑定，結果只有分離出一株菌株並將其命名為ML5。隨後測定其自聚集能力和生物膜形成，結果顯示ML5於pH 4.2下其自聚集能力顯著較強，但與其他乳酸菌和酵母菌之共聚集能力較弱。ML5於單株培養時所產生的生物膜較少，但與其他菌種共培養後則會形成較多生物膜。此特性得以假設聚集力較好之菌株共培養可形成小顆粒，因此進行共培養試驗，分為控制組 (7株優勢菌種) 與自聚集能力較強組 (Lactobacillus kefiranofaciens、Kazachstania turicensis與Lt. parakefiri)，分別共培養於牛奶和乳清中，結果並沒有顆粒形成，但7株優勢菌種共培養中可以觀察到微生物附著於牛奶凝乳塊之表面。從以上结果，我們假設牛奶中的天然物質，如凝乳塊可以作為克弗爾微生物附著之載體。之後便使用PMA real-time PCR定量克弗爾粒粒中各優勢菌種的活菌數，其中主要最優勢乳酸菌種為Lb. kefiranofaciens (~108 CFU/mL) 和Kluyveromyces marxianus (~107 CFU/mL)。根據定量結果，即Lb. kefiranofaciens接種8 log CFU/mL，Lt. kefir、Klu. marxianus接種7 log CFU/mL，而Leu. mesenteroides、Lc. lactis、Lt. parakefiri和Kaz. turicensis則接種6 log CFU/mL，以7天間隔連續發酵的方式共培養此7株優勢菌種於40 mL牛奶，共培養實驗分為四組:控制組包括4 g克弗爾粒 (G0) 與0.1 g克弗爾粒粒 (G1)、純菌株組 (G2)、純菌株 + 0.1 g克弗爾粒 (G3) 及純菌株 + 0.1 g顆粒狀凝乳塊 (G4)。與G1組相比，額外添加純菌株之G3組稍微增加了克弗爾粒粒的重量，但沒有顯著差異，但部分優勢菌種長得較快。通過掃描式電子顯微鏡觀察到G4組的顆粒狀凝乳塊微結構與克弗爾粒相近，且可作為克弗爾粒優勢菌種貼附之載體。但是此試驗顯示酵母於共培養中之生長迅速，與克弗爾粒中的實際情況不同，因此將Klu. marxianus接種量調整至6 log CFU/mL及Lc. lactis與Kaz. turicensis調整至4-5 log CFU/mL，結果顯示G4中所有優勢菌種活菌數都較為穩定，可產出穩定菌相的克弗爾產品。隨著發酵時間的增加，發酵乳中的胞外多醣也隨之增加。 綜上所述，我們的研究結果證實以純菌共培養無法形成小顆粒，但以天然載體供克弗爾微生物附著使其發酵過程中持續生長，此法有望最後形成如同克弗爾粒之菌元，但未來還需進一步探討克弗爾優勢菌之間的相互作用。

Kefir is a traditional fermented dairy product using kefir grains as starter cultures which are symbiotic mixtures of lactic acid bacteria (LAB) and yeasts embedded in a matrix of polysaccharides and proteins. Numerous studies elucidated that kefir provided health benefits. However, the mechanisms of kefir grain formation remained unclear. The present study aimed to produce kefir grains with different co-culture methods meanwhile understanding the relationship between dominant kefir species and other factors affected. Through further next-generation sequencing, Taiwanese kefir grains contained five dominant bacterial species and two dominant yeast species. There was one bacterial species, named Lentilactobacillus parakefiri not isolated and identified before. Hence, this species was isolated from Taiwanese whey kefir grains and identified through phenotypic and genotypic methods. We isolated only one strain and designated it as ML5. We then assessed its cell surface properties, conducting tests for auto-aggregation and biofilm formation. Results showed that ML5 exhibited strong auto-aggregation at pH 4.2 but had weaker co-aggregation abitlity with other LAB and yeast. It also produced less biofilm in monoculture but formed more biofilm when co-culture with other species. This distinctive characteristic has the potential to facilitate the formation of small granules. Therefore, we co-cultured one group with seven dominant species and one group with species that possessed stronger auto-aggregation ability, which is Lactobacillus kefiranofaciens, Kazachstania turicensis and Lt. parakefiri in milk and whey with Leuconostoc mesenteroides that benefited the growth of Lb. kefiranofaciens. Results indicated that no grain formed, but co-culture of all seven dominant kefir species could be observed in the attachment of microbes onto the surface of the milk curd. From these results, we hypothesized that natural elements in milk such as hard curd could be the vector for microbes to obtain kefir grains. Then, viable cells of each dominant species in kefir grains were quantified using PMA real-time PCR. The most dominant bacteria and yeast were Lb. kefiranofaciens (~108 CFU/mL) and Kluyveromyces marxianus (~107 CFU/mL), respectively. According to the quantifying microbial proportion of kefir grains, we co-cultured these seven dominant species in 40 mL milk using a 7-day interval subcultural fermentation to obtain small kefir grains. The co-culture experiment was divided into four groups: Control included 4 g kefir grains (G0) and 0.1 g kefir grain (G1), pure strains group (G2), pure strains + 0.1 g kefir grains (G3), and pure strains + 0.1 g granular curd (G4). Compared with control group, additional pure strains slightly increased the weight of kefir grain but no significant difference and the amount of each dominant species faster in kefir grain (G3). Through scanning electron microscope and real-time PCR, additional granular curd (G4) could provide a vector for dominant species adhering since the amount of kefir species elevated and its appearance looked similar to kefir grains. Nevertheless, yeasts exhibited accelerated growth in co-culture which was differed from the actual conditions observed in kefir grains. Thus, inoculated amount was adjusted for each species and it seemed that all species were constantly grew in G4, potential to produce stable kefir product. The EPS production also increased with fermentation time. In summary, our findings suggest that relying solely on pure strains co-cultured in milk is insufficient for grain formation. Introducing a natural vector represents a promising approach for kefir species to thrive and develop grains. In the future, further research will explore the interplay between kefir species.