Graduate School of Animal Husbandry, Obihiro University of Agriculture and Veterinary Medicine, 11, Nishi 2, Inadacho, Obihiro, Hokkaido 080-8555, Japan.
Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14, Hayashi-Cho, Takamatsu, Kagawa, Japan.
Int J Food Microbiol. 2019 Oct 2;306:108263. doi: 10.1016/j.ijfoodmicro.2019.108263. Epub 2019 Jul 9.
Ishizuchi-kurocha is a Japanese traditional fermented tea that is produced by primary aerobic and secondary fermentation steps. The secondary fermentation step of Ishizuchi-kurocha is mainly mediated through lactic acid bacteria. Here, we performed quantitative analyses of the culturable fungal communities at each step and identified several morphologically representative fungal isolates. While filamentous fungi (median, 3.2 × 10 CFU/g sample) and yeasts (median, 3.7 × 10 CFU/g) were both detected after the primary fermentation step, only yeasts (median, 1.6 × 10 CFU/g) were detected in the end of the secondary fermentation step, suggesting that the fungal community in tea leaves are dramatically changed between the two steps. Pichia kudriavzevii and Pichia manshurica, the prevalent fungal species at the end of the secondary fermentation step, grew well in exudate from the secondary fermentation step. P. kudriavzevii also grew well in media containing d- or l-lactate as the sole carbon source. The growth of the disruptant of cyb2A encoding a cytochrome b lactate dehydrogenase in P. kudriavzevii was severely impaired on medium supplemented with l-lactate, but not d-lactate, suggesting that Cyb2Ap plays a crucial role in the use of l-lactate, and P. kudriavzevii efficiently uses both l- and d-lactate as carbon sources. Thus, lactate assimilation seems to be a key phenotype to become a prevalent species in the secondary fermentation step, and Cyb2Ap has a pivotal role in l-lactate metabolism in P. kudriavzevii. Further understanding and engineering of P. kudriavzevii and P. manshurica will contribute to the control of lactic acid bacteria fermentation during the fermented tea production and also to other industrial uses.
石臼黑茶是一种日本传统发酵茶，它通过初级需氧和次级发酵两个步骤制成。石臼黑茶的次级发酵步骤主要通过乳酸菌进行介导。在这里，我们对每个步骤可培养真菌群落进行了定量分析，并鉴定了几个形态代表的真菌分离株。虽然在初级发酵步骤之后都检测到丝状真菌（中位数，3.2×10 CFU/g 样品）和酵母（中位数，3.7×10 CFU/g），但在次级发酵步骤结束时仅检测到酵母（中位数，1.6×10 CFU/g），这表明在这两个步骤之间，茶叶中的真菌群落发生了巨大变化。在次级发酵步骤结束时，普遍存在的真菌物种是毕赤酵母属（Pichia）的酒香酵母（Pichia kudriavzevii）和格氏假丝酵母（Pichia manshurica），它们在次级发酵渗出物中生长良好。P. kudriavzevii 也在含有 d-或 l-乳酸作为唯一碳源的培养基中生长良好。毕赤酵母属（Pichia）cyb2A 编码细胞色素 b 乳酸脱氢酶的 disruptant 在补充 l-乳酸的培养基中的生长严重受损，但在补充 d-乳酸的培养基中不受影响，这表明 Cyb2Ap 在利用 l-乳酸方面起着至关重要的作用，并且 P. kudriavzevii 可以有效地利用 l-和 d-乳酸作为碳源。因此，乳酸同化似乎是成为次级发酵步骤中优势种的关键表型，而 Cyb2Ap 在 P. kudriavzevii 的 l-乳酸代谢中起着关键作用。进一步了解和工程化 P. kudriavzevii 和 P. manshurica 将有助于控制发酵茶生产过程中的乳酸菌发酵，也有助于其他工业用途。
