Microsoft Word tb 熊君燕-研究报告

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1 微生物学通报 Microbiology China Aug. 20, 2017, 44(8): DOI: /j.microbiol.china 研究报告 清香型白酒中乳酸菌和酵母菌的相互作用 * 熊君燕李小东谢圣凯余培斌陈建新 ( ) 摘要 : 目的 探究清香型白酒中不同乳酸菌和酵母菌的相互作用, 了解不同菌株的发酵性能, 为更深入地认识白酒发酵机理 实现发酵过程优化提供理论基础 方法 利用程序控温和固态发酵模拟清香型白酒酿造环境, 测定纯培养和共培养中菌株的理化指标 活菌数以及主要代谢产物的变化 结果 Saccharomyces cerevisiae YJ1 糖消耗快产乙醇和酯类物质多, Lactobacillus plantarum JMRS4 糖消耗快产酸较多 共培养中乳酸菌对 Saccharomyces cerevisiae YJ1 的生长和产乙醇抑制较大, 对 Candida aaseri MJ7 产乙醇几乎无影响 乳酸菌对 Pichia kudriavzevii MJ14 的生物量和乙醇代谢抑制作用较小, 还对其产己酸乙酯 乙酸乙酯和异戊醇等代谢产物有促进作用 ; 而反过来 Pichia kudriavzevii MJ14 对 3 株乳酸菌产乳酸均有抑制作用, 对产乙酸则有促进作用 结论 建立了一种固态培养方法, 结合清香型白酒发酵温度变化规律, 有效模拟了实际发酵环境 Pichia kudriavzevii MJ14 在与乳酸菌共培养中受到的抑制较小并能有效抑制乳酸菌产乳酸,Saccharomyces cerevisiae YJ1 能代谢产生多种风味物质, 对清香型白酒酿造有重要意义 关键词 : 清香型白酒, 乳酸菌, 酵母菌, 相互作用, 固态发酵, 程序控温发酵 Interaction between lactic acid bacteria and yeasts in light-aroma liquor XIONG Jun-Yan LI Xiao-Dong XIE Sheng-Kai YU Pei-Bin CHEN Jian-Xin * (School of Biotechnology, Jiangnan University, National Engineering Laboratory for Cereal Fermentation Technology, Wuxi, Jiangsu , China) Abstract: [Objective] It can provide a theoretical basis for understanding the fermentation mechanism and optimizing the fermentation process that studying on the interaction between different lactic acid bacteria and yeast in light-style liquor and fermentation performance of different strains. [Methods] The light-style liquor fermentation environment was simulated by programmed temperature control and solid state fermentation. In pure culture and co-culture, the changes of physicochemical parameters, viable count and the main metabolites were analyzed. [Results] Saccharomyces cerevisiae YJ1 strain had a higher rate of sugar consumption and produced more ethanol and esters. Lactobacillus plantarum JMRS4 strains had higher rate of sugar consumption and more acid production. Lactic acid bacteria slightly inhibited Pichia kudriavzevii MJ14 biomass and *Corresponding author: Tel: ; jxchen@jiangnan.edu.cn Received: December 14, 2016; Accepted: February 21, 2017; Published online ( February 24, 2017 * 通讯作者 :Tel jxchen@jiangnan.edu.cn 收稿日期 : ; 接受日期 : ; 优先数字出版日期 (

2 1768 微生物学通报 Microbiol. China 2017, Vol.44, No.8 ethanol production, but promoted the production of ethyl hexanoate, ethyl acetate and isoamyl alcohol. In turn, Pichia kudriavzevii MJ14 inhibited three strains of lactic acid bacteria from producing lactic acid and promoted the production of acetic acid. [Conclusion] The actual fermentation process of light-style liquor was simulated by establishing the solid state culture method and simulating the temperature change during the fermentation process. Pichia kudriavzevii MJ14 was slightly inhibited by lactic acid bacteria and could effectively inhibit lactic acid bacteria from producing lactic acid when co-cultured. Saccharomyces cerevisiae YJ1 can produce a variety of flavor substances, which is of great significance to the production of light-style liquor. Keywords: Light-aroma liquor, Lactic acid bacteria, Yeast, Interaction, Solid state fermentation, Temperature controlled fermentation [1] [2] [3] [4] [5] [6] [7] [8-9] [10] [11] [12-13] [14] [15] [16] [16] [17] 1 材料与方法 1.1 材料 实验材料 : 菌株来源 : Pichia kudriavzevii MJ14 Saccharomyces cerevisiae YJ1 Candida aaseri MJ7 Pediococcus sp. JYA1 Lactobacillus plantarum MRS4 Leuconostoc lactis JMRS 培养基 : WL [18] 1 g/l YPD [18] MRS [19] 1 g/l

3 : 主要试剂和仪器 ( ) ( ) ( ) ( ) - (Trace 1300-ISQ QD) (Pico 17) Thermo Fisher Scientific (1260 infinity) Agilent (57328U) Supelco (BSP-250) (AL204) METTLER TOLEDO (W201D) (UV mini) SHIMADZU (No6812) 1.3 方法 菌株筛选 :(1) 10 g 7 90 ml WL 30 C WL (2) MRS 30 C MRS 菌种鉴定 :(1) CTAB [5] (2) PCR 10 Buffer 5 μl 2.5 mmol/l dntps 4 μl 1 μl 100 μmol/l 3 μl Taq 0.5 μl 33.5 μl 94 C 5 min 94 C 30 s 50 C 30 s ( ) 56 C 30 s ( ) 72 C 90 s 72 C 10 min [5][20] PCR NCBI 菌株形态和生理生化研究 :(1) (2) [21-22] 菌株模拟固态发酵 : CFU/mL 3 12 d 48 h 生长曲线的测定 :(1) CFU/mL YPD 25 C 2 h 1 ml YPD 28 C (2) g 90 ml 1 ml MRS YPD 30 C 理化指标的测定 : [23] 50 g 100 ml 250 ml 50 ml 100 ml 乳酸和乙酸含量测定 : HPLC 10 g 20 ml 4 C r/min 5 min 5 ml 0.5 ml (10.6%) 0.5 ml (30%) 4 C 0.22 µm [24] HPLC ph mmol/l 1 ml/min 210 nm 30 C 20 µl y=511x R 2 =

4 微生物学通报 Microbiol. China , Vol.44, No.8 y=678.59x R2= NCBI 比对筛选得到 22 株酵母菌和 15 株乳酸菌 如表 1 所示 挥发性成分测定 采用顶空固相微萃取和 气质联用测定 菌株的形态和生理生化特征 挑选 3 株酵 前处理 取 2 g 酒醅和 3 g 氯化钠于 10 ml 顶 母菌和 3 株乳酸菌作为目的菌株 经过外观形态和 空瓶中 加入 1 ml 内标乙酸正戊酯(1 g/l)和 7 ml 生理生化特征确定酵母为 P. kudriavzevii MJ14 超纯水 S. cerevisiae YJ1 C. aaseri MJ7 乳酸菌为 P. sp. 顶空固相微萃取条件 样品 50 C 预热 15 min 插入针头距离液面 1 cm 处 吸附 30 min 进样口 解析 5 min 色谱条件 起始温度 40 C 保留 2 min 以 2 C/min 升温至 80 C 保留 1 min 再以 5 C/min 升温至 230 C 保持 5 min 进样口温度为 250 C 质谱条件 离子源温度 230 C 质子扫描范 围 结果与讨论 2.1 菌株的筛选与鉴定 菌 株 筛 选 经 过 筛 选 纯 化 分 子 鉴 定 和 图1 Figure 1 JYA1 L. plantarum JMRS4 L. lactis JMRS1-2 菌 落和显微镜形态结构如图 1 所示 酵母菌的形态描 表1 酒醅中酵母菌和乳酸菌的分离结果 Table 1 The separation of yeast and lactic acid bacteria in the fermented grains 属名 Genus name Saccharomyces Pichia Candida Lactobacillus Pediococcus Leuconostoc 菌株数 Number of strains 酵母菌和乳酸菌在培养基上菌落及微观形态比较 Comparation of colonies morphology and micromorphologies of yeast and lactic acid bacteria 注 A C E 分别为 P. kudriavzevii MJ14 S. cerevisiae YJ1 C. aaseri MJ7 的菌落形态 B D F 分别为 P. kudriavzevii MJ14 S. cerevisiae YJ1 C. aaseri MJ7 的显微镜形态 G I K 分别为 P. sp. JYA1 L. plantarum JMRS4 L. lactis JMRS1-2 菌落形态 H J L 分别为 P. sp. JYA1 L. plantarum JMRS4 L. lactis JMRS1-2 显微镜形态. Note: A, C and E stand for the colony morphology of P. kudriavzevii MJ14, S. cerevisiae YJ1 and C. aaseri MJ7 respectively; B, D and F stand for the micromorphologies of P. kudriavzevii MJ14, S. cerevisiae YJ1 and C. aaseri MJ7 respectively; G, I and K stand for the colony morphology of P. sp. JYA1, L. plantarum JMRS4 and L. lactis JMRS1-2 respectively; H, J and L stand for the micromorphologies of P. sp. JYA1, L. plantarum JMRS4 and L. lactis JMRS1-2 respectively.

5 : 清香型白酒发酵条件探究 固态培养与液态培养下酵母菌活菌数的变化 : S. cerevisiae YJ h 4 [12] 控温培养与恒温培养对酵母菌生长的影响 : 25 C 3 S. cerevisiae YJ CFU/g 10 5 CFU/g C. aaseri MJ7 25 C C. aaseri MJ7 S. cerevisiae YJ1 P. kudriavzevii MJ14 Strains P. kudriavzevii MJ14 S. cerevisiae YJ1 表 2 酵母菌形态及生理生化反应 Table 2 Morphology and physiological and biochemical responses of yeast Fermentation test Colonies morphology Micromorphology Lactose Maltose Sucrose Glucose Starch Ethanol C. aaseri MJ7 +. Note: +: Positive; : Negative 表 3 乳酸菌形态及生理生化反应 Table 3 Morphology and physiological and biochemical responses of lactic acid bacteria Acid production Colonies Vancomycin Strains Micromorphology morphology tolerance Citrate Fructose Maltose Lactose Sucrose Glucose Starch P. sp. JYA1 d L. plantarum JMRS4 L. lactis JMRS1-2 d d + d. Note: +: Positive; : Negative; d: Not tested d

6 1772 微生物学通报 Microbiol. China 2017, Vol.44, No.8 Figure 2 图 2 S. cerevisiae YJ1 在液体和固体培养中的活菌数变化 Viable count of S. cerevisiae YJ1 in liquid culture medium and solid culture medium 图 3 酵母菌在恒温和控温培养条件下活菌数的变化情况 Figure 3 Viable count of yeast during constant temperature fermentation and variable temperature fermentation MJ14 P. kudriavzevii MJ14 YJ1 S. cerevisiae YJ1 MJ7 C. aaseri MJ7 MJ14+JYA1 P. kudriavzevii MJ14 P. sp. JYA1 YJ1+JYA1 S. cerevisiae YJ1 P. sp. JYA1 MJ7+JYA1 C. aaseri MJ7 P. sp. JYA1 H 25 C K. Note: MJ14: P. kudriavzevii MJ14; YJ1: S. cerevisiae YJ1; MJ7: C. aaseri MJ7; MJ14+JYA1: Co-culture of P. kudriavzevii MJ14 with P. sp. JYA1; YJ1+JYA1: Co-culture of S. cerevisiae YJ1 with P. sp. JYA1; MJ7+JYA1: Co-culture of C. aaseri MJ7 with P. sp. JYA1; H: 25 C constant temperature fermentation; K: Variable temperature fermentation. 2.3 酵母菌和乳酸菌单菌发酵理化指标的变化 4A S. cerevisiae YJ1 P. kudriavzevii MJ14 C. aaseri MJ7 3 S. cerevisiae YJ % vol C. aaseri MJ7 0.9% vol 3B 4A 3

7 : 1773 图 4 酵母菌和乳酸菌单菌发酵理化指标变化 Figure 4 Physicochemical indexes during single cultures of yeast and lactic acid bacteria A B JMRS4 L. plantarum JMRS4 JYA1 P. sp. JYA1 JMRS1-2 L. actis JMRS1-2 R AS AC. Note: A: Reducing sugar and alcohol strength in the fermentation process of yeast; B: Reducing sugar and acidity in the fermentation process of lactic acid bacteria; JMRS4: L. plantarum JMRS4; JYA1: P. sp. JYA1; JMRS1-2: L. actis JMRS1-2; R: Reducing sugar; AS: Alcohol degree; AC: Acidity. 4B 8 8 L. plantarum JMRS4 P. sp. JYA1 L. actis JMRS mg/100 g 2.4 共培养中乳酸菌对酵母菌产乙醇的影响 12 ( 5) S. cerevisiae YJ1 P. kudriavzevii MJ14 C. aaseri MJ7 3B P. sp. JYA1 P. sp. JYA1 [25] S. cerevisiae YJ1 P. kudriavzevii MJ14 ph [17] C. aaseri MJ7 图 5 酒精度含量 Figure 5 Content of alcoholic strength YJ1*-AS S. cerevisiae YJ1 MJ7*-AS C. aaseri MJ7 MJ14*-AS P. kudriavzevii MJ14. Note: YJ1*-AS: Alcoholic strength of S. cerevisiae YJ1 during single culture and co-culture conditions; MJ7*-AS: Alcoholic strength of C. aaseri MJ7 during single cultures and co-culture conditions; MJ14*-AS: Alcoholic strength of P. kudriavzevii MJ14 during single cultures and co-culture conditions.

8 1774 微生物学通报 Microbiol. China 2017, Vol.44, No 共培养中酵母菌对乳酸菌产乳酸和乙酸的影响 6A L. plantarum JMRS4 12 d 7.63 g/l P. sp. JYA1 L. lactis JMRS1-2 P. kudriavzevii MJ14 6B L. lactis JMRS g/l L. plantarum JMRS4 P. sp. JYA1 L. lactis JMRS1-2 [26] P. kudriavzevii MJ14 L. lactis JMRS 纯培养和共培养条件下特征代谢产物的差异 7 S. cerevisiae YJ1 P. kudriavzevii MJ14 C. aaseri MJ7 C. aaseri MJ7 C. aaseri MJ7 S. cerevisiae YJ1 S. cerevisiae YJ1 L. plantarum JMRS4 L. lactis JMRS1-2 图 6 乳酸和乙酸含量 Figure 6 The concentration of lactic acid and acetic acid JYA1-L P. sp. JYA1 JMRS4-L L. plantarum JMRS4 JMRS1-2-L L. lactis JMRS1-2 JYA1-A P. sp. JYA1 JMRS4-A L. plantarum JMRS4 JMRS1-2-A L. lactis JMRS1-2. Note: JYA1-L: Lactic acid content of P. sp. JYA1 during single cultures and co-culture conditions; JMRS4-L: Lactic acid content of L. plantarum JMRS4 during single cultures and co-culture conditions; JMRS1-2-L: Lactic acid content of L. lactis JMRS1-2 during single cultures and co-culture conditions; JYA1-A: Acetic acid content of P. sp. JYA1 during single cultures and co-culture conditions; JMRS4-A: Acetic acid content of L. plantarum JMRS4 during single cultures and co-culture conditions; JMRS1-2-A: Acetic acid content of L. lactis JMRS1-2 during single cultures and co-culture conditions.

9 : 1775 Figure 7 图 7 纯培养和共培养条件下特征代谢产物热图 Heat map of main metabolites during single cultures and co-culture conditions 3 结论 3 P. kudriavzevii MJ14 P. kudriavzevii MJ14 C. aaseri MJ7 S. cerevisiae YJ1 P. kudriavzevii MJ14 P. sp. JYA1 L. plantarum JMRS4 L. plantarum JMRS4 L. lactis JMRS1-2 P. kudriavzevii MJ14 S. cerevisiae YJ1 C. aaseri MJ7 参考文献 [1] Frey-Klett P, Burlinson P, Deveau A, et al. Bacterial-fungal interactions: hyphens between agricultural, clinical, environmental, and food microbiologists[j]. Microbiology and Molecular Biology Reviews, 2011, 75(4): [2] Tang J, Wang HY, Xu Y. Effect of mixed culture of Saccharomyces cerevisiae and Pichia anomala on fermentation

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