WO2024251311A2 - 一株耐乳酸胁迫且高产乙醇和风味化合物的拜耳接合酵母 - Google Patents

一株耐乳酸胁迫且高产乙醇和风味化合物的拜耳接合酵母 Download PDF

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WO2024251311A2
WO2024251311A2 PCT/CN2024/110474 CN2024110474W WO2024251311A2 WO 2024251311 A2 WO2024251311 A2 WO 2024251311A2 CN 2024110474 W CN2024110474 W CN 2024110474W WO 2024251311 A2 WO2024251311 A2 WO 2024251311A2
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fermentation
cctcc
bayer
lactic acid
yeast
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WO2024251311A3 (zh
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杜海
徐岩
魏军林
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Jiangnan University
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Jiangnan University
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Definitions

  • the invention relates to a strain of Bayer's zygosaccharomyces cerevisiae which is resistant to lactic acid stress and has high ethanol and flavor compound production, and belongs to the technical field of microorganisms.
  • microorganisms will encounter a variety of environmental pressures, such as high acid, high ethanol, and high temperature.
  • environmental pressures such as high acid, high ethanol, and high temperature.
  • lactic acid is a metabolite of microorganisms in many traditional fermented foods.
  • it contributes to the mellow taste of fermented foods.
  • due to its low volatility and other characteristics it continues to accumulate during the brewing process, causing the acidity in the fermentation system to increase, inhibiting the growth and metabolism of functional microorganisms.
  • Lactic acid accumulation causes problems such as low yield and flavor defects in fermentation.
  • these problems cannot be solved by changing the operating process.
  • Selecting strains with excellent traits and applying them to fermentation with low yields will be an effective method.
  • most of the current research focuses on analyzing the lactic acid tolerance characteristics of some functional microorganisms.
  • fermented foods are sensory products with flavor substances as the quantitative orientation.
  • These functional microorganisms must not only be able to tolerate the high acid environment during the fermentation process, but also play a role in producing flavor substances. Therefore, the selected strain must be applied to the fermentation process. It must have multiple capabilities such as lactic acid tolerance and flavor production, which is a relatively large challenge for screening strains.
  • the present invention screened a functional yeast strain, Zygosaccharomyces bailii LBM888, from the Maotai-flavor liquor brewing system.
  • This strain can not only tolerate lactic acid pressure, but also has the ability to degrade lactic acid.
  • this strain can also produce high ethanol and a variety of flavor substances under lactic acid stress.
  • This strain is made into a bacterial agent and applied to the Maotai-flavor liquor fermentation production process, which significantly increases the ethanol and flavor substance content in the fermented grains.
  • the strain of the present invention has a good practical application effect and can be applied to the actual production process of fermented food.
  • the present invention provides a strain of Zygosaccharomyces bailii LBM888 , which has been deposited in the China Center for Type Culture Collection on May 10, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 2023729.
  • the Baier yeast ( Zygosaccharomyces bailii ) CCTCC NO: M 2023729 has the following characteristics:
  • Morphological characteristics The colony on the YPD plate is white and opaque, with a certain luster, regular, smooth, round edges, and a convex middle. Under a transmission electron microscope, a single cell is a large oval, without flagella and hyphae;
  • Glucose can be utilized, but glycerol, erythritol, xylitol, arabinose, ribitol, sorbitol, mannitol, maltitol, xylose, D-ribose, D-arabinose, L-sorbose, L-rhamnose, D-galactose, D-trehalose, sucrose, stachyose, D-raffinose, D-melibiose, D-melezitose, maltotriose, maltose, gentiobiose, D-cellobiose, and dextrin cannot be utilized;
  • Ethanol production characteristics After 60 h of fermentation in sorghum juice medium, the glucose content decreased from the initial 40 g/L to 4.09 ⁇ 0.23 g/L at the end of fermentation, and the ethanol yield was 13.66 ⁇ 0.45 g/L. In the fermentation medium with 40 g/L lactic acid, the ethanol content reached 11.99 ⁇ 0.13 g/L at the end of fermentation.
  • Lactic acid tolerance and lactic acid degradation Able to tolerate 40 g/L lactic acid; can still grow well in fermentation medium containing 40 g/L lactic acid, with an OD 600 of 3.02 ⁇ 0.08; lactic acid content at the beginning of fermentation was 40 g/L, while 5.88 g/L was degraded at the end of fermentation; able to produce a high content of ethanol under lactic acid stress, with the ethanol content reaching 11.99 ⁇ 0.13 g/L at the end of fermentation;
  • the flavor compounds with a content of more than 0.1 g/L included ethyl acetate (1.02 g/L), isopentanol (1.52 g/L), phenylethanol (0.72 g/L), phenylethyl acetate (0.50 g/L), isobutanol (0.13 g/L), isopentanol (0.10 g/L) and isovaleric acid (0.10 g/L).
  • the flavor compounds with a content of less than 0.1 g/L included isopentanol, ethyl lactate and 1-octanol.
  • Application in liquor brewing can significantly reduce lactic acid content, increase ethanol content and volatile flavor substances in the mash.
  • the second object of the present invention is to provide a microbial agent containing the Zygosaccharomyces bailii CCTCC NO: M 2023729 of the present invention.
  • the microbial agent uses Zygosaccharomyces bailii CCTCC NO: M 2023729 as a main microorganism.
  • the viable count of Zygosaccharomyces bailii CCTCC NO: M 2023729 in the bacterial agent is 10 5 to 10 8 CFU/mL.
  • the bacterial concentration of Zygosaccharomyces bailii CCTCC NO: M 2023729 in the bacterial agent is in the range of 10 5 -10 8 CFU/mL.
  • the bacterial agent is obtained by preparing the seed solution of Zygosaccharomyces bailii CCTCC NO: M 2023729 and then expanding the culture.
  • the bacterial agent is a liquid bacterial agent or a solid bacterial agent containing the Zygosaccharomyces bailii CCTCC NO: M 2023729 strain of the present invention.
  • the microbial agent contains living cells of the Zygosaccharomyces bailii CCTCC NO: M 2023729 strain of the present invention, dry cells of the Zygosaccharomyces bailii CCTCC NO: M 2023729 strain of the present invention obtained by freeze-drying, immobilized cells of the Zygosaccharomyces bailii CCTCC NO: M 2023729 strain of the present invention, or the Zygosaccharomyces bailii CCTCC NO: M 2023729 strain of the present invention in any other form.
  • the bacterial agent also contains any strain of any species that can be used for fermented food or fermented food preparation, such as Bacillus licheniformis, Saccharomyces cerevisiae, Bacillus subtilis, etc.
  • the bacterial agent further contains any carrier that can be used for fermenting food.
  • the third object of the present invention is to provide the application of the Zygosaccharomyces bailii CCTCC NO: M 2023729 or a bacterial agent containing the Zygosaccharomyces bailii CCTCC NO: M 2023729, wherein the application includes one or more of the following: degrading lactic acid, increasing ethanol content, and increasing flavor substance content.
  • the specific application is to pick a single colony of Bayer's yeast CCTCC NO: M 2023729 and inoculate it in a sorghum juice culture medium to obtain a seed liquid after cultivation; then inoculate the Bayer's yeast CCTCC NO: M 2023729 seed liquid into a fermentation medium added with lactic acid for fermentation cultivation;
  • the sorghum juice culture medium is prepared by soaking sorghum and water at a ratio of 1:4 (M:V) overnight, then adding amylase to cook and liquefy it, then adding saccharifying enzyme at 60°C for saccharification, and finally filtering and centrifuging to adjust the sugar content to 5°Bx;
  • the fermentation culture medium is prepared by adjusting the pH of the sorghum juice culture medium to 3.2 with lactic acid.
  • the fourth object of the present invention is to provide an application of Zygosaccharomyces bailii CCTCC NO: M 2023729 or a bacterial agent containing the Zygosaccharomyces bailii CCTCC NO: M 2023729 in fermented food, wherein the application is to add Zygosaccharomyces bailii CCTCC NO: M 2023729 or a bacterial agent containing the Zygosaccharomyces bailii CCTCC NO: M 2023729 during the preparation of the fermented food.
  • the method is applied to liquor brewing.
  • the present invention provides a method for producing ethanol and flavor substances under lactic acid pressure.
  • the method comprises the following steps: picking a single colony of Zygosaccharomyces bailii CCTCC NO: M 2023729 and inoculating it into a sorghum juice culture medium, and obtaining a seed liquid after culturing; and then inoculating the Zygosaccharomyces bailii CCTCC NO: M 2023729 seed liquid into a fermentation culture medium added with lactic acid for fermentation and culturing.
  • the sorghum juice culture medium is prepared by soaking sorghum and water at a ratio of 1:4 (M:V) overnight, then adding amylase to cook and liquefy, adding saccharifying enzyme at 60°C, and finally filtering and centrifuging to adjust the sugar content to 5°Bx.
  • the present invention provides a method for producing ethanol and flavor substances under lactic acid pressure, the method comprising: picking a single colony of Bayer's yeast CCTCC NO: M 2023729 and inoculating it into a sorghum juice culture medium, and culturing it at 200 rpm and 30°C for 24 hours to obtain a seed solution; then inoculating 1 mL of Bayer's yeast CCTCC NO: M 2023729 seed solution into 100 mL of a fermentation culture medium to which 40 g/L of lactic acid was added, and culturing it at 30°C and 200 rpm for 72 hours.
  • the invention provides an application of a yeast agent in liquor brewing, wherein the application comprises: inoculating Bayer's Zygosaccharomyces CCTCC NO: M 2023729 into a seed culture medium to obtain a seed liquid; then centrifuging the seed liquid, discarding the supernatant, and resuspending the precipitate in water to obtain the agent;
  • the bacterial agent is applied to the fourth round of production of Maotai-flavor liquor fermentation; the specific operation is as follows:
  • the invention provides an application of a yeast agent in liquor brewing, wherein the application comprises: inoculating Bayer's Zygosaccharomyces CCTCC NO: M 2023729 into a seed culture medium, culturing for 48 h at 30°C and 200 rpm to obtain a seed solution of 10 8 cells/mL; then taking 10 mL of the seed solution and centrifuging it at 10,000 ⁇ g for 5 min, discarding the supernatant, and resuspending the precipitate in 1000 mL of sterile water to obtain a corresponding agent solution;
  • This bacterial agent liquid is applied to the fourth round of production of Maotai-flavor liquor fermentation; the specific operation is as follows:
  • the Bayer yeast CCTCC NO: M 2023729 of the present invention is screened from Chinese Maotai-flavor liquor Daqu.
  • This strain can not only tolerate lactic acid pressure, but also has the ability to degrade lactic acid. In addition, this strain can also produce high ethanol and various flavor substances under lactic acid stress.
  • This strain is made into a bacterial agent and applied to the fermentation production process of Maotai-flavor liquor, which significantly increases the content of ethanol and flavor substances in the mash.
  • the strain of the present invention has good practical application effect and can be applied to the actual production process of fermented food.
  • bailii Zygosaccharomyces CCTCC NO: M 2023729 taxonomic name Zygosaccharomyces bailii LBM888, was deposited in China Center for Type Culture Collection (CCTCC) on May 10, 2023, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2023729.
  • Figure 1A Colony morphology of Zygosaccharomyces bailii CCTCC NO: M 2023729 on YPD solid plates.
  • Figure 1B Morphology of Zygosaccharomyces bailii CCTCC NO: M 2023729 under transmission electron microscopy.
  • Figure 3 Carbon source utilization of Zygosaccharomyces bailii CCTCC NO: M 2023729.
  • Figure 4 Growth of Zygosaccharomyces bailii CCTCC NO: M 2023729 under lactic acid stress; ZB-0 indicates no lactic acid was added; ZB+Lac indicates 40 g/L lactic acid was added. *, P ⁇ 0.05, **, P ⁇ 0.01 (Tukey's test).
  • FIG5A Lactic acid degradation by Zygosaccharomyces bailii CCTCC NO: M 2023729 under lactic acid stress.
  • FIG5B Ethanol production ability of Zygosaccharomyces bailii CCTCC NO: M 2023729 under lactic acid stress.
  • Figure 6 Production of volatile flavor compounds by Zygosaccharomyces bailii CCTCC NO: M 2023729 under lactic acid stress.
  • Figure 7 Process diagram of the application of Zygosaccharomyces bailii CCTCC NO: M 2023729 in the fermentation process of Maotai-flavor liquor.
  • Figure 8A Effect of adding Zygosaccharomyces bailii CCTCC NO: M 2023729 inoculant on the lactic acid content during the fermentation process of Maotai-flavor liquor.
  • Figure 8B Effect of adding Zygosaccharomyces bailii CCTCC NO: M 2023729 inoculant on the ethanol content during the fermentation process of Maotai-flavor liquor.
  • YPD Screening medium
  • Seed culture medium glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L.
  • Sorghum juice medium Soak sorghum and water at a ratio of 1:4 (M:V) overnight, then add amylase to cook and liquefy, add saccharifying enzyme at 60°C, finally filter and centrifuge, and adjust the sugar content to 5°Bx.
  • the fermentation broth of the simulation experiment was filtered with a 0.22 ⁇ m aqueous filter membrane, and the filtrate was transferred to a liquid phase vial.
  • 5 g of mash was weighed and added to 20 mL of ultrapure water, shaken and mixed, and ultrasonicated in an ice bath for 30 min, then the ultrasonicated mixture was centrifuged at 4°C and 8000 ⁇ g for 5 min, the supernatant was filtered with a 0.22 ⁇ m aqueous filter membrane, and the filtrate was transferred to a liquid phase vial.
  • Liquid phase system Waters 2695; chromatographic column: Bio-Rad Aminex HPX-87H Ion Exclusion Column, column temperature: 60°C; detector: UV detector (PDA), detection wavelength 210 nm; mobile phase: 5 mmol L-1 H2SO4, flow rate: 0.6 mL min-1.
  • the fermentation broth of the simulation experiment was filtered with a 0.22 ⁇ m aqueous filter membrane, and the filtrate was transferred to a liquid phase vial.
  • 5 g of mash was weighed and added to 20 mL of ultrapure water, shaken and mixed, and ultrasonicated on ice for 30 min. The ultrasonicated mixture was then centrifuged at 4°C and 8000 ⁇ g for 5 min. The supernatant was filtered with a 0.22 ⁇ m aqueous filter membrane, and the filtrate was transferred to a liquid phase vial.
  • Liquid phase system Waters 2695; Chromatographic column: Bio-Rad Aminex HPX-87H Ion Exclusion Column, column temperature: 60°C; Detector: Differential refractive index detector (RID); Mobile phase: 5 mmol L-1 H2SO4, flow rate: 0.6 mL min-1.
  • the fermentation broth of the simulation experiment was centrifuged at 4°C and 8000 ⁇ g for 5 min, and 8 mL of the supernatant was added to the headspace bottle.
  • 5 g of fermented grains were weighed and added to 20 mL of ultrapure water, and 0.85% NaCl and 1% CaCl2 were added.
  • the mixture was ultrasonicated in an ice bath for 30 min; then the ultrasonicated mixture was centrifuged at 4°C and 8000 ⁇ g for 5 min.
  • 8 mL of the supernatant was added to the headspace bottle (20 mL, 3 g NaCl was added in advance), and then 20 ⁇ L of L-menthol (100 ⁇ g ⁇ mL-1) was added.
  • HS-SPME-GC-MS GC 6890N and MS 5975
  • DB-Wax column (30 m ⁇ 0.25 mm, 0.25 ⁇ m) were used to detect volatile compounds.
  • a strain of lactic acid-tolerant Bayer yeast was screened from the fermentation process of Maotai-flavor liquor. The process is as follows:
  • Collect a sample of fermented mash from a famous Maotai-flavor liquor then take 10g of the sample, add 90 mL of 0.9% sterile saline, shake at 30°C and 200rpm for 1h, dilute the suspension 10 times, take 100uL of the diluted liquid and evenly spread it on a YPD solid plate. After culturing for 3 days, select a single colony for purification. The obtained single bacteria was added to a sorghum juice medium with a pH of 3.2 adjusted with lactic acid, and cultured at 30°C and 200rpm for 3 days. After the culture was completed, the growth amount and the lactic acid, ethanol and volatile compounds in the fermentation broth were measured.
  • the activated strain obtained by screening in Example 1 was streaked and inoculated on YPD solid medium and cultured statically at 30° C. for 2 days. The morphology of the colonies on the plate was observed. At the same time, a single colony was picked and placed in sorghum juice liquid medium and observed under a transmission electron microscope after culturing for 2 days.
  • the strain was inoculated into sorghum juice medium and cultured at 30°C and 200 rpm for 84 h. Samples were taken every 12 h to measure OD 600 and observe its growth characteristics.
  • the strain reached a stable phase after 60 h of growth in sorghum juice medium, and the OD600 reached 3.36 ⁇ 0.05 at the end of fermentation.
  • the glucose content in the sorghum juice medium at the beginning of fermentation was 40 g/L.
  • the results in Table 1 showed that the residual glucose was 4.09 ⁇ 0.23 g/L at the end of fermentation, and the ethanol production of the strain was 13.66 ⁇ 0.45 g/L.
  • the carbon source utilization experiment of the strain was determined as follows: a single colony was picked and inoculated into sorghum juice medium, and the seed solution was obtained after culturing at 200 rpm and 30°C for 24 hours. Then 10 mL of seed solution was centrifuged at 10,000 ⁇ g for 5 minutes, the supernatant was discarded, the cell pellet was suspended with an equal amount of sterile saline, and then the suspension was diluted to 10 ⁇ 4 with the same saline, and 100 uL was inoculated to the biolog YT microplate. According to the instructions, the microplate was statically cultured in a 30°C constant temperature incubator for 36 hours, and the OD 590 was measured with an enzyme marker. When OD 590 > 0.2, it indicates that the strain can utilize this carbon source.
  • PCR amplification was performed using ITS universal primers (ITS1 and ITS4). PCR amplification conditions: 94°C 5 min, 94°C 30 s, 55°C 30 s, 72°C 1 min, 30 cycles; 72°C 10 min.
  • the PCR amplification product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing after passing the 1% agarose gel test.
  • This strain has been deposited in the China Center for Type Culture Collection on May 10, 2023, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2023729.
  • Example 3 Ethanol production and flavor production ability of strain CCTCC NO: M 2023729 under lactic acid pressure
  • the pH of sorghum juice medium was adjusted to 3.2 with lactic acid and used as fermentation medium to evaluate the ability of the experimental strain Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) to produce ethanol and flavor compounds under lactic acid pressure.
  • a single colony of Zygosaccharomyces bailii CCTCC NO: M 2023729 was picked and inoculated into sorghum juice medium, and cultured at 200 rpm and 30°C for 24 h to obtain seed liquid.
  • lactic acid content in the fermentation broth showed that the main mechanism of lactic acid tolerance of Zygosaccharomyces bailii ZB was its ability to degrade lactic acid in the system (Figure 5A).
  • the lactic acid content at the beginning of the fermentation was 40 g/L, while at the end of the fermentation it was 34.12 ⁇ 1.07 g/L, degrading 5.88 g/L.
  • the results of ethanol determination showed that Zygosaccharomyces bailii ZB was not only able to adapt to 40 g/L lactic acid stress, but also able to produce a higher content of ethanol under lactic acid stress, with the ethanol content reaching 11.99 ⁇ 0.13 g/L at the end of the fermentation (Figure 5B).
  • the results in Figure 6 show that Zygosaccharomyces bailii CCTCC NO: M 2023729 can produce a variety of flavor substances under lactic acid stress.
  • the flavor compounds with a content of more than 0.1 g/L include ethyl acetate (1.02 g/L), isopentanol (1.52 g/L), phenylethanol (0.72 g/L), phenylethyl acetate (0.50 g/L), isobutanol (0.13 g/L), isopentanol (0.10 g/L) and isovaleric acid (0.10 g/L).
  • the flavor compounds with a content of less than 0.1 g/L include isopentanol, ethyl lactate and 1-octanol.
  • Bayer's yeast CCTCC NO: M 2023729 can adapt well to the lactic acid environment. It can not only reduce the lactic acid content in the system, but also produce ethanol and flavor compounds.
  • Bayer's Zygosaccharomyces CCTCC NO: M 2023729 (hereinafter referred to as Bayer's Zygosaccharomyces ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then 10 mL of the seed solution was centrifuged at 10,000 ⁇ g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution. This bacterial agent was applied to the fourth round of production of Maotai-flavor liquor fermentation.
  • the lactic acid content of the control group was 85.07 ⁇ 3.52 g/kg, while that of the bacterial agent-added group (cellar ZB) was 54.51 ⁇ 2.08 g/kg, a decrease of 35.92%.
  • the ethanol content of the control group was 6.37 ⁇ 1.45 g/kg, while the ethanol content of the group with the inoculant addition was 12.30 ⁇ 1.01 g/kg, an increase of 92.93%.
  • the ethanol content of the control group was 28.00 ⁇ 0.65 g/kg, while that of the group with the inoculant addition was 29.98 ⁇ 0.34 g/kg, an increase of 7.07%.
  • the addition of the experimental inoculant significantly increased the volatile flavor substances in the mash, mainly esters, alcohols and aromatic compounds, as shown in Table 2.
  • the significantly increased ester substances include ethyl acetate, ethyl propionate, isoamyl acetate, ethyl isobutyrate, furanyl acetate, ethyl phenylacetate and phenylethyl acetate.
  • the significantly increased alcohol substances include isobutanol, phenylethyl alcohol, isopentanol and 2-heptanol.
  • the significantly increased aromatic compounds include 2-ethyl-6-methylpyrazine, 2,6-diethylpyrazine and tetramethylpyrazine.
  • Flavor compounds initial Empty cellar ZB Out of the cellar blank Out of the cellar ZB Esters (mg/kg) Ethyl acetate 2.18 ⁇ 0.04 3.78 ⁇ 0.06 4.74 ⁇ 0.35 9.36 ⁇ 2.47 11.24 ⁇ 1.18 Ethyl propionate 0.02 ⁇ 0.00 0.11 ⁇ 0.00 0.61 ⁇ 0.05 0.19 ⁇ 0.02 0.88 ⁇ 0.05 Ethyl 2-methylpropionate 0.00 ⁇ 0.00 0.00 ⁇ 0.00 ⁇ 0.00 ⁇ 0.00 0.22 ⁇ 0.01 Isoamyl acetate 0.09 ⁇ 0.02 0.21 ⁇ 0.04 0.30 ⁇ 0.04 0.55 ⁇ 0.22 0.72 ⁇ 0.23 Ethyl isobutyrate 0.00 ⁇ 0.00 0.00 ⁇ 0.00 0.00 0.00 ⁇ 0.00 0.22 ⁇ 0.05 Furoxyl acetate 0.00 ⁇ 0.00 0.06 ⁇ 0.01 0.20 ⁇ 0.00 0.10 ⁇ 0.00 0.45 ⁇ 0.02 2-Methoxyacetic acid 3-methylbutyl ester
  • Example 5 Application of yeast in liquor brewing
  • Bayer's Zygosaccharomyces CCTCC NO: M 2023729 (hereinafter referred to as Bayer's Zygosaccharomyces ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then, 10 mL of the seed solution was centrifuged at 10,000 ⁇ g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • the prepared bacterial agent liquid is mixed with the grain that has been moistened, steamed and cooled, and then mixed and fermented.
  • the bacterial agent liquid is 10-20% of the feed amount (by mass), and is put into the tank for fermentation. After steaming and roasting, the Daqu light-fragrance liquor is prepared (for the specific Daqu production process, please refer to the paper: Research Progress on Brewing Microorganisms and Flavor Substances of Light-fragrance Liquor).
  • the prepared bacterial agent liquid is mixed with the steamed and cooled grains, piled for saccharification for 1 day, and then mixed and put into the tank truck for fermentation for 14 days.
  • the solid bacteria dosage is 1% of the feed amount (by mass).
  • the Xiaoqu light-fragrance liquor is prepared by steaming and roasting (for the specific Xiaoqu production process, please refer to the paper: Metabolic characteristics of microbial flora synthesizing higher alcohols in the fermentation process of Xiaoqu light-fragrance liquor).
  • the steamed and cooled grains are mixed, and the mash is re-steamed and cooled.
  • the prepared bacterial agent liquid is mixed in the mash, and the amount of bacterial agent liquid added is 25% of the amount of feed (by mass), and the mash is fermented in a tank for 40 days.
  • the Lao Bai Gan flavor liquor is prepared by steaming and mixing (the specific production process of Lao Bai Gan flavor liquor refers to the paper: Optimization of the formula of multi-grain Lao Bai Gan flavor liquor).
  • Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) into the seed culture medium and culture at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then take 10 mL of the seed solution and centrifuge at 10,000 ⁇ g for 5 min, discard the supernatant, and resuspend the precipitate in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • (2) Preparation of yeast First, prepare the saccharification liquid. Soak the glutinous rice in a rice soaking tank for 30 minutes. After discarding the rice slurry, steam the rice for 30-40 minutes. Cool the steamed rice to 60°C for later use. Add 2 ⁇ saccharification enzyme and 1 ⁇ liquefaction enzyme, stir evenly, and place in a water bath for constant temperature saccharification at 60°C for more than 4 hours. Stir every 1 hour or so to ensure uniform saccharification. Filter the saccharification liquid with gauze to obtain the sugar solution, and then dilute it with water to 13 ⁇ 0.5°Bx (20°C).
  • Yellow wine fermentation Add the following ingredients for yellow wine fermentation: 1000 g glutinous rice, 1500 g cooked rice, 1100 mL brewing water, 110 g Daqu, and 100 mL yeast;
  • the total fermentation time is about 20 days. Pay attention to raking during the fermentation process.
  • the temperature of the pre-fermentation is controlled below 33°C, and the temperature of the post-fermentation is controlled at about 15 ⁇ 2°C.
  • the temperature of the pre-fermentation is 28°C, and the total fermentation time is 5 days; raking twice a day; the fermentation temperature of the post-fermentation is 15°C, and the fermentation time is 15 days; raking once every 5 days.
  • Example 7 Application of yeast agent in soy sauce brewing
  • Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) into the seed culture medium and culture at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then take 10 mL of the seed solution and centrifuge at 10,000 ⁇ g for 5 min, discard the supernatant, and resuspend the precipitate in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • soy sauce koji Add salt water to the prepared soy sauce koji at a ratio of 1:1.5 (m/v), stir them evenly to make a fermented soy sauce, and ferment it for 3-6 months, turning the sauce every 15 days. After the fermentation is completed, the soy sauce mash is squeezed, filtered to remove impurities and sterilized to obtain the finished soy sauce.
  • Example 8 Application of yeast agent in vinegar brewing
  • Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) into the seed culture medium and culture at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then take 10 mL of the seed solution and centrifuge at 10,000 ⁇ g for 5 min, discard the supernatant, and resuspend the precipitate in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • Alcohol fermentation after saccharification, add 30 kg of water and 6 kg of bacterial liquid, and ferment at 28°C for 144-168 hours to obtain mature mash;
  • Acetic acid fermentation After every 24 hours of fermentation, turn the mash, add rice husks to keep warm and moist after each turning. No more rice husks will be added from the 11th day of fermentation. Turn the mash to cool the product temperature. After 18-20 days, when the acidity no longer rises, add 4 kg of salt and seal for 30-45 days.
  • pouring vinegar and frying vinegar Take the vinegar mash that has finished aging, add fried beige in proportion, soak for several hours, and use the leaching method to circulate the vinegar. Add sugar to the vinegar juice, and fry the vinegar after it has been clarified. After the temperature of the product drops to 75-80°C, it can be sealed and stored.
  • Example 9 Application of yeast agent in sake brewing
  • Bayer's Zygosaccharomyces CCTCC NO: M 2023729 (hereinafter referred to as Bayer's Zygosaccharomyces ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then, 10 mL of the seed solution was centrifuged at 10,000 ⁇ g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • Polishing Use a polishing machine to remove 20%-30% of the raw rice mass.
  • the broken rice grains should be as small as possible, the shape of polished rice and brown rice should be similar, and the germ and groove of the raw rice should be completely removed;
  • the water temperature for soaking rice is room temperature, the water absorption rate is about 28-30%, and the soaking time is 9-12 hours;
  • Steamed rice The rice grains are distinct, hard on the outside and soft on the inside, with no white core, loose and not mushy, cooked but not mushy, uniform and consistent, and the yield of glutinous rice is generally around 140%-150%;
  • Fermentation The initial fermentation temperature is controlled at 28-30°C for 3 days, then fermented at around 20°C for 22 days, and the total fermentation time is 25 days.
  • Example 10 Application of yeast agent in liquor brewing
  • Bayer's Zygosaccharomyces CCTCC NO: M 2023729 (hereinafter referred to as Bayer's Zygosaccharomyces ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then, 10 mL of the seed solution was centrifuged at 10,000 ⁇ g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • Sorghum 40-50%), wheat (6-10%), corn (5-10%), glutinous rice (10-12%) and rice (15-20%) are weighed in proportion, soaked in hot water (55-65°C) for 24-32 hours, rice bran (20-35% of the total weight of grain) is steamed at 90-100°C for 30-40 minutes, and then cooled for later use;
  • Cooling and fermentation The re-steamed mash is cooled to 30-35°C, and the rice bran treated in step (1) and 18-28% of liquor koji are added, followed by 5%-10% of bacterial agent solution (calculated according to the weight of grain), and the mixture is stirred evenly for fermentation in a cellar;
  • Taking wine out of the cellar Take the fermented mash out of the cellar and use a steamer to distill the wine.
  • the flow rate is 1.5-2.5 kg/min and the flow temperature is 25-30 °C.
  • the liquor yield of the above process is 28%-33%, and the high-quality rate is 88%-91%.
  • the brewed liquor is colorless or slightly yellow and transparent, with prominent ester aroma and burnt aroma, clean and refreshing in the mouth, sweet and smooth, with rich multi-grain puffing fragrance, natural fusion of multiple aromas, relatively mellow body, and typical style of salty, elegant and mellow.
  • the obtained Taorong-type liquor reaches the first-class wine standard.
  • Bayer's Zygosaccharomyces CCTCC NO: M 2023729 (hereinafter referred to as Bayer's Zygosaccharomyces ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then, 10 mL of the seed solution was centrifuged at 10,000 ⁇ g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • the rice is taken as the main raw material in proportion and soaked at room temperature for about 8-12 hours until it fully absorbs water; the water of the soaked rice grains is drained and steamed at normal pressure for about 30-40 minutes until there is no white core in the rice; after the rice is steamed, pour cold boiled water over the rice to about 25-28°C, transfer it to a fermentation tank, add 0.4%-0.8% (mass ratio) of bacterial agent liquid in proportion, inoculate 0.5%-1.5% (mass ratio) of brewer's yeast, stir evenly, add drinking water 1-1.5 times the mass of raw rice, and ferment at a constant temperature of 28-30°C for 48-72 hours.
  • the obtained sweet rice brewing precision is 2-4% (v/v), the aroma is coordinated, rich, the texture is uniform, and the taste is sweet and sour.
  • Bayer's Zygosaccharomyces CCTCC NO: M 2023729 (hereinafter referred to as Bayer's Zygosaccharomyces ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then, 10 mL of the seed solution was centrifuged at 10,000 ⁇ g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • the rice is taken as the main raw material in proportion and soaked at room temperature for about 8-12 hours until it fully absorbs water, and it is required to be easy to be crushed by hand after soaking and there is no hard rice core; the water of the soaked rice grains is drained, and the rice is steamed at normal pressure for about 30-40 minutes until there is no white core in the rice; after the rice is steamed, it is cooled to about 25-28°C with cold boiled water, and 5%-10% (mass ratio) of bacterial agent liquid is added in proportion for saccharification.
  • rice is added, and 5%-10% (mass ratio) of brewer's yeast is inoculated, stirred evenly, and drinking water 1-1.5 times the mass of the original rice is added. Fermentation is carried out at 28-30°C for 3-5 days, and the temperature is reduced to 10-15°C for fermentation for 10-15 days. After the fermentation is completed, it is squeezed and filtered to obtain rice wine.
  • the alcohol content of rice wine is 5-10% (v/v), rich in rice and fruity aroma, harmonious aroma, rich flavor, and uniform texture.
  • the results show that the bacterial agent of the present invention can increase the flavor substances of rice wine.
  • Example 13 Application of yeast in fermented dairy products
  • Bayer's Zygosaccharomyces CCTCC NO: M 2023729 (hereinafter referred to as Bayer's Zygosaccharomyces ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then, 10 mL of the seed solution was centrifuged at 10,000 ⁇ g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • lactobacillus milk beverages It can be used to make lactobacillus milk beverages, and the specific preparation process is as follows:
  • the raw skim milk is sterilized at 95°C for 20 min, then cooled to 4°C, and then a bacterial agent solution is added to make the bacterial concentration reach above 10 6 CFU/mL.
  • the milk beverage is obtained by refrigerating and storing at 4°C.
  • Example 14 Application of yeast in fermented dairy products
  • Bayer's Zygosaccharomyces CCTCC NO: M 2023729 (hereinafter referred to as Bayer's Zygosaccharomyces ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then, 10 mL of the seed solution was centrifuged at 10,000 ⁇ g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • Soak soybeans in soft water the water volume is three times the volume of the original soybeans, soak for 1-2 hours at a temperature of 80°C, and then remove the soybean skin. Then, drain the soaking water, add boiling water to grind, and keep warm for 10-15 minutes at a temperature above 80°C.
  • the slurry is filtered with a 150-mesh filter membrane and centrifuged to obtain the crude soy milk. Then, heat it to a temperature of 140-150°C, and then quickly introduce the hot crude soy milk into a vacuum cooling chamber for vacuuming.
  • the odorous substances in the crude soy milk are quickly discharged with the water vapor. After vacuum degassing, the temperature is reduced to about 37°C, and then the bacterial agent liquid is added to make the concentration reach more than 10 6 CFU/mL, and the soy milk is obtained by refrigerating and storing at 4°C.
  • Example 15 Application of yeast in fermented fruit and vegetable beverages
  • Bayer's Zygosaccharomyces CCTCC NO: M 2023729 (hereinafter referred to as Bayer's Zygosaccharomyces ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then, 10 mL of the seed solution was centrifuged at 10,000 ⁇ g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • Fresh vegetables (such as one or more of cucumber, carrot, beet, celery or cabbage) are selected, washed and juiced, and then sterilized at high temperature for 2 seconds at 140°C, and then immediately cooled to about 37°C, and then added with the bacterial agent solution of the present invention to make the concentration reach 10 6 CFU/mL or more, and then refrigerated and stored at 4°C to obtain a fruit and vegetable beverage.
  • the results show that the bacterial agent of the present invention can increase the flavor substances of fruit and vegetable beverages.
  • the main steps include:
  • Bayer's Zygosaccharomyces CCTCC NO: M 2023729 (hereinafter referred to as Bayer's Zygosaccharomyces ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain a seed solution of 1 ⁇ 10 8 cells/mL. Then, 10 mL of the seed solution was centrifuged at 10,000 ⁇ g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.
  • Saccharification Mix the crushed malt with hot water at a temperature of 63.5°C, and then saccharify to obtain wort;
  • Fermentation After the wort is cooled, it is pumped into the fermentation tank and 20% (v/v) bacterial agent is added for fermentation. The fermentation time is 56 to 60 hours.
  • Distillation The fermented juice is distilled twice in a copper Holstein distiller; First distillation: Add the distillate to the still, when the juice in the still is heated to 80°C, it begins to boil and enters the distillation stage, the cooling water temperature is less than 18°C, the head and tail of the wine are not removed in one distillation, the alcohol content of the initial distillate is 20 ⁇ 28%vol, and the distillation time is controlled at 8 ⁇ 9h; Second distillation: The initial distillate is distilled for the second time to separate the head, heart and tail of the wine; Keep the heart and reduce the alcohol content to 63.5%vol as component A for standby use; the head and tail of the wine are used for the next distillation, and the second distillation time is about 10 ⁇ 12h.
  • Blending and filtration Reduce the alcohol content by blending to 40 degrees to obtain a semi-finished product, and filter to obtain the finished product.

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Abstract

本发明公开了一株耐乳酸胁迫且高产乙醇和风味化合物的拜耳接合酵母，属于微生物技术领域。本发明的拜耳接合酵母CCTCC NO：M 2023729是从中国酱香型白酒大曲中筛选获得的，不仅能够耐受乳酸压力，还具有降解乳酸的能力，还能在乳酸胁迫下高产乙醇和多种风味物质。本发明菌株可以制成菌剂应用到酱香型白酒发酵生产过程中，能够显著提高酒醅中的乙醇和风味物质含量。

Description

一株耐乳酸胁迫且高产乙醇和风味化合物的拜耳接合酵母 技术领域
本发明涉及一株耐乳酸胁迫且高产乙醇和风味化合物的拜耳接合酵母，属于微生物技术领域。
 背景技术
传统发酵食品酿造过程中微生物会遭遇多种环境压力，如高酸、高乙醇、高温等。其中，乳酸是很多传统发酵食品中微生物的代谢产物，其一方面贡献了发酵食品醇厚的口感，另一方面由于其难挥发等特性在酿造过程中持续积累，造成发酵体系中的酸度增加，抑制功能微生物的生长与代谢。例如，在多批次重复发酵的酱香型白酒酿造过程中，其独特的多批次发酵使得乳酸在批次中持续积累，含量可以达到40 g/kg。一些产量低下的批次中乳酸含量更高，严重超过了白酒中正常的浓度范围，造成风味物质的下降以及缺失。
乳酸积累使得发酵存在产量低下及风味缺陷等问题。但是由于开放式的自然接种发酵的特点，无法通过改变操作工艺解决这些问题。选育具有优良性状的菌种并将之应用到产量低下的发酵中将是一项行之有效的方法。然而，目前大部分研究主要集中于解析一些功能微生物的乳酸耐受特性。但是发酵食品是感官属性产品，以风味物质为量化导向，这些功能微生物不仅要能耐受发酵过程中的高酸环境，还要发挥作用产风味物质等。因此，所选的菌株要应用到发酵过程中，必须要集乳酸耐受和产风味物质等多种能力于一体，这对于筛选菌种是一项比较大的挑战。
 发明内容
为了解决以上问题，本发明从酱香型白酒酿造体系中筛选到一株功能酵母拜耳接合酵母（ Zygosaccharomyces bailii）LBM888，此菌株不仅能够耐受乳酸压力，还具有降解乳酸的能力。此外，此菌株还能在乳酸胁迫下高产乙醇和多种风味物质。将此菌株制成菌剂应用到酱香型白酒发酵生产过程中，显著提高了酒醅中的乙醇和风味物质含量。本发明的菌株实际应用效果良好，可以应用到发酵食品实际生产过程中。
本发明提供了一株拜耳接合酵母（ Zygosaccharomyces bailii）LBM888，所述拜耳接合酵母（ Zygosaccharomyces bailii）LBM888已于2023年5月10日保藏于中国典型培养物保藏中心，保藏地址为中国武汉，武汉大学，保藏编号为CCTCC NO：M 2023729。
所述拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729，具有如下特性：
形态特征：在YPD平板上的菌落形态呈白色不透明，有一定的光泽，边缘规则、光滑、呈圆形，中间凸起。在透射电镜下，单个细胞为较大的卵圆形，无鞭毛，无菌丝；
生长特性：于高粱汁培养基中生长60 h后达到稳定期，发酵结束时OD600达到3.36 ± 0.05；在有40 g/L的乳酸的发酵培养基中胁迫下生长，OD 600为3.02±0.08；
碳源利用特性：可以利用葡萄糖，不能利用甘油、赤藓醇、木糖醇、阿拉伯淳、核糖醇、山梨醇、甘露醇、麦芽糖醇、木糖、D-核糖、D-阿拉伯糖、L-山梨糖、L-鼠李糖、D-半乳糖、D-海藻糖、蔗糖、水苏糖、D-棉子糖、D-蜜二糖、D-松三糖、麦芽三糖、麦芽糖、龙胆二糖、D-纤维二糖、糊精；
产乙醇特性：高粱汁培养基中发酵60 h后，葡萄糖从初始的40 g/L降至发酵结束时的4.09 ± 0.23 g/L，乙醇产量为13.66 ± 0.45 g/L；在40 g/L的乳酸的发酵培养基中，发酵结束时乙醇含量达到11.99 ± 0.13 g/L。
耐乳酸和降解乳酸：能够耐受40 g/L的乳酸；在含40 g/L的乳酸的发酵培养基中仍然能够很好生长，OD 600为3.02±0.08；发酵初始时的乳酸含量为40 g/L，而发酵结束时降解了5.88 g/L；能够在乳酸胁迫下产生较高含量的乙醇，发酵结束时乙醇含量达到11.99 ± 0.13 g/L；
在乳酸胁迫下产生多种风味物质：含量超过0.1g/L的风味化合物有乙酸乙酯（1.02 g/L）、异戊醇（1.52 g/L）、苯乙醇（0.72 g/L）、乙酸苯乙酯（0.50 g/L）、异丁醇（0.13 g/L）、乳酸异戊酯（0.10 g/L）和异戊酸（0.10 g/L）。含量小于0.1g/L的风味化合物有乙酸异戊酯、乳酸乙酯和1-辛醇等；
在白酒酿造中的应用：可以显著降低乳酸含量、提高乙醇含量和酒醅中挥发性风味物质。
本发明的第二个目的是提供一种微生物菌剂，含有本发明的拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729。
在本发明的一种实施方式中，所述微生物菌剂以拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729为主要微生物。
在本发明的一种实施方式中，所述菌剂中拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729的活菌数为10 5～10 8 CFU/mL。
在本发明的一种实施方式中，所述菌剂中拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729的菌浓范围为10 5~10 8 CFU/mL。
在本发明的一种实施方式中，所述菌剂，是制备拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729的种子液，然后扩培得到的。
在本发明的一种实施方式中，所述菌剂为含有本发明的拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729菌株的液体菌剂或者固体菌剂
在本发明的一种实施方式中，所述微生物菌剂含有本发明的拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729菌株的菌体活细胞、冷冻干燥得到的本发明的拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729菌株的干菌体、固定化的本发明的拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729菌株的细胞，或者以其他任何形式存在的本发明的拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729菌株。
在本发明的一种实施方式中，所述菌剂中还含有任何可以应用于发酵食品或者发酵食品制备的任意种属的菌株，比如地衣芽孢杆菌、酿酒酵母、枯草芽孢杆菌等等。
在本发明的一种实施方式中，所述菌剂中还含有任意能用于发酵食品的载体。
本发明的第三个目的是提供所述拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729或含有所述拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729的菌剂的应用，所述应用包括如下一种或者多种：降解乳酸、提高乙醇含量、提高风味物质含量。具体应用为，挑取拜耳接合酵母CCTCC NO：M 2023729单菌落接种于高粱汁培养基中，培养后获得种子液；然后向添加了乳酸的发酵培养基中接入所述拜耳接合酵母CCTCC NO：M 2023729种子液进行发酵培养；所述高粱汁培养基为，将高粱与水以1:4（M:V）浸泡过夜，然后加淀粉酶蒸煮液化后，在60℃加糖化酶糖化，最后过滤离心，调节糖度至5°Bx；所述发酵培养基为，用乳酸将高粱汁培养基的pH调节至3.2。
本发明的第四个目的是提供拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729或含有所述拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729的菌剂在发酵食品中的应用，所述应用为，在制备发酵食品的过程中，添加拜耳接合酵母CCTCC NO：M 2023729或含有所述拜耳接合酵母CCTCC NO：M 2023729的菌剂。
在本发明的一种实施方式中，所述应用于白酒酿造。
本发明提供了一种在乳酸压力下产乙醇和风味物质的方法，所述方法为，挑取拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729单菌落接种于高粱汁培养基中，培养后获得种子液；然后向添加了乳酸的发酵培养基中接入所述拜耳接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729种子液进行发酵培养。
在本发明的一种实施方式中，所述高粱汁培养基为，将高粱与水以1:4（M:V）浸泡过夜，然后加淀粉酶蒸煮液化后，在60℃加糖化酶糖化，最后过滤离心，调节糖度至5°Bx。
本发明提供了一种在乳酸压力下产乙醇和风味物质的方法，所述方法为，挑取拜耳接合酵母CCTCC NO：M 2023729单菌落接种于高粱汁培养基中，200 rpm，30°C条件培养24h后获得种子液；然后向100 mL添加了40 g/L的乳酸的发酵培养基中接入1mL 拜耳接合酵母CCTCC NO：M 2023729种子液，于30℃、200 rpm的条件下培养72 h。
本发明提供了一种酵母菌剂在白酒酿造中的应用，所述应用为，将拜耳接合酵母CCTCC NO：M 2023729接到种子培养基中，获得种子液；然后将种子液离心，弃掉上清液，将沉淀重悬于水中，得到菌剂；
将所述菌剂应用到酱香型白酒发酵的第四轮次生产过程中；具体操作如下：
酒醅经蒸馏、摊晾及拌曲后，取出酒醅，将菌剂均匀洒到酒醅中，拌匀；最后将对应的酒醅装到竹篮中，放入发酵堆和窖池中进行原位条件发酵。
本发明提供了一种酵母菌剂在白酒酿造中的应用，所述应用为，将拜耳接合酵母CCTCC NO：M 2023729接到种子培养基中，于30℃、200 rpm 的条件下培养48 h获得10 8 cells/mL的种子液；然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液；
将此菌剂液应用到酱香型白酒发酵第四轮次生产过程中；具体操作如下：
酒醅经蒸馏、摊晾及拌曲后，取出30 kg酒醅，将1000 mL菌液均匀洒到30 kg酒醅中，拌匀；最后将对应的酒醅装到竹篮中，放入发酵堆和窖池中进行原位条件发酵。
本发明的有益效果：
本发明的拜耳接合酵母CCTCC NO：M 2023729是从中国酱香型白酒大曲中筛选获得的。此菌株不仅能够耐受乳酸压力，还具有降解乳酸的能力。此外，此菌株还能在乳酸胁迫下高产乙醇和多种风味物质。将此菌株制成菌剂应用到酱香型白酒发酵生产过程中，显著提高了酒醅中的乙醇和风味物质含量。本发明的菌株实际应用效果良好，可以应用到发酵食品实际生产过程中。
生物材料保藏
拜耳接合酵母CCTCC NO：M 2023729，分类学命名为 Zygosaccharomyces bailii LBM888，2023年5月10日保藏于中国典型培养物保藏中心（CCTCC），保藏地址为中国武汉，武汉大学，保藏编号为CCTCC NO：M 2023729。
附图说明
图1A：拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729在YPD固体平板上的菌落形态。
图1B：拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729在透射电镜下的形态。
图2：拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729的生长曲线（n = 3）。
图3：拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729的碳源利用情况。
图4：拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729在乳酸胁迫下的生长；ZB-0表明没有添加乳酸；ZB+Lac表明添加了40 g/L乳酸胁迫。*,  P < 0.05，**,  P < 0.01 (Tukey’s test)。
图5A：拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729在乳酸胁迫下的乳酸降解。
图5B：拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729在乳酸胁迫下的产乙醇能力。
图6：拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729在乳酸胁迫下的挥发性风味物质产量。
图7：拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729应用在酱香型白酒发酵过程中的过程图。
图8A：添加拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729菌剂对酱香型白酒发酵过程中的乳酸含量的影响。
图8B：添加拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729菌剂对酱香型白酒发酵过程中的乙醇含量的影响。
图9：酱香型白酒生产工艺。
 具体实施方式
（一）培养基
筛选培养基（YPD）：葡萄糖20 g/L，蛋白胨20 g/L，酵母膏10 g/L，琼脂粉20 g/L。
种子培养基：葡萄糖20 g/L，蛋白胨20 g/L，酵母膏10 g/L。
高粱汁培养基：将高粱与水以1:4（M:V）浸泡过夜，然后加淀粉酶蒸煮液化后，在60℃加糖化酶糖化，最后过滤离心，调节糖度至5°Bx。
（二）检测乳酸含量的方法
对于模拟实验的发酵液，用0.22µm水相滤膜对发酵液进行过滤，将滤液转移至液相小瓶。对于发酵酒醅，称取5 g酒醅加入20 mL超纯水中，震荡混匀后，冰浴超声30 min，然后将超声后的混合液于4℃，8000×g离心5 min，将上清液用0.22 μm的水相滤膜过滤，滤液转移至液相小瓶中。液相系统：Waters 2695；色谱柱：Bio-Rad Aminex HPX-87H Ion Exclusion Column，柱温：60℃；检测器：紫外检测器（PDA），检测波长210 nm；流动相：5 mmol L-1 H2SO4，流速：0.6 mL min-1。
（三）检测乙醇含量的方法
对于模拟实验的发酵液，用0.22µm水相滤膜对发酵液进行过滤，将滤液转移至液相小瓶。对于发酵酒醅，称取5 g酒醅加入20 mL超纯水中，震荡混匀后，冰浴超声30 min，然后将超声后的混合液于4℃，8000×g离心5 min，将上清液用0.22µm的水相滤膜过滤，滤液转移至液相小瓶中。液相系统：Waters 2695；色谱柱：Bio-Rad Aminex HPX-87H Ion Exclusion Column，柱温：60℃；检测器：示差折光检测器（RID）；流动相：5 mmol L-1 H2SO4，流速：0.6 mL min-1。
（四）检测挥发性化合物的方法
对于模拟实验的发酵液，将发酵液于4℃，8000×g离心5 min后，取8 mL上清液加入顶空瓶中。称取5 g酒醅加入20 mL超纯水中，加入0.85%的NaCl和1%的CaCl2，震荡混匀后，冰浴超声30 min；然后将超声后的混合液于4℃，8000×g离心5 min。取8 mL上清液加入顶空瓶（20 mL，预先加入3 g NaCl）中，然后加入20 μL的L-薄荷醇（100 μg×mL-1）。采用HS-SPME-GC-MS（GC 6890N和MS 5975）和DB-Wax色谱柱（30 m×0.25 mm，0.25μm）检测挥发性化合物。
实施例1：菌株的筛选
从酱香型白酒发酵过程中筛选得到了一株耐乳酸的拜耳接合酵母，过程如下：
采集某著名酱香型白酒发酵酒醅样品，然后取10g样品，加入90 mL 0.9%无菌生理盐水，于30℃、200rpm震荡1h后，悬浮液进行10倍梯度稀释，取100uL稀释后的液体均匀涂布于YPD固体平板上，培养3d后，挑选单菌落进行纯化。将所得的单菌加入到用乳酸调节的pH为3.2的高粱汁培养基中，进行30℃、200rpm培养3d。培养结束后，测定生长量及发酵液中的乳酸、乙醇和挥发性化合物。
最终挑选出一株能够降解乳酸且高产乙醇和风味化合物的菌株，将该菌株划线到YPD斜面培养基中进行保藏，同时保藏在甘油管中。
实施例2：菌株的鉴定
（1）菌落特征及菌体形态
将已活化好的实施例1筛选获得的菌株划线接种于YPD固体培养基上，30℃静止培养2d。观察菌落在平板上的形态。同时，挑取单菌落于高粱汁液体培养基中，培养2d后进行透射电镜观察。
图1结果表明菌株在YPD平板上的菌落形态呈白色不透明，有一定的光泽，边缘规则、光滑、呈圆形，中间凸起。在透射电镜下，单个细胞为较大的卵圆形，无鞭毛，无菌丝。
（2）菌株的生长特性
将菌株接种于高粱汁培养基中，30℃、200 rpm培养84h，每隔12 h取样测定OD 600观察其生长特性。
图2可以看出，菌株在高粱汁培养基中生长60 h后达到稳定期，发酵结束时OD600达到3.36 ± 0.05。发酵初始高粱汁培养基中的葡萄糖含量为40 g/L。表1的结果表明发酵结束时剩余葡萄糖为4.09 ± 0.23 g/L，菌株的乙醇产量为13.66 ± 0.45 g/L。
表1 发酵结束时发酵液中的葡萄糖和乙醇含量（n = 3）
物质 含量（g/L）
葡萄糖 4.09±0.23
乙醇 13.66±0.45
（3）生理生化及碳源利用实验：
对菌株进行了碳源利用实验测定，步骤如下：挑取单菌落接种于高粱汁培养基中，200 rpm，30°C条件培养24h后获得种子液。然后取10mL种子液10,000 ×g离心5min，弃掉上清液，细胞沉淀用等量无菌生理盐水悬浮，然后将悬浮液用相同的生理盐水稀释至10 −4后，取100uL接至biolog YT微孔版。按照说明书将微孔板进行30℃恒温培养箱静止培养36h，用酶标仪测定OD 590。OD 590 > 0.2时表明菌株可以利用此碳源。
图3结果表明此菌株的碳源利用谱比较窄，在测定的这些碳源中，仅能利用葡萄糖，OD 590值为0.96。而在其他碳源中，其值都小于0.2。
（4）分子生物学鉴定
接种菌株的单菌落至YPD液体培养基中，培养48h后，提取该菌的总DNA。采用ITS通用引物（ITS1和ITS4）进行PCR扩增。PCR扩增条件：94℃ 5 min, 94℃ 30 s, 55℃ 30 s, 72 ℃ 1 min, 循环30次；72℃ 10 min。PCR扩增产物经1%琼脂糖凝胶检验合格后送往苏州金唯智生物科技有限公司进行测序。将测序结果上传至美国国立生物技术信息中心（national center for biotechnology information，NCBI）数据库中进行BLAST比对，发现该菌株与 Zygosaccharomyces bailii的同源性最高，因此鉴定为 Zygosaccharomyces bailii ，命名为 Zygosaccharomyces bailii LBM888。
该菌株已经于2023年5月10日保藏于中国典型培养物保藏中心，保藏地址为中国武汉，武汉大学，保藏编号为CCTCC NO：M 2023729。
实施例3： 菌株CCTCC NO：M 2023729在乳酸压力下的产乙醇和风味物质能力
用乳酸将高粱汁培养基的pH调节至3.2，作为发酵培养基，对实验菌株拜尔接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）进行乳酸压力下产乙醇和风味化合物的能力评估。
具体步骤如下：
挑取拜尔接合酵母（ Zygosaccharomyces bailii）CCTCC NO：M 2023729单菌落接种于高粱汁培养基中，200 rpm，30°C条件培养24 h后获得种子液。
然后向100 mL添加了40 g/L的乳酸的发酵培养基（用乳酸将高粱汁培养基的pH调节至3.2）中接入1mL 拜尔接合酵母ZB种子液，于30℃、200 rpm的条件下培养72 h，每隔12 h取样，测定菌株的生长（OD 600）及发酵液中的乳酸、乙醇与挥发性化合物。
同时，以不添加乳酸的发酵培养基进行培养作为对照。
结果显示，菌株拜尔接合酵母ZB能够适应乳酸胁迫，在乳酸胁迫下仍然能够很好生长，如图4所示。尽管在发酵前48 h 拜尔接合酵母ZB在乳酸胁迫下的生长量显著（ P < 0.05）低于无乳酸胁迫，但是发酵结束时生物量无显著差异（ P > 0.05）, 此时有乳酸胁迫的OD 600为3.02±0.08，而无乳酸胁迫的为3.36±0.05。
发酵液中乳酸含量的测定表明拜尔接合酵母ZB耐受乳酸的主要机制为其能够降解体系中的乳酸（图5A）。发酵初始时的乳酸含量为40 g/L，而发酵结束时为34.12±1.07g/L，降解了5.88 g/L。乙醇测定结果表明拜尔接合酵母ZB不仅能够适应40 g/L的乳酸胁迫，而且还能够在乳酸胁迫下产生较高含量的乙醇，发酵结束时乙醇含量达到11.99 ± 0.13 g/L（图5B）。
图6结果表明拜耳接合酵母CCTCC NO：M 2023729能够在乳酸胁迫下产生多种风味物质。其中含量超过0.1 g/L的风味化合物有乙酸乙酯（1.02 g/L）、异戊醇（1.52 g/L）、苯乙醇（0.72 g/L）、乙酸苯乙酯（0.50 g/L）、异丁醇（0.13 g/L）、乳酸异戊酯（0.10 g/L）和异戊酸（0.10 g/L）。含量小于0.1g/L的风味化合物有乙酸异戊酯、乳酸乙酯和1-辛醇等。
以上结果表明，拜耳接合酵母CCTCC NO：M 2023729能够很好地适应乳酸环境，不仅能够降低体系中的乳酸含量，还能够产乙醇与风味化合物。
实施例4： 酵母菌剂在白酒酿造中的应用
从下沙投料到最后一个轮次的取酒丢糟，酱香型白酒的生产遵循季节演替规律， 一个生产周期接近一年的时间，要经过下沙和造沙两个投料轮次，九次蒸煮，八次发酵和七次取酒，其生产工艺如图9所示。
将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。将此菌剂应用到酱香型白酒发酵第四轮次生产过程中。
具体操作如下：
酒醅经蒸馏、摊晾及拌曲后，取出30 kg酒醅，将1000 mL菌液均匀洒到30 kg酒醅中，拌匀。空白对照为将1000 mL无菌水均匀洒到30 kg酒醅中。
每个处理三个平行，最后将对应的酒醅装到竹篮中，放入发酵堆和窖池中进行原位条件发酵，操作如图7所示。
在堆积发酵结束和窖内发酵结束后取样，测定酒醅中的乳酸、乙醇与挥发性风味物质。
结果显示：
乳酸与乙醇的测定结果如图8所示。可以看到酱香型白酒发酵第四轮次的初始乳酸含量高于60 g/kg，表明乳酸积累是制约酱香型白酒产量与质量的重要因素。
实验菌剂的添加有效降低了发酵过程中的乳酸含量，如图8A所示。在堆积结束时，不添加菌剂组（入窖空白）的乳酸含量为73.84 ± 3.65 g/kg，而菌剂添加组（入窖ZB）的乳酸为35.45 ± 7.09 g/kg，降低了51.99%。
在窖内发酵结束时，对照组的乳酸（出窖空白）含量为85.07 ± 3.52 g/kg，而菌剂添加组（出窖ZB）的乳酸为54.51 ± 2.08 g/kg，降低了35.92%。
对于乙醇而言，实验菌剂的添加显著（ P < 0.05）提升了堆积发酵阶段的乙醇产量（图8B），堆积结束时，对照组的乙醇含量为6.37 ± 1.45 g/kg，而菌剂添加组的乙醇含量为12.30 ± 1.01 g/kg，提升了92.93%。窖内发酵结束时，对照组的乙醇含量为28.00 ± 0.65 g/kg，而菌剂添加组的29.98 ± 0.34 g/kg，提高了7.07%。
此外，实验菌剂的添加显著提高了酒醅中的挥发性风味物质，主要为酯类、醇类和芳香族化合物，如表2所示。显著提升的酯类物质包括乙酸乙酯、丙酸乙酯、乙酸异戊酯、异丁酸乙酯、醋酸呋喃甲酯、苯乙酸乙酯和乙酸苯乙酯等。显著提升的醇类物质包括异丁醇、苯乙醇、异戊醇和2-庚醇。显著提升的芳香族化合物包括2-乙基-6-甲基吡嗪、2,6-二乙基吡嗪和四甲基吡嗪。
表2 添加CCTCC NO：M 2023729菌剂对酱香型白酒发酵过程中的挥发性风味化合物影响
风味化合物 初始 入窖空白 入窖ZB 出窖空白 出窖ZB
酯类（mg/kg）
乙酸乙酯 2.18 ± 0.04 3.78 ± 0.06 4.74 ± 0.35 9.36 ± 2.47 11.24 ± 1.18
丙酸乙酯 0.02± 0.00 0.11± 0.00 0.61± 0.05 0.19± 0.02 0.88± 0.05
2-甲基丙酸乙酯 0.00± 0.00 0.00± 0.00 0.00± 0.00 0.00± 0.00 0.22± 0.01
乙酸异戊酯 0.09 ± 0.02 0.21 ± 0.04 0.30 ± 0.04 0.55 ± 0.22 0.72 ± 0.23
异丁酸乙酯 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.22 ± 0.05
醋酸呋喃甲酯 0.00± 0.00 0.06± 0.01 0.20± 0.00 0.10± 0.00 0.45± 0.02
2-甲氧基乙酸3-甲基丁酯 0.03± 0.00 0.19± 0.00 0.21± 0.00 0.56± 0.04 1.21± 0.09
醋酸呋喃甲酯 0.00± 0.00 0.00± 0.00 0.14± 0.00 0.05± 0.00 0.24± 0.00
苯乙酸乙酯 0.14± 0.02 1.67± 0.10 1.92± 0.05 6.08± 0.09 7.22± 0.30
戊二酸二乙酯 0.00± 0.00 0.00± 0.00 0.00± 0.00 0.09± 0.00 0.14± 0.00
乳酸丁酯 0.00 ± 0.00 0.00 ± 0.00 0.03 ± 0.00 0.00 ± 0.00 0.12 ± 0.12
3-苯丙酸乙酯 0.00± 0.00 0.05± 0.00 0.12± 0.00 0.11± 0.00 0.55± 0.00
棕榈酸乙酯 0.40± 0.03 1.81± 0.09 2.83± 0.10 5.25± 0.21 6.55± 0.22
十六碳烯酸乙酯 0.00± 0.00 0.08± 0.00 0.10± 0.00 0.15± 0.01 0.26± 0.00
乙酸苯乙酯 0.09± 0.00 0.63± 0.02 1.33± 0.10 0.91± 0.07 2.44± 0.11
亚油酸乙酯 0.20± 0.05 0.43± 0.02 0.74± 0.06 1.19± 0.09 1.54± 0.05
醇类（mg/kg）
异丁醇 0.00 ± 0.00 0.05± 0.00 0.30± 0.01 0.10± 0.01 0.31± 0.03
苯乙醇 2.98 ± 0.09 5.53 ± 2.73 11.06 ± 1.40 7.97 ± 2.45 29.23 ± 2.87
异戊醇 0.98 ± 0.04 1.09 ± 0.32 3.82 ± 0.47 1.98 ± 0.58 4.60 ± 1.09
2-庚醇 0.00 ± 0.00 0.00 ± 0.00 0.10 ± 0.01 0.00 ± 0.00 0.13 ± 0.01
芳香族（mg/kg）
2-乙基-6-甲基吡嗪 0.00 ± 0.00 0.00 ± 0.00 0.06 ± 0.01 0.04 ± 0.01 0.19 ± 0.00
2,6-二乙基吡嗪 0.00 ± 0.00 0.00 ± 0.00 0.04 ± 0.00 0.09 ± 0.02 0.20 ± 0.01
四甲基吡嗪 0.07 ± 0.00 0.17 ± 0.09 0.51 ± 0.06 1.01 ± 0.08 1.51 ± 0.02
实施例5： 酵母菌剂在白酒酿造中的应用
具体步骤如下：
将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
（1）大曲清香型白酒：
将制备的菌剂液混在经过润糁蒸熟摊凉的粮食中，并进行混匀配糟发酵。其中用菌剂液为投料量的10~20%（按质量计），入缸发酵。清蒸清烧，制备得到大曲清香型白酒（具体大曲的生产工艺参照论文：清香型白酒酿造微生物和风味物质的研究进展）。
（2）小曲清香型白酒：
将制备的菌剂液混在经过蒸煮、摊凉的粮食中，堆积糖化1 d后进行混匀配糟入槽车发酵14 d。其中用固态菌剂量为投料量的1%（按质量计）。清蒸清烧，制备得到小曲清香型白酒（具体小曲的生产工艺参照论文：小曲清香型白酒发酵过程微生物菌群合成高级醇代谢特征）。
（3）老白干香型白酒
经过蒸煮、摊凉的粮食进行配料，粮醅混匀复蒸、摊凉。将制备的菌剂液混在粮醅中，其中用菌剂液的添加量为投料量的25%（按质量计），入缸发酵40 d。清蒸混烧，制备得到老白干香型白酒（具体老白干香型白酒的生产工艺参照论文：多粮老白干香型白酒配方优化）。
结果显示：本发明的菌剂能够增加白酒的风味物质。
实施例6：酵母菌剂在黄酒（料酒）中的应用
（1）将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
（2）酒母制备：首先制备糖化液，将糯米放入浸米罐中浸米30 min，将米浆水倒掉后蒸饭30-40 min，将蒸好的米饭冷却至温度为60℃备用，加入2‰糖化酶和1‰液化酶，搅拌均匀后放入水浴锅60℃恒温糖化约4 h以上，每隔1 h左右搅拌一次，使其糖化均匀。将糖化液用纱布过滤得到糖液后加水稀释至13±0.5°Bx（20℃）。灌入瓶中加封口膜，放入灭菌锅121℃杀菌20 min，取出冷却待用。将经培养后的菌剂液，按照5%（v/v）的接种比例转接到大米糖化液中，再恒温培养36-48 h，即可用于投料。
（3）黄酒发酵：按照黄酒酿造配料进行落料：糯米1000 g、米饭1500 g、酿造用水1100 mL、大曲110 g、酒母100 mL；
总发酵时间为20天左右。发酵过程注意开耙，前发酵温度控制在33℃以下，后发酵温度控制15±2℃左右。前发酵的温度为28℃，总共发酵5 d；每天开耙两次；后发酵的发酵温度为15℃，发酵15 d；5 d开耙一次。
结果显示：本发明的菌剂能够增加黄酒（料酒）的风味物质。
实施例7：酵母菌剂在酱油酿造上的应用
（1）将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
（2）挑选饱满干净的大豆，洗净后加水浸泡5-10 h，等豆粒完全吸水膨胀后沥水后加压蒸熟；热豆出锅后，冷却至70-80℃左右，加入面粉30-40%（按照大豆质量计），搅拌均匀，继续冷却至30-40℃，然后加入2%-5%（v/v）的菌剂液，翻拌均匀，保持品温28-30℃左右进行发酵，发酵中间由于微生物代谢产热，需要不间断翻曲通风降温，经72- 96h后获得酱油曲；将制好的酱油曲按照1：1.5（m/v）加入盐水，搅拌均匀进行制醅，发酵3-6个月，每15天翻酱一次；发酵结束后压榨酱油醅，过滤除杂及灭菌就可以得到成品酱油。
结果显示：本发明的菌剂能够增加酱油的风味物质。
实施例8：酵母菌剂在食醋酿造上的应用
（1）将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
（2）选料处理：选择优质糯米100 kg，加水200 kg浸米15-24 h，然后淋米至无白浆后沥干，蒸熟淋水冷却至25-30℃；
拌料入缸：糯米入缸前先拌入酒药1.5-2 kg，低温糖化72-96 h；
酒精发酵：糖化结束后加水30 kg，加入菌剂液6 kg，28℃下发酵144-168h，即得成熟酒醅; 
制醅：在发酵池内投入麸皮150 kg，摊平，将发酵成熟的酒醅打入池内并搅拌均匀，取稻壳5 kg均匀摊于池内上层，再取发酵成熟的醋醅5 kg，搅拌均匀，覆盖5 kg稻壳，撒匀即完成制醅；
醋酸发酵：每发酵24 h后进行翻醅，每次翻醅完都加稻壳进行保温保湿，发酵第11天起不再添加稻壳，翻醅使品温冷却，18-20天后检测酸度不再上升时，加盐4 kg，密封30-45天；
淋醋与煎醋：取陈酿结束的醋醅，按比例加入炒米色，浸泡数小时后，采用套淋法循环泡淋，得到的醋汁加入食糖调配，澄清后进行煎醋，等品温降至75-80℃后罐装密封保存。
结果显示：本发明的菌剂能够增加食醋的风味物质。
实施例9：酵母菌剂在清酒酿造上的应用
将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
（1）抛光：利用抛光机除去原料米质量的20%-30%。碎米粒要尽量的少，精白米与糙米形状相似，原料米的胚芽和沟完全去除；
（2）浸米：浸米水温为室温，吸水率在28-30%左右，浸米时间为9-12 h； 
（3）蒸饭：米饭颗粒分明，外硬内软，内无白心，疏松不糊，熟而不烂，均匀一致，糯米出饭率一般在140%-150%左右；
（4）落料：落料时米饭温度冷却到25-30℃，其中米100（Kg）、米饭140（Kg）、米曲、菌剂液20（Kg）、酒母3（Kg）、水120 L。
（5）发酵：起始发酵温度控制在28-30℃，时间为3天，之后在20℃左右发酵，时间为22天，总发酵时间为25天。
结果显示：本发明的菌剂能够增加清酒的风味物质。
实施例10：酵母菌剂在白酒酿造上的应用
将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
（1）将高粱（40-50%），小麦（6-10%），玉米（5-10%），糯米（10-12%）和大米（15-20%）按照比例称好，热水(55-65℃)浸泡24-32h，稻糠（按照粮食总重量20-35%）在90-100 ℃蒸30-40 min，摊凉备用；
（2）初蒸：将泡好的玉米、高粱小麦等控干表面水分，进行原粮的初蒸，时间为30-60 min；
（3）焖水：初蒸结束后，加入40-50℃的温水，水面高出粮面15 cm以上，闷粮10-30 min，之后将水沥干；
（4）复蒸：初蒸完的原粮，加入步骤（1）润好水的大米、糯米，充分拌匀后入蒸粮锅进行复蒸，时间为60-70 min；
（5）降温下曲：复蒸糟醅冷却到30-35℃，加入步骤（1）处理后的稻糠和加入18-28%白酒大曲，加入5%-10%的菌剂液（按照粮食重量计算），搅拌均匀进行入窖发酵；
（6）封窖发酵：糟醅入窖池后进行盖糟，盖糟高于地面至少40 cm，窖泥封窖，窖泥厚度为15-20 cm，发酵45-70 d；
（7）出池摘酒：将发酵好的糟醅出窖，利用装甑锅进行蒸馏出酒，流酒速度为1.5-2.5kg/min，流酒温度25-30 ℃。
上述工艺制备的白酒出酒率28%-33%，优质品率88-91%，所酿造的白酒无色或微黄透明，酯香、焦糊香突出，入口净爽、甜醹柔润，多粮膨化香氤氲，多香自然融合，酒体较醇厚，具有醹、雅、融典型风格，所得陶融型白酒到达了一级酒标准。
实施例11：酵母菌剂在在甜米酿的应用
将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
以大米为主要原料按比例称取后，在常温下浸泡8-12 h左右至充分吸水；将浸泡后的米粒水分沥干，常压下蒸煮30-40min左右至米饭内无白心；米饭蒸熟后用冷开水淋饭至25-28℃左右，转移至发酵罐内，按比例添加0.4%-0.8%（质量比）菌剂液，接种0.5%-1.5%（质量比）酿酒酵母，搅拌均匀，加入生米质量1-1.5倍的饮用水，在28-30°C条件下的恒温发酵48-72h。所得甜米酿酒精度2-4%（v/v），香气协调、浓郁、质地均匀，口感酸甜适口。
结果显示：本发明的菌剂能够增加甜米酿的风味物质。
实施例12：酵母菌剂在米酒中的应用
将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
以大米为主要原料按比例称取后，在常温下浸泡8-12 h左右至充分吸水，要求浸后手捏易碎，无硬米芯为止；将浸泡后的米粒水分沥干，常压下蒸煮30-40 min左右至米饭内无白心；米饭蒸熟后用冷开水淋冷至25-28℃左右，按比例添加5%-10%（质量比）菌剂液进行糖化，糖化结束后补加米饭，接种5%-10%（质量比）接种酿酒酵母，搅拌均匀，加入原米质量1-1.5倍的饮用水，在28-30°C条件下的发酵3-5天，降低温度至10-15°C范围发酵10-15天，发酵结束后，压榨、过滤得到米酒。米酒酒精度5-10%（v/v），富含米香果香、香气协调、浓郁、质地均匀。
结果显示：本发明的菌剂能够增加米酒的风味物质。
实施例13：酵母菌剂在发酵乳制品中的应用
将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
可用于制造乳杆菌乳饮料，具体制备过程如下：
将原料乳脱脂奶在95℃热杀菌20 min，然后冷却至4℃，再加入菌剂液，使菌体浓度达到10 6 CFU/mL以上，在4℃冷藏保存即得到乳饮料。
结果显示：本发明的菌剂能够增加乳饮料的风味物质。
实施例14：酵母菌剂在发酵乳制品中的应用
将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
可用于制造豆奶，具体制备过程如下：
采用软水浸泡大豆，水量为原大豆量三倍体积，在温度80℃下浸泡1~2h，再去除大豆皮。接着，沥去浸泡水，另加沸水磨浆，并在温度高于80℃的条件下保温10~15min。浆体用150目滤膜过滤后离心，得到的离心液即为粗豆奶，再将它加热到温度140~150℃，然后将热的粗豆奶迅速导入真空冷却室进行抽真空，所述粗豆奶中的异味物质随着水蒸汽迅速排出。经过真空脱气后，将其温度降至37℃左右，再接入菌剂液，使浓度达到10 6CFU/mL以上，在4℃冷藏保存即得到豆奶。
结果显示：本发明的菌剂能够增加豆奶的风味物质。
实施例15：酵母菌剂在发酵果蔬饮料中的应用
将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
可用于制造果蔬饮料，具体制备过程如下：
选用新鲜蔬菜(例如黄瓜、胡萝卜、甜菜、芹菜或圆白菜中的一种或多种)洗净后榨汁，接着进行高温瞬间灭菌，在温度140℃下高温热杀菌2s后，立即降温到37℃左右，再接入本发明的菌剂液，使其浓度达到10 6CFU/mL以上，在4℃冷藏保存即得到果蔬饮料。
结果显示：本发明的菌剂能够增加果蔬饮料的风味物质。
实施例16：酵母菌剂在威士忌中的应用
主要包括以下步骤：
将拜耳接合酵母CCTCC NO：M 2023729（以下简称为拜尔接合酵母ZB）接到种子培养基中，于30℃、200 rpm 的条件下培养48 h，获得1×10 8 cells/mL的种子液。然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液。
粉碎:分别将称取的麦芽机械粉碎，麦芽加工成粗颗粒粉状；
糖化：将粉碎后的麦芽和热水进行混合，热水温度为63.5℃，然后糖化得到麦芽汁；
发酵：麦芽汁冷却后泵入发酵槽中，加入20%（v/v）菌剂液进行发酵，发酵时间56～60小时；
蒸馏：发酵后的酒汁在铜制霍斯坦蒸馏器中进行两次蒸馏；第一次蒸馏：将蒸馏液加到蒸馏釜内，当釜内酒汁升温至80℃时，开始沸腾，进入蒸馏阶段，冷却水温度小于18℃，一次蒸馏不去酒头酒尾，初馏液酒精度为20~28％vol，一次蒸馏时间控制在8~9h；第二次蒸馏：将初馏液进行第二次蒸馏，分馏出酒头、酒心、酒尾；留取酒心并降低酒精度至63.5％vol作为组分A，备用；酒头、酒尾用于下一次蒸溜，二次蒸馏时间约为10~12h。
陈酿：蒸馏后陈酿。
调配和过滤：降低酒精度调配至40度得半成品，过滤得成品。
结果显示：本发明的菌剂能够增加威士忌的风味物质。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (16)

  1. 一株拜耳接合酵母（ Zygosaccharomyces bailii），其特征在于，所述拜耳接合酵母已于2023年5月10日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCC NO：M 2023729。
  2. 一种微生物菌剂，其特征在于，含有权利要求1所述的拜耳接合酵母CCTCC NO：M 2023729。
  3. 根据权利要求2所述的微生物菌剂，其特征在于，所述微生物菌剂以拜耳接合酵母CCTCC NO：M 2023729为主要微生物。
  4. 根据权利要求3所述的微生物菌剂，其特征在于，所述菌剂中拜耳接合酵母CCTCC NO：M 2023729的活菌数为10 5~10 8 CFU/mL。
  5. 根据权利要求3所述的微生物菌剂，其特征在于，所述菌剂中拜耳接合酵母CCTCC NO：M 2023729的菌浓范围为10 5~10 8 CFU/mL。
  6. 根据权利要求2所述的微生物菌剂，其特征在于，所述微生物菌剂为液体菌剂或者固体菌剂。
  7. 根据权利要求2所述的微生物菌剂，其特征在于，所述微生物菌剂含有权利要求1的拜耳接合酵母CCTCC NO：M 2023729菌株的菌体活细胞、冷冻干燥得到的干菌体、固定化的细胞。
  8. 权利要求1所述的拜耳接合酵母CCTCC NO：M 2023729或含有权利要求1所述的拜耳接合酵母CCTCC NO：M 2023729的菌剂的应用，其特征在于，所述应用为如下一种或者多种：降解乳酸、提高乙醇含量、提高风味物质含量；具体应用为，挑取拜耳接合酵母CCTCC NO：M 2023729单菌落接种于高粱汁培养基中，200 rpm，30°C条件培养24h后获得种子液；然后向100 mL添加了40 g/L的乳酸的发酵培养基中接入1mL 拜耳接合酵母CCTCC NO：M 2023729种子液，于30℃、200 rpm的条件下培养72 h；所述高粱汁培养基为，将高粱与水以1:4（M/V）浸泡过夜，然后加淀粉酶蒸煮液化后，在60℃加糖化酶糖化，最后过滤离心，调节糖度至5°Bx；所述发酵培养基为，用乳酸将高粱汁培养基的pH调节至3.2。
  9. 权利要求1所述的拜耳接合酵母CCTCC NO：M 2023729或含有权利要求1所述的拜耳接合酵母CCTCC NO：M 2023729的菌剂在发酵食品中的应用，其特征在于，所述应用为，在制备发酵食品的过程中，添加拜耳接合酵母CCTCC NO：M 2023729或含有所述拜耳接合酵母CCTCC NO：M 2023729的菌剂。
  10. 根据权利要求9所述的应用，其特征在于，所述应用为：在黄酒、酱油、食醋、清酒、料酒、甜米酿、米酒、乳制品或果蔬饮品的制备过程中添加拜耳接合酵母CCTCC NO：M 2023729或含有所述拜耳接合酵母CCTCC NO：M 2023729的菌剂。
  11. 一种在乳酸压力下产乙醇和风味物质的方法，其特征在于，所述方法为，挑取拜耳接合酵母CCTCC NO：M 2023729单菌落接种于高粱汁培养基中，培养后获得种子液；然后向添加了乳酸的发酵培养基中接入所述拜耳接合酵母CCTCC NO：M 2023729种子液进行发酵培养。
  12. 根据权利要求11所述的方法，其特征在于，所述高粱汁培养基为，将高粱与水以1:4（M:V）浸泡过夜，然后加淀粉酶蒸煮液化后，在60℃加糖化酶糖化，最后过滤离心，调节糖度至5°Bx；所述发酵培养基为，用乳酸将高粱汁培养基的pH调节至3.2。
  13. 一种在乳酸压力下产乙醇和风味物质的方法，其特征在于，所述方法为，挑取拜耳接合酵母CCTCC NO：M 2023729单菌落接种于高粱汁培养基中，200 rpm，30°C条件培养24h后获得种子液；然后向100 mL添加了40 g/L的乳酸的发酵培养基中接入1mL 拜耳接合酵母CCTCC NO：M 2023729种子液，于30℃、200 rpm的条件下培养72 h。
  14. 根据权利要求13所述的方法，其特征在于，所述高粱汁培养基为，将高粱与水以1:4（M:V）浸泡过夜，然后加淀粉酶蒸煮液化后，在60℃加糖化酶糖化，最后过滤离心，调节糖度至5°Bx；所述发酵培养基为，用乳酸将高粱汁培养基的pH调节至3.2。
  15. 一种酵母菌剂在白酒酿造中的应用，其特征在于，所述应用为，将拜耳接合酵母CCTCC NO：M 2023729接到种子培养基中，获得种子液；然后将种子液离心，弃掉上清液，将沉淀重悬于水中，得到菌剂；
    将所述菌剂应用到酱香型白酒发酵的第四轮次生产过程中；具体操作如下：
    酒醅经蒸馏、摊晾及拌曲后，取出酒醅，将菌剂均匀洒到酒醅中，拌匀；最后将对应的酒醅装到竹篮中，放入发酵堆和窖池中进行原位条件发酵。
  16. 一种酵母菌剂在白酒酿造中的应用，其特征在于，所述应用为，将拜耳接合酵母CCTCC NO：M 2023729接到种子培养基中，于30℃、200 rpm 的条件下培养48 h获得10 8 cells/mL的种子液；然后取10 mL种子液在10,000 × g 条件下离心 5 min，弃掉上清液，将沉淀重悬于1000 mL无菌水中，得到对应的菌剂液；
    将此菌剂液应用到酱香型白酒发酵第四轮次生产过程中；具体操作如下：
    酒醅经蒸馏、摊晾及拌曲后，取出30 kg酒醅，将1000 mL菌液均匀洒到30 kg酒醅中，拌匀；最后将对应的酒醅装到竹篮中，放入发酵堆和窖池中进行原位条件发酵。在此处键入权利要求项1。
PCT/CN2024/110474 2023-06-08 2024-08-07 一株耐乳酸胁迫且高产乙醇和风味化合物的拜耳接合酵母 Ceased WO2024251311A2 (zh)

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