Strain of lactic-acid-stress-tolerant zygosaccharomyces bailii capable of achieving high-yield production of ethanol and flavor compounds

Abstract

A strain of lactic-acid-stress-tolerant Zygosaccharomyces bailii capable of achieving high-yield production of ethanol and flavor compounds, belonging to the technical field of microorganisms. The Zygosaccharomyces bailii CCTCC NO: M 2023729 is screened from Chinese Maotai-flavor liquor yeast, and the strain is not only tolerant of lactic acid stress, but also has lactic acid degrading capability, and can also achieve high-yield production of ethanol and various flavor compounds under the stress of lactic acid. The strain can be prepared into a microbial agent for use in the fermentation production process of Maotai-flavor liquor, and can significantly increase the content of ethanol and flavor compounds in fermented grains.

Description

A strain of Zygosaccharomyces bailii that is tolerant to lactic acid stress and produces high amounts of ethanol and flavor compounds Technical Field

[0001] The invention relates to a strain of Zygosaccharomyces bayeris that is resistant to lactic acid stress and has high ethanol and flavor compound production, and belongs to the technical field of microorganisms. Background Art

[0002] During the brewing process of traditional fermented foods, microorganisms encounter various environmental stresses, such as high acidity, high ethanol levels, and high temperatures. Lactic acid is a metabolite of many microorganisms in traditional fermented foods. On the one hand, it contributes to the mellow taste of fermented foods. On the other hand, due to its low volatility and other characteristics, it continues to accumulate during the brewing process, causing an increase in acidity in the fermentation system and inhibiting the growth and metabolism of functional microorganisms. For example, in the brewing process of Maotai-flavor liquor, which involves repeated fermentation in multiple batches, the unique multi-batch fermentation process causes lactic acid to accumulate continuously within the batch, with the content reaching 40 g / kg. The lactic acid content in some low-yield batches is even higher, seriously exceeding the normal concentration range in liquor, resulting in a decrease and loss of flavor substances.

[0003] Lactic acid accumulation leads to problems such as low fermentation yields and flavor defects. However, due to the characteristics of open-type natural inoculation fermentation, these problems cannot be solved by changing the operating process. Breeding strains with superior traits and applying them to low-yield fermentations would be an effective approach. However, most current research focuses on analyzing the lactic acid tolerance of some functional microorganisms. However, fermented foods are sensory products, with flavor substances as the quantification guide. These functional microorganisms must not only be able to tolerate the high acid environment during fermentation but also play a role in producing flavor substances. Therefore, the selected strains must combine multiple capabilities such as lactic acid tolerance and flavor production to be applied in the fermentation process, which poses a considerable challenge for strain selection. Summary of the Invention

[0004] To address these challenges, the present invention identified a functional yeast strain, Zygosaccharomyces bailii LBM888, from the Maotai-flavor liquor brewing system. This strain not only tolerates lactic acid stress but also possesses the ability to degrade it. Furthermore, this strain can produce high levels of ethanol and various flavor compounds under lactic acid stress. This strain, formulated into a microbial inoculant and applied to the Maotai-flavor liquor fermentation process, significantly increased the ethanol and flavor content in the mash. The strain has demonstrated excellent practical application and can be applied to the production of fermented foods.

[0005] The present invention provides a strain of Zygosaccharomyces bailii LBM888. The Zygosaccharomyces bailii LBM888 was deposited in the China Center for Type Culture Collection on May 10, 2023, at Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M 2023729.

[0006] The Zygosaccharomyces bailii CCTCC NO: M 2023729 has the following characteristics:

[0007] Morphological characteristics: The colonies on YPD plates are white and opaque with a certain gloss, regular, smooth, rounded edges, and a raised center. Under transmission electron microscopy, individual cells are large oval, without flagella or hyphae.

[0008] Growth characteristics: After 60 h of growth in sorghum juice medium, the plant reached the stationary phase, and the OD600 reached 3.36 ± 0.05 at the end of fermentation. When grown under stress in fermentation medium with 40 g / L lactic acid, the OD 600 is 3.02±0.08;

[0009] Carbon source utilization characteristics: can utilize glucose, but cannot utilize 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;

[0010] 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.

[0011] Lactic acid resistance and lactic acid degradation: able to tolerate 40 g / L lactic acid; still able to grow well in fermentation medium containing 40 g / L lactic acid, OD 600 The lactic acid content at the beginning of fermentation was 40 g / L, while it was degraded to 5.88 g / L at the end of fermentation. It was able to produce a higher content of ethanol under lactic acid stress, and the ethanol content reached 11.99 ± 0.13 g / L at the end of fermentation.

[0012] Under lactic acid stress, a variety of flavor compounds were produced: flavor compounds with content exceeding 0.1 g / L included ethyl acetate (1.02 g / L), isoamyl alcohol (1.52 g / L), phenylethanol (0.72 g / L), phenylethyl acetate (0.50 g / L), isobutanol (0.13 g / L), isoamyl lactate (0.10 g / L), and isovaleric acid (0.10 g / L). Flavor compounds with content less than 0.1 g / L included isoamyl acetate, ethyl lactate, and 1-octanol.

[0013] Application in liquor brewing: It can significantly reduce lactic acid content, increase ethanol content and volatile flavor substances in the mash.

[0014] 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.

[0015] In one embodiment of the present invention, the microbial agent uses Zygosaccharomyces bailii CCTCC NO: M 2023729 as a main microorganism.

[0016] In one embodiment of the present invention, the viable count of Zygosaccharomyces bailii CCTCC NO: M 2023729 in the bacterial agent is 10 5 ~10 8 CFU / mL.

[0017] In one embodiment of the present invention, 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.

[0018] In one embodiment of the present invention, the bacterial agent is obtained by preparing the seed solution of Zygosaccharomyces bailii CCTCC NO: M 2023729 and then expanding the culture.

[0019] In one embodiment of the present invention, 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.

[0020] In one embodiment 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.

[0021] In one embodiment of the present invention, the bacterial agent further contains any strain of any species that can be used in fermented foods or fermented food preparation, such as Bacillus licheniformis, Saccharomyces cerevisiae, Bacillus subtilis, etc.

[0022] In one embodiment of the present invention, the bacterial agent further contains any carrier that can be used for fermenting food.

[0023] A third object of the present invention is to provide applications of the Zygosaccharomyces bailii CCTCC NO: M 2023729 or a bacterial agent containing the Zygosaccharomyces bailii CCTCC NO: M 2023729, wherein the applications include one or more of the following: degrading lactic acid, increasing ethanol content, and increasing flavor substance content. The specific application is as follows: a single colony of Bayer's yeast CCTCC NO: M 2023729 is picked and inoculated into a sorghum juice medium to obtain a seed liquid after cultivation; the Bayer's yeast CCTCC NO: M 2023729 seed liquid is then inoculated into a fermentation medium supplemented with lactic acid for fermentation and cultivation; the sorghum juice 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, adding saccharifying enzyme at 60°C, and finally filtering and centrifuging to adjust the sugar content to 5°Bx; the fermentation medium is prepared by adjusting the pH of the sorghum juice medium to 3.2 with lactic acid.

[0024] A fourth object of the present invention is to provide a use of Zygosaccharomyces bailii CCTCC NO: M 2023729 or a bacterial agent containing the same in fermented foods. The use comprises adding Zygosaccharomyces bailii CCTCC NO: M 2023729 or a bacterial agent containing the same during the preparation of the fermented foods.

[0025] In one embodiment of the present invention, the method is applied to liquor brewing.

[0026] 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, culturing to obtain a seed liquid; and then inoculating the Zygosaccharomyces bailii CCTCC NO: M 2023729 seed liquid into a fermentation culture medium supplemented with lactic acid for fermentation and culture.

[0027] In one embodiment of the present invention, 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.

[0028] 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 culturing it at 200 rpm and 30°C for 24 hours to obtain a seed solution; then, inoculating 1 mL of the Zygosaccharomyces bailii CCTCC NO: M 2023729 seed solution into 100 mL of a fermentation culture medium supplemented with 40 g / L of lactic acid, and culturing it at 30°C and 200 rpm for 72 hours.

[0029] The present invention provides an application of a yeast agent in liquor brewing, wherein the application comprises the following steps: inoculating Zygosaccharomyces bayeris 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;

[0030] The bacterial agent is applied to the fourth round of production of Maotai-flavor liquor fermentation; the specific operation is as follows:

[0031] After the mash is distilled, spread out to dry and mixed with koji, the mash is taken out and the bacterial agent is evenly sprinkled on the mash and mixed well; finally, the corresponding mash is put into a bamboo basket and placed in a fermentation pile and cellar for in-situ fermentation.

[0032] The present invention provides an application of a yeast agent in liquor brewing, wherein the application comprises inoculating Bayer yeast CCTCC NO: M 2023729 into a seed culture medium, culturing at 30°C and 200 rpm for 48 h to obtain 10 8 cells / mL seed solution; 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;

[0033] This bacterial agent liquid was applied to the fourth round of production of Maotai-flavor liquor fermentation; the specific operation was as follows:

[0034] After the mash is distilled, spread out and mixed with koji, 30 kg of mash is taken out, 1000 mL of bacterial liquid is evenly sprinkled into the 30 kg of mash and mixed well; finally, the corresponding mash is put into a bamboo basket and placed in the fermentation pile and cellar for in situ fermentation.

[0035] Beneficial effects of the present invention:

[0036] The present invention's Zygosaccharomyces bailii strain, CCTCC No. M 2023729, was screened from Daqu, a Chinese Maotai-flavor liquor. This strain not only tolerates lactic acid stress but also possesses the ability to degrade lactic acid. Furthermore, this strain can produce high levels of ethanol and various flavor compounds under lactic acid stress. When formulated into a microbial inoculant and applied to the fermentation process of Maotai-flavor liquor, this strain significantly increased the ethanol and flavor compound content in the mash. The strain has demonstrated excellent practical application and can be applied to the production of fermented foods.

[0037] Biomaterial Deposit

[0038] Zygosaccharomyces bailii CCTCC NO: M 2023729, taxonomically named Zygosaccharomyces bailii LBM888, was deposited in the China Center for Type Culture Collection (CCTCC) on May 10, 2023, at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 2023729. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A: Colony morphology of Zygosaccharomyces bailii CCTCC NO: M 2023729 on YPD solid plates.

[0040] Figure 1B: Morphology of Zygosaccharomyces bailii CCTCC NO: M 2023729 under transmission electron microscopy.

[0041] Figure 2: Growth curve of Zygosaccharomyces bailii CCTCC NO: M 2023729 (n = 3).

[0042] Figure 3: Carbon source utilization of Zygosaccharomyces bailii CCTCC NO: M 2023729.

[0043] Figure 4: Growth of Zygosaccharomyces bailii (CCTCC NO: M 2023729) under lactic acid stress; ZB-0 indicates no lactic acid addition; ZB+Lac indicates 40 g / L lactic acid addition. *, P < 0.05, **, P < 0.01 (Tukey's test).

[0044] FIG5A : Lactic acid degradation by Zygosaccharomyces bailii CCTCC NO: M 2023729 under lactic acid stress.

[0045] FIG5B shows the ethanol production ability of Zygosaccharomyces bailii CCTCC NO: M 2023729 under lactic acid stress.

[0046] Figure 6: Volatile flavor compound production by Zygosaccharomyces bailii CCTCC NO: M 2023729 under lactic acid stress.

[0047] Figure 7: Process diagram of the application of Zygosaccharomyces bailii CCTCC NO: M 2023729 in the fermentation process of Maotai-flavor liquor.

[0048] Figure 8A: Effect of adding Zygosaccharomyces bailii CCTCC NO: M 2023729 inoculant on the lactic acid content during the fermentation of Maotai-flavor liquor.

[0049] Figure 8B: Effect of adding Zygosaccharomyces bailii CCTCC NO: M 2023729 inoculant on the ethanol content during the fermentation of Maotai-flavor liquor.

[0050] Figure 9: Production process of Maotai-flavor liquor. DETAILED DESCRIPTION

[0051] (1) Culture medium

[0052] Screening medium (YPD): glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, agar powder 20 g / L.

[0053] Seed culture medium: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L.

[0054] Sorghum juice medium: Soak sorghum with water at a ratio of 1:4 (M:V) overnight, then add amylase to cook and liquefy it, add saccharifying enzyme at 60℃, and finally filter and centrifuge to adjust the sugar content to 5°Bx.

[0055] (2) Method for detecting lactic acid content

[0056] For the simulated fermentation broth, the fermentation broth was filtered through a 0.22 µm aqueous membrane filter, and the filtrate was transferred to a liquid chromatography vial. For the fermented mash, 5 g of mash was added to 20 mL of ultrapure water, mixed by vortexing, and sonicated on ice for 30 min. The sonicated mixture was then centrifuged at 8000 × g for 5 min at 4°C. The supernatant was filtered through a 0.22 µm aqueous membrane filter, and the filtrate was transferred to a liquid chromatography vial. The liquid chromatography system used was a Waters 2695; the chromatographic column was a Bio-Rad Aminex HPX-87H Ion Exclusion Column, the column temperature was 60°C; the detector was a UV detector (PDA), the detection wavelength was 210 nm; the mobile phase was 5 mmol L⁻¹ H₂SO₄, the flow rate was 0.6 mL min⁻¹.

[0057] (III) Methods for detecting ethanol content

[0058] For the simulated fermentation broth, the fermentation broth was filtered through a 0.22 µm aqueous filter membrane, and the filtrate was transferred to a liquid chromatography vial. For the fermented mash, 5 g of mash was added to 20 mL of ultrapure water, mixed by vortexing, and sonicated on ice for 30 min. The sonicated mixture was then centrifuged at 8000 × g for 5 min at 4°C. The supernatant was filtered through a 0.22 µm aqueous filter membrane, and the filtrate was transferred to a liquid chromatography vial. The liquid chromatography system used was a Waters 2695; the chromatographic column was a Bio-Rad Aminex HPX-87H Ion Exclusion Column, the column temperature was 60°C, and the detector was a differential refractive index detector (RID). The mobile phase was 5 mmol L⁻¹ H₂SO₄, and the flow rate was 0.6 mL min⁻¹.

[0059] (IV) Methods for detecting volatile compounds

[0060] For the simulated fermentation broth, 8 mL of the supernatant was collected and added to a headspace vial. Five grams of fermented mash was weighed and added to 20 mL of ultrapure water, followed by the addition of 0.85% NaCl and 1% CaCl₂. After vortexing and mixing, the mixture was sonicated on ice for 30 minutes. The sonicated mixture was then centrifuged at 8000 × g for 5 minutes at 4°C. Eight mL of the supernatant was collected and added to a 20 mL headspace vial (pre-spiked with 3 g NaCl), followed by the addition of 20 μL of L-menthol (100 μg mL⁻¹). Volatile compounds were analyzed using HS-SPME-GC-MS (GC 6890N and MS 5975) using a DB-Wax column (30 m × 0.25 mm, 0.25 μm).

[0061] Example 1: Screening of strains

[0062] A lactic acid-tolerant strain of Bayer yeast was screened from the fermentation process of Maotai-flavor liquor. The process is as follows:

[0063] A sample of fermented mash from a renowned Maotai-flavor liquor was collected. A 10g sample was added to 90 mL of 0.9% sterile saline solution and shaken at 30°C and 200 rpm for 1 hour. The suspension was then serially diluted 10-fold. 100 μL of the diluted solution was evenly spread onto a YPD plate. After 3 days of incubation, a single colony was selected for purification. The resulting single colony was added to sorghum juice medium adjusted to pH 3.2 with lactic acid and incubated at 30°C and 200 rpm for 3 days. After incubation, the growth rate and the levels of lactic acid, ethanol, and volatile compounds in the fermentation broth were measured.

[0064] Finally, a strain that could degrade lactic acid and produce high levels of ethanol and flavor compounds was selected, and the strain was streaked onto YPD slant medium for preservation and also stored in glycerol tubes.

[0065] Example 2: Identification of strains

[0066] (1) Colony characteristics and bacterial morphology

[0067] The activated strain obtained from Example 1 was streaked onto YPD solid medium and incubated statically at 30°C for 2 days. The morphology of the colonies on the plate was observed. Simultaneously, a single colony was picked and inoculated into sorghum juice liquid medium and observed under a transmission electron microscope after 2 days of incubation.

[0068] The results in Figure 1 show that the bacterial colonies on YPD plates are white and opaque with a slight sheen, regular, smooth, rounded edges, and a raised center. Under a transmission electron microscope, individual cells are large oval in shape, lacking flagella and hyphae.

[0069] (2) Growth characteristics of strains

[0070] 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 the OD 600 Observe its growth characteristics.

[0071] As shown in Figure 2, the strain reached stationary phase after 60 hours 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 was 40 g / L at the beginning of fermentation. The results in Table 1 indicate that the residual glucose content at the end of fermentation was 4.09 ± 0.23 g / L, and the ethanol production of the strain was 13.66 ± 0.45 g / L.

[0072] Table 1 Glucose and ethanol contents in the fermentation broth at the end of fermentation (n = 3)

[0073] Substance content (g / L) Glucose 4.09±0.23 Ethanol 13.66±0.45

[0074] (3) Physiological, biochemical and carbon source utilization experiments:

[0075] Carbon source utilization was determined by the following assay: a single colony was inoculated into sorghum juice medium and cultured at 200 rpm and 30°C for 24 h to obtain seed solution. 10 mL of seed solution was then centrifuged at 10,000 × g for 5 min, the supernatant discarded, and the cell pellet suspended in an equal volume of sterile saline. The suspension was then diluted to 10% with the same saline. −4 Then, 100uL was taken and transferred to the biolog YT microplate. The microplate was incubated in a 30℃ constant temperature incubator for 36h according to the instructions, and the OD was measured with a microplate reader. 590 OD 590 When the value is > 0.2, it indicates that the strain can utilize this carbon source.

[0076] The results in Figure 3 show that the carbon source utilization spectrum of this strain is relatively narrow. Among the carbon sources tested, it can only utilize glucose. 590 The value is 0.96. In other carbon sources, the values ​​are all less than 0.2.

[0077] (4) Molecular biological identification

[0078] A single colony of the strain was inoculated into YPD liquid medium and cultured for 48 hours. Total DNA was then extracted. PCR amplification was performed using universal ITS primers (ITS1 and ITS4). PCR amplification conditions were: 94°C for 5 minutes, 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, 30 cycles, and 72°C for 10 minutes. PCR products that passed 1% agarose gel analysis were sent to Suzhou GeneWeiZhi Biotechnology Co., Ltd. for sequencing. Sequencing results were uploaded to the National Center for Biotechnology Information (NCBI) database for BLAST comparison. The strain showed the highest homology with Zygosaccharomyces bailii and was therefore identified as Zygosaccharomyces bailii and named Zygosaccharomyces bailii LBM888.

[0079] This strain 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.

[0080] Example 3: Ethanol and flavor production capacity of strain CCTCC NO: M 2023729 under lactic acid pressure

[0081] The pH of sorghum juice medium was adjusted to 3.2 with lactic acid and used as fermentation medium. The ability of the experimental strain Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Z. bailii ZB) to produce ethanol and flavor compounds under lactic acid pressure was evaluated.

[0082] The specific steps are as follows:

[0083] A single colony of Zygosaccharomyces bailii CCTCC NO: M 2023729 was picked and inoculated into sorghum juice medium. The culture was carried out at 200 rpm and 30°C for 24 h to obtain seed liquid.

[0084] Then, 1 mL of Zygosaccharomyces bailii ZB seed solution was inoculated into 100 mL of fermentation medium supplemented with 40 g / L lactic acid (the pH of the sorghum juice medium was adjusted to 3.2 with lactic acid) and cultured at 30°C and 200 rpm for 72 h. Samples were taken every 12 h to measure the growth of the strain (OD 600 ) and lactic acid, ethanol and volatile compounds in the fermentation broth.

[0085] At the same time, a fermentation medium without lactic acid was used as a control.

[0086] The results showed that the strain Zygosaccharomyces bailii ZB was able to adapt to lactic acid stress and still grow well under lactic acid stress, as shown in Figure 4. Although the growth of Zygosaccharomyces bailii ZB under lactic acid stress was significantly lower than that without lactic acid stress in the first 48 h of fermentation (P < 0.05), there was no significant difference in biomass at the end of fermentation (P > 0.05). 600 was 3.02±0.08, while that without lactic acid stress was 3.36±0.05.

[0087] Measurement of lactic acid content in the fermentation broth indicated that the primary mechanism of lactic acid tolerance for Z. bailii ZB was its ability to degrade lactic acid (Figure 5A). The lactic acid content at the start of fermentation was 40 g / L, while it reached 34.12 ± 1.07 g / L by the end, representing a degradation of 5.88 g / L. Ethanol assays demonstrated that Z. bailii ZB not only adapted to 40 g / L lactic acid stress but also produced high levels of ethanol under this condition, reaching 11.99 ± 0.13 g / L by the end of fermentation (Figure 5B).

[0088] The results in Figure 6 show that Zygosaccharomyces bailii CCTCC NO: M 2023729 can produce a variety of flavor compounds under lactic acid stress. Among these, the flavor compounds with levels exceeding 0.1 g / L include ethyl acetate (1.02 g / L), isoamyl alcohol (1.52 g / L), phenylethanol (0.72 g / L), phenylethyl acetate (0.50 g / L), isobutanol (0.13 g / L), isoamyl lactate (0.10 g / L), and isovaleric acid (0.10 g / L). Flavor compounds with levels below 0.1 g / L include isoamyl acetate, ethyl lactate, and 1-octanol.

[0089] The above results show that Zygosaccharomyces bailii CCTCC NO: M 2023729 can adapt well to the lactic acid environment, not only reducing the lactic acid content in the system, but also producing ethanol and flavor compounds.

[0090] Example 4: Application of yeast agent in liquor brewing

[0091] From the addition of raw materials to the final round of wine removal and discarding of lees, the production of sauce-flavor liquor follows the law of seasonal succession. A production cycle takes nearly a year and involves two rounds of adding raw materials (adding raw materials) and making lees, nine rounds of steaming, eight rounds of fermentation, and seven rounds of wine removal. The production process is shown in Figure 9.

[0092] The Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 cells / mL of seed solution. Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 minutes, the supernatant discarded, and the pellet resuspended in 1000 mL of sterile water to obtain the corresponding inoculum solution. This inoculum was applied to the fourth production cycle of Maotai-flavor liquor fermentation.

[0093] The specific operations are as follows:

[0094] After distillation, airing, and mixing with koji, remove 30 kg of the mash and evenly sprinkle 1000 mL of the bacterial solution onto the 30 kg mash. Mix thoroughly. For a blank control, sprinkle 1000 mL of sterile water evenly onto the 30 kg mash.

[0095] Each treatment was performed in parallel three times. Finally, the corresponding mash was placed in a bamboo basket and placed in the fermentation pile and cellar for in-situ fermentation. The operation is shown in Figure 7.

[0096] Sampling was conducted after the completion of pile fermentation and cellar fermentation to determine the lactic acid, ethanol and volatile flavor substances in the mash.

[0097] The results show:

[0098] The results of lactic acid and ethanol determination are shown in Figure 8. It can be seen that the initial lactic acid content of the fourth round of fermentation of Maotai-flavor liquor is higher than 60 g / kg, indicating that lactic acid accumulation is an important factor restricting the yield and quality of Maotai-flavor liquor.

[0099] The addition of the experimental inoculant effectively reduced lactic acid content during fermentation, as shown in Figure 8A. At the end of the fermentation, the lactic acid content of the group without inoculant addition (cellar blank) was 73.84 ± 3.65 g / kg, while that of the group with inoculant addition (cellar ZB) was 35.45 ± 7.09 g / kg, a decrease of 51.99%.

[0100] At the end of fermentation in the cellar, the lactic acid content of the control group (cellar blank) was 85.07 ± 3.52 g / kg, while that of the microbial agent-added group (cellar ZB) was 54.51 ± 2.08 g / kg, a decrease of 35.92%.

[0101] The addition of the experimental inoculant significantly (P < 0.05) increased ethanol yield during the bulk fermentation phase (Figure 8B). At the end of bulk fermentation, the ethanol content in the control group was 6.37 ± 1.45 g / kg, while that in the inoculant-added group was 12.30 ± 1.01 g / kg, a 92.93% increase. At the end of cellar fermentation, the ethanol content in the control group was 28.00 ± 0.65 g / kg, while that in the inoculant-added group was 29.98 ± 0.34 g / kg, a 7.07% increase.

[0102] Furthermore, the addition of the experimental inoculant significantly increased the volatile flavor compounds in the mash, primarily esters, alcohols, and aromatic compounds, as shown in Table 2. Significantly increased esters included ethyl acetate, ethyl propionate, isoamyl acetate, ethyl isobutyrate, furyl acetate, ethyl phenylacetate, and phenylethyl acetate. Significantly increased alcohols included isobutanol, phenylethyl alcohol, isopentanol, and 2-heptanol. Significantly increased aromatic compounds included 2-ethyl-6-methylpyrazine, 2,6-diethylpyrazine, and tetramethylpyrazine.

[0103] Table 2 Effects of adding CCTCC NO: M 2023729 inoculant on volatile flavor compounds during the fermentation of Maotai-flavor liquor

[0104] Flavor compounds Initial entry Blank entry ZB exit Blank exit 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 ± 0.000.00 ± 0.000.00 ± 0.000.00 ± 0.000.22 ± 0.01 Isoamyl acetate 0.09 ± 0.02 0.21 ± 0.04 0.30 ± 0.04 0.55 ± 0.22 0.72 ± 0.23Ethyl isobutyrate0.00 ± 0.000.00 ± 0.000.00 ± 0.000.00 ± 0.000.22 ± 0.05Methyl furanoate0.00± 0.000.06± 0.010.20± 0.000.10± 0.000.45± 0.023-Methylbutyl 2-methoxyacetate0.03± 0.000.19± 0.000.21± 0.000.56± 0.041.21± 0.09Methyl furanoate0.00± 0.000.00± 0.000.14± 0.000.05± 0.000.24± 0.00Ethyl phenylacetate0.14± 0.021.67± 0.101.92± 0.056.08± 0.097.22± 0.30Diethyl glutarate0.00± 0.000.00± 0.000.00± 0.000.09± 0.000.14± 0.00Butyl lactate0.00 ± 0.000.00 ± 0.000.03 ± 0.000.00 ± 0.000.12 ± 0.12Ethyl 3-phenylpropionate0.00± 0.000.05± 0.000.12± 0.000.11± 0.000.55± 0.00Ethyl palmitate0.40± 0.031.81± 0.092.83± 0.105.25± 0.216.55± 0.22Hexadecenoic acid ethyl ester0.00± 0.000.08± 0.000.10± 0.000.15± 0.010.26± 0.00Phenylethyl acetate0.09± 0.000.63± 0.021.33± 0.100.91± 0.072.44± 0.11Linoleic acid ethyl ester0.20± 0.050.43± 0.020.74± 0.061.19± 0.091.54± 0.05Alcohols (mg / kg)Isobutanol0.00 ± 0.000.05± 0.000.30± 0.010.10± 0.010.31± 0.03Phenylethanol2.98 ± 0.095.53 ± 2.7311.06 ± 1.407.97 ± 2.4529.23 ± 2.87Isoamyl alcohol0.98 ± 0.041.09 ± 0.323.82 ± 0.471.98 ± 0.584.60 ± 1.092-Heptanol0.00 ± 0.000.00 ± 0.000.10 ± 0.010.00 ± 0.000.13 ± 0.01Aromatics (mg / kg)2-Ethyl-6-methylpyrazine0.00 ± 0.000.00 ± 0.000.06 ± 0.010.04 ± 0.010.19 ± 0.002,6-Diethylpyrazine0.00 ± 0.000.00 ± 0.000.04 ± 0.000.09 ± 0.020.20 ± 0.01Tetramethylpyrazine0.07 ± 0.000.17 ± 0.090.51 ± 0.061.01 ± 0.081.51 ± 0.02.

[0105] Example 5: Application of yeast agent in liquor brewing

[0106] The specific steps are as follows:

[0107] The Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0108] (1) Daqu Fenxiang Liquor:

[0109] The prepared inoculant liquid is mixed with the steamed, cooled grains, mixed evenly, and then fermented with the mash. The inoculant liquid accounts for 10-20% of the total grain weight (by mass). The fermentation is then added to the vat. The mixture is then steamed and roasted to produce the Daqu-flavored Baijiu (for detailed Daqu production technology, refer to the paper: Research Progress on Brewing Microorganisms and Flavoring Substances in Light-Fragrance Baijiu).

[0110] (2) Xiaoqu Fenxiang Liquor:

[0111] The prepared inoculant solution was mixed with the steamed and cooled grains. After saccharification for one day, the mixture was mixed evenly with the grains and placed in a tank truck for fermentation for 14 days. The solid bacteria dosage was 1% of the total grain weight. The mixture was steamed and roasted to produce a Xiaoqu-flavored baijiu (for detailed Xiaoqu production technology, see the paper "Metabolism Characteristics of Microbial Communities Synthesizing Higher Alcohols During the Fermentation Process of Xiaoqu-flavored Baijiu").

[0112] (3) Laobaigan-flavored liquor

[0113] The steamed and cooled grains are then mixed with the remaining ingredients, and the mash is then re-steamed and cooled. The prepared inoculant solution is then added to the mash, with the amount of solution added accounting for 25% of the total weight of the grains. The mixture is then fermented in a vat for 40 days. The mixture is then steamed and co-roasted to produce Lao Baigan-flavored liquor. (For the specific production process of Lao Baigan-flavored liquor, please refer to the paper: Optimization of the Formula for Multi-grain Lao Baigan-flavored Liquor.)

[0114] The results showed that the bacterial agent of the present invention can increase the flavor substances of liquor.

[0115]

[0116] Example 6: Application of yeast agent in yellow rice wine (cooking wine)

[0117] (1) Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0118] (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℃ for later use. Add 2‰ saccharifying enzyme and 1‰ liquefying enzyme, stir evenly, and place in a water bath at 60℃ for saccharification for more than 4 hours. Stir every 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℃). Pour it into a bottle, add sealing film, and sterilize it in a sterilizer at 121℃ for 20 minutes. Remove and cool it for later use. Transfer the cultured bacterial agent liquid to the rice saccharification liquid at an inoculation ratio of 5% (v / v), and then culture it at a constant temperature for 36-48 hours before it can be used for feeding.

[0119] (3) Yellow rice wine fermentation: the fermentation materials were prepared according to the yellow rice wine brewing ingredients: 1000 g glutinous rice, 1500 g cooked rice, 1100 mL brewing water, 110 g Daqu, and 100 mL yeast;

[0120] The total fermentation time is approximately 20 days. During the fermentation process, pay attention to harrowing. Maintain the temperature of the pre-fermentation below 33°C, and the post-fermentation at approximately 15±2°C. The pre-fermentation temperature is 28°C, and the fermentation lasts for 5 days, with harrowing twice daily. The post-fermentation temperature is 15°C, and the fermentation lasts for 15 days, with harrowing every 5 days.

[0121] The results showed that the bacterial agent of the present invention can increase the flavor substances of yellow rice wine (cooking wine).

[0122] Example 7: Application of yeast agent in soy sauce brewing

[0123] (1) Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0124] (2) Select plump and clean soybeans, wash them, and soak them in water for 5-10 hours. After the beans have completely absorbed water and expanded, drain them and steam them under pressure. After the hot beans are taken out of the pot, cool them to about 70-80℃, add 30-40% flour (based on the mass of soybeans), stir them evenly, continue to cool them to 30-40℃, then add 2%-5% (v / v) of bacterial agent liquid, stir them evenly, and keep the product temperature at about 28-30℃ for fermentation. During the fermentation, due to the heat generated by microbial metabolism, it is necessary to continuously stir the koji and ventilate to cool it down. After 72-96 hours, soy sauce koji is obtained. Add salt water to the prepared soy sauce koji at a ratio of 1:1.5 (m / v), stir them evenly and make mash. Ferment for 3-6 months, stirring the mash every 15 days. After the fermentation is completed, press the soy sauce mash, filter to remove impurities and sterilize it to obtain the finished soy sauce.

[0125] The results showed that the bacterial agent of the present invention can increase the flavor substances of soy sauce.

[0126] Example 8: Application of yeast agent in vinegar brewing

[0127] (1) Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0128] (2) Material selection and processing: Select 100 kg of high-quality glutinous rice, add 200 kg of water and soak the rice for 15-24 hours, then rinse the rice until there is no white pulp, drain it, steam it, rinse it with water and cool it to 25-30℃;

[0129] Mix the ingredients and put them into the vat: before putting the glutinous rice into the vat, mix 1.5-2 kg of yeast into the rice wine, and saccharify at low temperature for 72-96 hours;

[0130] Alcohol fermentation: After saccharification, add 30 kg of water and 6 kg of bacterial agent solution, and ferment at 28°C for 144-168 hours to obtain mature mash;

[0131] Making mash: put 150 kg of bran into the fermentation tank and spread it flat. Pour the fermented mash into the tank and stir it evenly. Take 5 kg of rice husk and spread it evenly on the upper layer of the tank. Then take 5 kg of fermented vinegar mash, stir it evenly, cover it with 5 kg of rice husk, and spread it evenly to complete the mash making.

[0132] Acetic acid fermentation: After every 24 hours of fermentation, turn the mash, adding rice husks to keep it warm and moist. No more rice husks will be added from the 11th day of fermentation. Turn the mash to cool it down. After 18-20 days, when the acidity no longer increases, add 4 kg of salt and seal it for 30-45 days.

[0133] Drizzling vinegar and frying vinegar: Take the vinegar mash that has finished aging, add fried beige in proportion, soak for several hours, and use the rinsing method to circulate the soaking. Add sugar to the obtained vinegar juice and fry the vinegar after clarification. After the product temperature drops to 75-80℃, seal and store it in cans.

[0134] The results showed that the bacterial agent of the present invention can increase the flavor substances of vinegar.

[0135] Example 9: Application of yeast agent in sake brewing

[0136] The Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0137] (1) 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 should be similar to that of brown rice, and the germ and groove of the raw rice should be completely removed;

[0138] (2) Rice soaking: 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;

[0139] (3) Steamed rice: The rice grains are distinct, hard on the outside and soft on the inside, with no white core, loose but not mushy, cooked but not mushy, uniform and consistent, and the glutinous rice yield is generally around 140%-150%;

[0140] (4) Dropping: When dropping, the temperature of the cooked rice is cooled to 25-30℃, including 100 kg of cooked rice, 140 kg of cooked rice, 20 kg of rice koji, 20 kg of bacterial liquid, 3 kg of yeast, and 120 L of water.

[0141] (5) Fermentation: The initial fermentation temperature is controlled at 28-30°C for 3 days, and then fermented at around 20°C for 22 days. The total fermentation time is 25 days.

[0142] The results showed that the bacterial agent of the present invention can increase the flavor substances of sake.

[0143] Example 10: Application of yeast agent in liquor brewing

[0144] The Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0145] (1) 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℃) for 24-32h, and rice bran (20-35% of the total weight of grain) is steamed at 90-100℃ for 30-40min, and then cooled for later use;

[0146] (2) Initial steaming: Drain the surface moisture of soaked corn, sorghum, wheat, etc. and steam the raw grains for 30-60 minutes;

[0147] (3) Stewing: After the initial steaming, add warm water at 40-50℃, with the water level at least 15 cm above the grain surface, and stew the grain for 10-30 minutes, then drain the water;

[0148] (4) Re-steaming: Add the rice and glutinous rice moistened with water in step (1) to the raw grains after initial steaming, mix thoroughly, and then put them into the steaming pot for re-steaming for 60-70 minutes;

[0149] (5) Cooling and fermentation: Cool the re-steamed mash to 30-35°C, add the rice bran treated in step (1) and 18-28% of liquor koji, add 5%-10% of microbial agent solution (calculated according to the weight of grain), stir evenly and put into the cellar for fermentation;

[0150] (6) Cellar sealing and fermentation: After the mash is placed in the cellar, it is covered with a lid at least 40 cm above the ground and sealed with cellar mud to a thickness of 15-20 cm. The fermentation is continued for 45-70 days.

[0151] (7) Take the wine out of the cellar: Take the fermented mash out of the cellar and use the steamer to distill the wine. The flow rate is 1.5-2.5kg / min and the flow temperature is 25-30℃.

[0152] The liquor produced by the above process has a liquor yield of 28%-33% and a high-quality rate of 88%-91%. The liquor is colorless or slightly yellow and transparent, with prominent ester aroma and burnt aroma. It tastes clean and refreshing, sweet and smooth, with a dense multi-grain puffed aroma and a natural fusion of multiple aromas. The liquor has a mellow body and has the typical style of being salty, elegant and mellow. The resulting Taorong-type liquor reaches the first-class liquor standard.

[0153] Example 11: Application of yeast agent in sweet rice wine

[0154] The Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0155] Rice is weighed in appropriate proportions and soaked at room temperature for approximately 8-12 hours until fully absorbed. The rice is then drained and steamed at normal pressure for approximately 30-40 minutes until no white core remains. Once cooked, the rice is poured with cold water until the temperature reaches approximately 25-28°C. The rice is then transferred to a fermentation tank, where 0.4%-0.8% (by weight) of a microbial inoculant solution and 0.5%-1.5% (by weight) of brewer's yeast are added. The mixture is stirred thoroughly, and drinking water (1-1.5 times the weight of the raw rice) is added. The fermentation is then carried out at a constant temperature of 28-30°C for 48-72 hours. The resulting sweet rice wine has a precision of 2-4% (v / v), a harmonious and rich aroma, a uniform texture, and a pleasant sweet and sour taste.

[0156] The results showed that the bacterial agent of the present invention can increase the flavor substances of sweet rice wine.

[0157] Example 12: Application of yeast agent in rice wine

[0158] The Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0159] Rice is weighed in appropriate proportions and soaked at room temperature for approximately 8-12 hours until fully absorbed, reaching a point where it is easily crushed by hand and free of hard cores. The rice is then drained and steamed at normal pressure for approximately 30-40 minutes until no white core remains. Once cooked, the rice is poured with cold water to a temperature of approximately 25-28°C. A 5%-10% (by weight) bacterial inoculum solution is then added for saccharification. After saccharification, additional rice is added and inoculated with 5%-10% (by weight) brewer's yeast. The mixture is stirred thoroughly, and drinking water (1-1.5 times the original weight of the rice) is added. The rice is fermented at 28-30°C for 3-5 days, then the temperature is lowered to 10-15°C for 10-15 days. After fermentation, the rice is pressed and filtered to produce rice wine. This wine, with an alcohol content of 5-10% (v / v), boasts a rich, fruity, and harmonious aroma and a uniform texture.

[0160] The results showed that the bacterial agent of the present invention can increase the flavor substances of rice wine.

[0161] Example 13: Application of yeast in fermented dairy products

[0162] The Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0163] It can be used to make lactobacillus milk beverages. The specific preparation process is as follows:

[0164] The raw skim milk was sterilized at 95℃ for 20 min, then cooled to 4℃, and then the bacterial solution was added to make the bacterial concentration reach 106 CFU / mL or more, and stored at 4°C to obtain a milk beverage.

[0165] The results showed that the bacterial agent of the present invention can increase the flavor substances of milk beverages.

[0166] Example 14: Application of yeast in fermented dairy products

[0167] The Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0168] It can be used to make soy milk. The specific preparation process is as follows:

[0169] Soak soybeans in soft water three times the volume of the original soybeans, soak at 80°C for 1-2 hours, and then remove the soybean skins. Next, drain the soaking water, add boiling water to grind the pulp, and keep it warm at a temperature above 80°C for 10-15 minutes. Filter the slurry with a 150-mesh filter membrane and centrifuge. The centrifuge liquid obtained is crude soy milk, which is then heated to 140-150°C. The hot crude soy milk is then quickly introduced into a vacuum cooling chamber for vacuuming. The odorous substances in the crude soy milk are quickly discharged along with the water vapor. After vacuum degassing, the temperature is lowered to about 37°C, and then the bacterial solution is added to make the concentration reach 10 6 CFU / mL or more, and stored at 4°C to obtain soy milk.

[0170] The results showed that the bacterial agent of the present invention can increase the flavor substances of soy milk.

[0171] Example 15: Application of yeast in fermented fruit and vegetable beverages

[0172] The Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0173] It can be used to make fruit and vegetable beverages. The specific preparation process is as follows:

[0174] Fresh vegetables (such as one or more of cucumber, carrot, beet, celery or cabbage) are 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 the bacterial agent of the present invention is added to the juice to make the concentration reach 10 6 CFU / mL or more, and store in a refrigerator at 4°C to obtain a fruit and vegetable beverage.

[0175] The results showed that the bacterial agent of the present invention can increase the flavor substances of fruit and vegetable beverages.

[0176] Example 16: Application of yeast in whiskey

[0177] The main steps include:

[0178] The Zygosaccharomyces bailii CCTCC NO: M 2023729 (hereinafter referred to as Zygosaccharomyces bailii ZB) was inoculated into the seed culture medium and cultured at 30°C and 200 rpm for 48 h to obtain 1×10 8 Then, 10 mL of the seed solution was centrifuged at 10,000 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial agent solution.

[0179] Crushing: Mechanically crush the weighed malt to form coarse granular powder;

[0180] Saccharification: Mix the crushed malt with hot water at a temperature of 63.5°C and then saccharify to obtain wort;

[0181] Fermentation: After the wort is cooled, it is pumped into a fermentation tank and fermented with 20% (v / v) bacterial agent. The fermentation time is 56 to 60 hours.

[0182] Distillation: The fermented wine is distilled twice in a copper Holstein still; First distillation: The distillate is added to the still. When the temperature of the wine in the still reaches 80℃, it begins to boil and enters the distillation stage. The cooling water temperature is less than 18℃. 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 8-9 hours; Second distillation: The initial distillate is distilled for the second time to separate the head, heart and tail; the heart is retained and the alcohol content is reduced to 63.5% vol as component A for standby use; the head and tail are used for the next distillation, and the second distillation time is about 10-12 hours.

[0183] Aging: Aging after distillation.

[0184] Blending and filtration: Reduce the alcohol content to 40 degrees to obtain a semi-finished product, and filter to obtain the finished product.

[0185] The results showed that the bacterial agent of the present invention can increase the flavor substances of whiskey.

[0186] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Zygosaccharomyces bailii, characterized in that: The Bayer yeast was 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.

2. A microbial agent, characterized in that: Contains the bail yeast CCTCC NO: M 2023729 as claimed in claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent uses Bayer's zygosaccharomyces CCTCC NO: M 2023729 as a main microorganism.

4. The microbial agent according to claim 3, characterized in that: The viable count of Bayer's Zygosaccharomyces CCTCC NO: M 2023729 in the bacterial agent is 10 5 ~10 8 CFU / mL.

5. The microbial agent according to claim 3, characterized in that: The bacterial concentration range of Bayer's yeast CCTCC NO: M 2023729 in the bacterial agent is 10 5 ~10 8 CFU / mL.

6. The microbial agent according to claim 2, characterized in that: The microbial agent is a liquid agent or a solid agent.

7. The microbial agent according to claim 2, characterized in that: The microbial agent contains living cells of the bacillus yeast CCTCC NO: M 2023729 strain of claim 1, dry cells obtained by freeze-drying, and immobilized cells.

8. Use of the bacillus zygosaccharomyces CCTCC NO: M 2023729 according to claim 1 or a bacterial agent containing the bacillus zygosaccharomyces CCTCC NO: M 2023729 according to claim 1, characterized in that: The application is 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, and culture it at 200 rpm and 30°C for 24 hours to obtain a seed solution; then inoculate 1 mL of Bayer's yeast CCTCC NO: M 2023729 seed solution into 100 mL of fermentation culture medium added with 40 g / L of lactic acid, and culture it at 30°C and 200 rpm for 72 hours; 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, 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.

9. Use of the bacillus zygosaccharomyces CCTCC NO: M 2023729 according to claim 1 or a bacterial agent containing the bacillus zygosaccharomyces CCTCC NO: M 2023729 according to claim 1 in fermented food, characterized in that: The application is to add Bayer's Zygosaccharomyces CCTCC NO: M 2023729 or a bacterial agent containing Bayer's Zygosaccharomyces CCTCC NO: M 2023729 during the process of preparing fermented food.

10. The use according to claim 9, characterized in that: The application is: adding Bayer's zygosaccharomyces CCTCC NO: M 2023729 or a bacterial agent containing Bayer's zygosaccharomyces CCTCC NO: M 2023729 during the preparation of rice wine, soy sauce, vinegar, sake, cooking wine, sweet rice wine, rice wine, dairy products or fruit and vegetable drinks.

11. A method for producing ethanol and flavor substances under lactic acid pressure, characterized in that: The method comprises the following steps: picking a single colony of Bayer's yeast CCTCC NO: M 2023729 and inoculating it into a sorghum juice culture medium to obtain a seed solution after culture; and then inoculating the Bayer's yeast CCTCC NO: M 2023729 seed solution into a fermentation culture medium added with lactic acid for fermentation culture.

12. The method according to claim 11, characterized in that 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, adding saccharifying enzyme at 60°C, 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.

13. A method for producing ethanol and flavor substances under lactic acid pressure, characterized in that: The method comprises the following steps: picking a single colony of Bayer's yeast CCTCC NO: M 2023729 and inoculating it into a sorghum juice 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 medium supplemented with 40 g / L of lactic acid, and culturing it at 30°C and 200 rpm for 72 hours.

14. The method according to claim 13, characterized in that 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, adding saccharifying enzyme at 60°C, 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.

15. Application of a yeast agent in liquor brewing, characterized in that: The application is to inoculate Bayer's yeast CCTCC NO: M 2023729 into a seed culture medium to obtain a seed solution; then centrifuge the seed solution, discard the supernatant, and resuspend the precipitate in water to obtain a bacterial agent; The bacterial agent is applied to the fourth round of production of Maotai-flavor liquor fermentation; the specific operation is as follows: After the mash is distilled, spread out and mixed with koji, take it out, sprinkle the bacterial agent evenly on it and mix it well; finally, put the corresponding mash into bamboo baskets and put it into fermentation piles and cellars for in-situ fermentation.

16. Application of a yeast agent in liquor brewing, characterized in that: The application is to inoculate Bayer's yeast CCTCC NO: M 2023729 into a seed culture medium, and culture it at 30°C and 200 rpm for 48 hours to obtain 10 8 cells / mL of seed solution; then take 10 mL of 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; This bacterial agent liquid is applied to the fourth round of production of Maotai-flavor liquor fermentation; the specific operation is as follows: After the mash is distilled, spread and mixed with koji, 30 kg of mash is taken out, 1000 mL of bacterial liquid is evenly sprinkled into the 30 kg of mash and mixed well; finally, the corresponding mash is put into a bamboo basket and placed in a fermentation pile and cellar for in-situ fermentation. Type claim 1 here.

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