A Bayer conjugated yeast strain that is lactic acid stress resistant and produces ethanol and flavor compounds in high yields.

Abstract

This invention discloses a Bayer conjugation yeast strain that is resistant to lactic acid stress and produces ethanol and flavor compounds in high yields, and belongs to the field of microbiology. The Bayer conjugation yeast CCTCC NO:M2023729 of this invention was selected from Daqu, a Chinese jiangxiang type baijiu (Chinese liquor), and this strain not only tolerates lactic acid stress but also has the ability to decompose lactic acid. Furthermore, it can produce ethanol and various flavor substances in high yields under lactic acid stress. When this strain was prepared as a fungal agent and applied to the fermentation process of jiangxiang type baijiu, the content of ethanol and flavor substances in the jiubei (Chinese liquor) could be significantly improved.

Description

[Technical Field]

[0001] The present invention relates to a Bayer conjugated yeast that is resistant to lactic acid stress and produces ethanol and flavor compounds in high yield, and belongs to the field of microbial technology. Background technology

[0002] In the brewing process of traditional fermented foods, microorganisms are exposed to various environmental stresses such as high acidity, high ethanol concentration, and high temperature. Of these, lactic acid is a metabolite of microorganisms in many traditional fermented foods. While it gives fermented foods a mellow flavor, its low volatility means it continuously accumulates during the brewing process, increasing the acidity within the fermentation system and suppressing the growth and metabolism of functional microorganisms. For example, in the brewing process of soy sauce-flavored baijiu, which employs multi-stage repeated fermentation, lactic acid continuously accumulates in each batch due to the unique multi-stage fermentation, and its content can reach 40 g / kg. In batches with lower production volumes, the lactic acid content becomes even higher, significantly exceeding the normal concentration range in baijiu, leading to a decrease or absence of flavor compounds. [Background technology]

[0003] Lactic acid accumulation leads to problems in fermentation, such as reduced yield and flavor defects. However, due to the nature of open, naturally inoculated fermentation, these problems cannot be solved by changing the operational process. Selecting strains with superior properties and applying them to low-yield fermentation is an effective method. However, most research currently focuses on analyzing the lactic acid tolerance characteristics of functional microorganisms. Yet, fermented foods are products that emphasize sensory attributes, and flavor substances are used as quantitative evaluation indicators. Therefore, these functional microorganisms need to not only withstand the highly acidic environment during fermentation but also exhibit the ability to produce flavor substances. Consequently, in order to apply selected strains to the fermentation process, they need to possess multiple capabilities, such as lactic acid tolerance and flavor substance production ability, which presents a significant challenge in strain selection. [Overview of the Initiative]

[0004] JPEG0007900872000001.jpg75170

[0005] This invention provides the Bayer conjugation yeast (Zygosaccharomyces bailii) LBM888. This Bayer conjugation yeast (Zygosaccharomyces bailii) LBM888 was deposited with the China Typical Culture Depository Center on May 10, 2023, at Wuhan University in Wuhan City, China, with deposit number CCTCC NO:M2023729.

[0006] The aforementioned Bayer zygosity yeast (Zygosaccharomyces bailii) CCTCC NO: M2023729 has the following characteristics.

[0007] Morphological characteristics: Colonies formed on YPD plates are white and opaque, with a certain sheen, regular and smooth margins, circular in shape, and a raised center. Under a transmission electron microscope, single cells are relatively large and oval in shape, lacking flagella and hyphae.

[0008] Growth characteristics: After 60 hours of cultivation in sorghum juice medium, the plants reached a stable stage, and the OD600 value at the end of fermentation was 3.36 ± 0.05. Under stress conditions in a fermentation medium containing 40 g / L of lactic acid, the OD600 value was 3.02 ± 0.08.

[0009] Carbon source utilization characteristics: Glucose can be used, but glycerol, erythritol, xylitol, arabitol, 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, genthiobiose, D-cellobiose, and dextrin cannot be used.

[0010] Ethanol production characteristics: After 60 hours of fermentation in sorghum medium, the glucose concentration decreased from an initial 40 g / L to 4.09 ± 0.23 g / L at the end of fermentation, and the ethanol production was 13.66 ± 0.45 g / L. In a fermentation medium containing 40 g / L of lactic acid, the ethanol content at the end of fermentation reached 11.99 ± 0.13 g / L.

[0011] Lactic acid tolerance and lactic acid breakdown: It can tolerate 40 g / L of lactic acid. It grows well in a fermentation medium containing 40 g / L of lactic acid, with an OD600 value of 3.02 ± 0.08. While the lactic acid content is 40 g / L at the beginning of fermentation, 5.88 g / L of lactic acid is broken down by the end of fermentation. It can produce high-content ethanol under lactic acid stress, with an ethanol content reaching 11.99 ± 0.13 g / L at the end of fermentation.

[0012] Production of various flavor substances under lactic acid stress: Flavor compounds with a content exceeding 0.1 g / L include ethyl acetate (1.02 g / L), isoamyl alcohol (1.52 g / L), phenethyl alcohol (0.72 g / L), phenethyl acetate (0.50 g / L), isobutyl alcohol (0.13 g / L), isoamyl lactate (0.10 g / L), and isovaleric acid (0.10 g / L). Flavor compounds with a content of less than 0.1 g / L include isoamyl acetate, ethyl lactate, and 1-octanol.

[0013] Applications in baijiu brewing: It can significantly reduce lactic acid content and improve ethanol content and volatile flavor substances in the liquor.

[0014] A second object of the present invention is to provide a microbial agent containing the Bayer zygote (Zygosaccharomyces bailii) CCTCC NO: M2023729 of the present invention.

[0015] In one embodiment of the present invention, the microbial agent mainly consists of Bayer zygosaccharomyces bailii CCTCC NO:M2023729.

[0016] In one embodiment of the present invention, the number of viable cells of Bayer zygosaccharomyces bailii CCTCC NO:M2023729 in the fungal agent is 10 5 ~10 8 The concentration is CFU / mL.

[0017] In one embodiment of the present invention, the bacterial concentration range of Bayer zygosaccharomyces bailii CCTCC NO:M2023729 in the bacterial agent is 10 5 ~10 8 The concentration is CFU / mL.

[0018] In one embodiment of the present invention, the fungal agent is obtained by preparing a starter culture of Bayer zygosaccharomyces bailii CCTCC NO:M2023729 and then culturing and growing it.

[0019] In one embodiment of the present invention, the microbial agent is a liquid or solid microbial agent containing the Bayer zygote (Zygosaccharomyces bailii) CCTCC NO:M2023729 strain of the present invention.

[0020] In one embodiment of the present invention, the microbial agent contains active cellular cells of the Zygosaccharomyces bailii CCTCC NO:M2023729 strain of the present invention, dried cellular cells of the Zygosaccharomyces bailii CCTCC NO:M2023729 strain of the present invention obtained by freeze-drying, immobilized cells of the Zygosaccharomyces bailii CCTCC NO:M2023729 strain of the present invention, or the Zygosaccharomyces bailii CCTCC NO:M2023729 strain of the present invention existing in any other form.

[0021] In one embodiment of the present invention, the microbial agent further contains strains of any genus applicable to fermented foods or their production, such as Bacillus licheniformis, Saccharomyces cerevisiae, Bacillus subtilis, and the like.

[0022] In one embodiment of the present invention, the microbial agent further contains any carrier that can be used in fermented foods.

[0023] The third object of the present invention is to provide the application of the Zygosaccharomyces bailii CCTCC NO: M2023729 or the microbial agent containing the Zygosaccharomyces bailii CCTCC NO: M2023729, and the application includes one or more selected from the decomposition of lactic acid, the improvement of ethanol content, and the improvement of flavor substance content. The specific application is as follows. Take a single colony of Zygosaccharomyces bailii CCTCC NO: M2023729, inoculate it into a sorghum juice medium and culture it to obtain a seed culture solution. Then, inoculate the seed culture solution of Zygosaccharomyces bailii CCTCC NO: M2023729 into a fermentation medium added with lactic acid and perform fermentation culture. The sorghum juice medium is obtained by soaking sorghum and water in a ratio of 1:4 (M:V) overnight, adding amylase and steaming and liquefying, adding glucoamylase at 60°C for saccharification, and finally performing filtration and centrifugation to adjust the sugar degree to 5°Bx. The fermentation medium is obtained by adjusting the pH of the sorghum juice medium to 3.2 using lactic acid.

[0024] The fourth object of the present invention is to provide the application of Zygosaccharomyces bailii CCTCC NO: M2023729 or a bacterial agent containing the Zygosaccharomyces bailii CCTCC NO: M2023729 in fermented foods, and the application is to add Zygosaccharomyces bailii CCTCC NO: M2023729 or a bacterial agent containing the Zygosaccharomyces bailii CCTCC NO: M2023729 in the manufacturing process of fermented foods.

[0025] In one embodiment of the present invention, the application is applicable to the brewing of Chinese liquor

[0026] The present invention provides a method for producing ethanol and flavor substances under lactic acid stress. The method is as follows. Take a single colony of Zygosaccharomyces bailii CCTCC NO: M2023729, inoculate it into a sorghum juice medium and culture it, then obtain a seed solution, and then inoculate the seed solution of the Zygosaccharomyces bailii CCTCC NO: M2023729 into a fermentation medium added with lactic acid for fermentation culture.

[0027] In one embodiment of the present invention, the sorghum juice medium is obtained by soaking sorghum and water at a ratio of 1:4 (M:V) overnight, adding amylase and steaming and liquefying, adding glucoamylase at 60 °C for saccharification, and finally performing filtration and centrifugation to adjust the sugar content to 5 °Bx.

[0028] The present invention provides a method for producing ethanol and flavor substances under lactic acid stress. The method is as follows. Take a single colony of Zygosaccharomyces bailii CCTCC NO: M2023729, inoculate it into a sorghum juice medium, culture it under the conditions of 200 rpm and 30 °C for 24 hours to obtain a seed solution, and then inoculate 1 mL of the seed solution of the Zygosaccharomyces bailii CCTCC NO: M2023729 into 100 mL of a fermentation medium added with 40 g / L of lactic acid, and culture it under the conditions of 30 °C and 200 rpm for 72 hours.

[0029] The present invention provides an application of a yeast preparation in the brewing of baijiu, the application of which is as follows: Bayer conjugating yeast CCTCC NO:M2023729 is inoculated into a culture medium to obtain a starter solution, then the starter solution is centrifuged, the supernatant is discarded, and the precipitate is resuspended in water to obtain a yeast preparation.

[0030] The aforementioned fungal agent is applied to the fourth cycle production process of the soy sauce-flavored baijiu fermentation. The specific procedure is as follows:

[0031] After distilling the liquor, cooling it by spreading it out, and mixing it with koji, the liquor is removed, and the fungicide is evenly sprinkled onto it and mixed thoroughly. Finally, the liquor is placed in bamboo baskets and placed in fermentation mounds and cellars to carry out on-site fermentation.

[0032] The present invention provides an application of a yeast preparation in the brewing of baijiu, and the application is as follows: Bayer conjugating yeast CCTCC NO:M2023729 is inoculated into a starter culture medium and cultured at 30°C and 200 rpm for 48 hours to obtain a starter solution of 108 cells / mL. Then, 10 mL of the starter solution is taken and centrifuged at 10000 × g for 5 minutes, the supernatant is discarded, and the precipitate is resuspended in 1000 mL of sterile water to obtain the corresponding fungal solution.

[0033] The microbial solution will be applied to the fourth cycle of the fermentation process for soy sauce-flavored baijiu. The specific procedure is as follows:

[0034] After distilling the liquor, cooling it by spreading it out, and mixing it with koji, 30 kg of liquor is taken out, and 1000 mL of microbial solution is evenly sprayed onto the 30 kg of liquor and mixed evenly. Finally, the liquor is placed in bamboo baskets and placed in fermentation mounds and cellars to carry out on-site fermentation.

[0035] Beneficial Effects of the Present Invention The Bayer conjugated yeast CCTCC NO:M2023729 of the present invention was selected from koji blocks of Chinese soy sauce-flavored baijiu. This strain not only tolerates lactic acid stress but also has the ability to decompose lactic acid. Furthermore, this strain can produce ethanol and various flavor substances in high yields under lactic acid stress. When this strain was prepared as a fungal agent and applied to the fermentation production process of soy sauce-flavored baijiu, the content of ethanol and flavor substances in the liquor was significantly improved. The strain of the present invention has good practical application effects and can be applied to the actual production process of fermented foods.

[0036] Deposit of biological materials The Bayer conjugating yeast CCTCC NO:M2023729, with the taxonomic name Zygosaccharomyces bailii LBM888, was deposited with the China Center for Typical Cultures Depository (CCTCC) on May 10, 2023. The depositary is Wuhan University in Wuhan, China, and the depositary number is CCTCC NO:M2023729. [Brief explanation of the drawing]

[0037] [Figure 1A] This shows the colony morphology of Bayer's zygosylvestre (Zygosaccharomyces bailii) CCTCC NO:M2023729 on a YPD solid plate. [Figure 1B] This is the morphology of Bayer's zygosity yeast (Zygosaccharomyces bailii) CCTCC NO:M2023729 under a transmission electron microscope. [Figure 2] This is the growth curve (n=3) of the Bayer zygote yeast (Zygosaccharomyces bailii) CCTCC NO:M2023729. [Figure 3] This shows the carbon source utilization status of the Bayer zygosylvestre yeast (Zygosaccharomyces bailii) CCTCC NO: M2023729. [Figure 4]This shows the growth of Bayer's conjugated yeast (Zygosaccharomyces bailii) CCTCC NO:M2023729 under lactic acid stress. ZB-0 indicates no lactic acid added, and ZB+Lac indicates stress due to 40 g / L lactic acid added. *: P<0.05, **: P<0.01 (Tukey's test) [Figure 5A] This is lactic acid degradation in the Bayer zygote yeast (Zygosaccharomyces bailii) CCTCC NO:M2023729 under lactic acid stress. [Figure 5B] This is the ethanol production capacity of Bayer's zygosaccharomyces bailii CCTCC NO:M2023729 under lactic acid stress. [Figure 6] This represents the production of volatile flavor substances in Bayer's zygote yeast (Zygosaccharomyces bailii) CCTCC NO: M2023729 under lactic acid stress. [Figure 7] This is a diagram illustrating the application of Bayer's conjugating yeast (Zygosaccharomyces bailii) CCTCC NO: M2023729 in the fermentation process of soy sauce-flavored baijiu (Chinese liquor). [Figure 8A] This study investigates the effect of a fungal preparation containing Bayer's zygote yeast (Zygosaccharomyces bailii) CCTCC NO: M2023729 on the lactic acid content during the fermentation process of soy sauce-flavored baijiu (Chinese liquor). [Figure 8B] This study investigates the effect of a fungal preparation containing Bayer's zygote yeast (Zygosaccharomyces bailii) CCTCC NO: M2023729 on the ethanol content during the fermentation process of soy sauce-flavored baijiu (Chinese liquor). [Figure 9] This is the production process for soy sauce-flavored baijiu (Chinese liquor). [Modes for carrying out the invention]

[0038] (1) Culture medium Selection medium (YPD): Glucose 20g / L, Peptone 20g / L, Yeast extract 10g / L, Agar powder 20g / L

[0039] Incubator culture medium: Glucose 20g / L, Peptone 20g / L, Yeast extract 10g / L Sorghum medium: Sorghum and water were immersed overnight in a 1:4 (M:V) ratio, then amylase was added to liquefy the mixture by steaming, and saccharifying enzymes were added at 60°C to saccharify it. Finally, the mixture was filtered and centrifuged to adjust the sugar content to 5°Bx.

[0040] (ii) Method for detecting lactic acid content For the simulated fermentation broth, filter the broth through a 0.22 μm aqueous filter and transfer the filtrate to a vial for liquid chromatography. For the fermented sake liquor, weigh out 5 g of sake liquor, add 20 mL of ultrapure water, shake to mix, then sonicate in an ice bath for 30 minutes. After sonication, centrifuge the mixture at 4°C and 8000 × g for 5 minutes, filter the supernatant through a 0.22 μm aqueous filter, and transfer the filtrate to a vial for liquid chromatography. Liquid chromatography system: Waters 2695; Column: Bio-Rad Aminex HPX-87HIon Exclusion Column, Column temperature: 60°C; Detector: Ultraviolet detector (PDA), detection wavelength 210 nm; Mobile phase: 5 mmol / L H2SO4, Flow rate: 0.6 mL / min.

[0041] (3) Method for detecting ethanol content For the simulated fermentation broth, filter the broth through a 0.22 μm aqueous filter and transfer the filtrate to a vial for liquid chromatography. For the fermented sake liquor, weigh out 5 g of sake liquor, add 20 mL of ultrapure water, shake to mix, then sonicate in an ice bath for 30 minutes. After sonication, centrifuge the mixture at 4°C and 8000 × g for 5 minutes, filter the supernatant through a 0.22 μm aqueous filter, and transfer the filtrate to a vial for liquid chromatography. Liquid chromatography system: Waters 2695; Column: Bio-Rad Aminex HPX-87HIon Exclusion Column, Column temperature: 60°C; Detector: Differential refractive index detector (RID); Mobile phase: 5 mmol / L H2SO4, Flow rate: 0.6 mL / min.

[0042] (iv) Method for detecting volatile compounds For the simulated experiment's fermentation solution, the fermentation solution is centrifuged at 4°C and 8000×g for 5 minutes, then 8 mL of the supernatant is taken and placed in a headspace vial. 5 g of alcohol is weighed out, 20 mL of ultrapure water is added, and then 0.85% NaCl and 1% CaCl2 are added. After shaking to mix, the mixture is sonicated in an ice bath for 30 minutes; then the mixture after sonication is centrifuged at 4°C and 8000×g for 5 minutes. 8 mL of the supernatant is placed in a headspace vial (20 mL, with 3 g of NaCl added beforehand), followed by L-menthol (100 μg / mL) -1 Add 20 μL of the solution. Volatile compounds are detected using HS-SPME-GC-MS (GC6890N and MS5975) and a DB-Wax column (30 m × 0.25 mm, 0.25 μm).

[0043] Example 1: Selection of bacterial strains A strain of Bayer conjugated yeast with lactic acid tolerance was selected from the fermentation process of a soy sauce-flavored baijiu, and the process is as follows.

[0044] A sample of fermented baijiu (Chinese liquor) from a famous soy sauce-flavored baijiu was taken. 10 g of the sample was added to 90 mL of 0.9% sterile physiological saline and shaken at 30°C and 200 rpm for 1 hour. The suspension was then diluted tenfold in a stepwise manner. 100 μL of the diluted liquid was uniformly spread onto a YPD solid plate and cultured for 3 days. Single colonies were selected and purified. The resulting single colonies were inoculated into sorghum juice medium adjusted to pH 3.2 with lactic acid and cultured at 30°C and 200 rpm for 3 days. After the culture period, the viable cell count and the levels of lactic acid, ethanol, and volatile compounds in the fermentation liquid were measured.

[0045] Finally, one strain was selected that could decompose lactic acid and produce ethanol and flavor compounds in high yields. This strain was inoculated into YPD slant medium using the streak method and stored, as well as stored in glycerol stocks.

[0046] Example 2: Identification of bacterial strains (1) Colony characteristics and bacterial morphology The activated strains selected in Example 1 were inoculated into YPD solid medium using the streak method and incubated statically at 30°C for 2 days. Colony morphology on the plates was observed. Simultaneously, single colonies were inoculated into sorghum juice liquid medium and observed with a transmission electron microscope after 2 days of incubation.

[0047] As shown in Figure 1, the colony morphology of this strain on the YPD plate was white and opaque, with a certain sheen, regular and smooth margins, circular in shape, and a raised central portion. Under a transmission electron microscope, the single cells were large and oval in shape, and no flagella or hyphae were observed.

[0048] (2) Growth characteristics of the fungal strain The bacterial strains were inoculated into sorghum juice medium and cultured at 30°C and 200 rpm for 84 hours. Samples were taken every 12 hours, and the OD600 was measured to observe their growth characteristics.

[0049] As is clear from Figure 2, the strain reached a stable phase after 60 hours of incubation in sorghum medium, and the OD600 at the end of fermentation reached 3.36 ± 0.05. The glucose content in the sorghum medium at the start of fermentation was 40 g / L. As shown in Table 1, the residual glucose 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.

[0050] Table 1. Glucose and ethanol content in the fermentation liquid at the end of fermentation (n=3) JPEG0007900872000002.jpg42170

[0051] (3) Experiments on physiological biochemistry and carbon source utilization An experiment was conducted to measure the utilization of carbon sources by the bacterial strain, and the procedure was as follows: A single colony was selected and inoculated into sorghum juice medium, and cultured for 24 hours at 200 rpm and 30°C to obtain a starter culture. Next, 10 mL of the starter culture was centrifuged at 10000 × g for 5 minutes, the supernatant was discarded, the cell precipitate was suspended in an equal volume of sterile physiological saline, and then the suspension was diluted with the same physiological saline for 10 minutes. -4After dilution, 100 μL was inoculated into a biologYT microplate. Following the attached instructions, the microplate was incubated statically in a 30°C incubator for 36 hours, and the OD590 was measured using an enzyme labeling analyzer. An OD590 value greater than 0.2 indicates that the strain can utilize its carbon source.

[0052] As shown in Figure 3, the carbon source utilization spectrum of this strain was relatively narrow, and of the measured carbon sources, only glucose was available for use, with an OD590 value of 0.96. For all other carbon sources, the values ​​were less than 0.2.

[0053] (4) Molecular biological identification A single colony of the bacterial strain was inoculated into YPD liquid medium and cultured for 48 hours, after which the total DNA of the bacterium was extracted. PCR amplification was performed using ITS general-purpose primers (ITS1 and ITS4). The 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. The PCR amplification product was tested on a 1% agarose gel, and after passing the test, it was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The sequencing results were uploaded to the National Center for Biotechnology Information (NCBI) database and BLAST comparison was performed. It was revealed that the strain had the highest homology with Zygosaccharomyces bailii, and therefore it was identified as Zygosaccharomyces bailii and named Zygosaccharomyces bailii LBM888.

[0054] The strain in question was deposited with the China Center for Typical Cultures Depository on May 10, 2023, at Wuhan University in Wuhan City, China, with the deposit number CCTCC NO:M2023729.

[0055] Example 3: Production capacity of ethanol and flavor substances of strain CCTCC NO:M2023729 under lactic acid stress The pH of sorghum juice medium was adjusted to 3.2 using lactic acid, and this was used as a fermentation medium to evaluate the production capacity of ethanol and flavor compounds of the experimental strain, Bayer conjugated yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugated yeast ZB), under lactic acid stress.

[0056] The specific steps are as follows:

[0057] A single colony of Bayer's zygosylvestre (Zygosaccharomyces bailii) CCTCC NO: M2023729 was selected, inoculated into sorghum medium, and cultured for 24 hours at 200 rpm and 30°C to obtain a starter culture.

[0058] Next, 1 mL of Bayer conjugated yeast ZB starter culture was inoculated into 100 mL of fermentation medium (sorghum medium with pH adjusted to 3.2 using lactic acid) to which 40 g / L of lactic acid had been added, and the culture was incubated at 30°C and 200 rpm for 72 hours. Sampling was performed every 12 hours, and the growth rate of the strain (OD600) and the levels of lactic acid, ethanol, and volatile compounds in the fermentation liquid were measured.

[0059] At the same time, cultivation in a fermentation medium without added lactic acid was used as a control.

[0060] The results showed that the Bayer conjugate yeast ZB strain is adaptable to lactic acid stress and can grow well even under lactic acid stress (see Figure 4). During the first 48 hours of fermentation, the growth rate of Bayer conjugate yeast ZB under lactic acid stress was significantly lower than that of the system without lactic acid stress (P<0.05), but no significant difference in biomass was observed at the end of fermentation (P>0.05). At this point, the OD600 under lactic acid stress was 3.02±0.08, while it was 3.36±0.05 in the system without lactic acid stress.

[0061] Measurements of lactic acid content in the fermentation liquid revealed that the main mechanism by which Bayer conjugated yeast ZB exhibits lactic acid tolerance is its ability to decompose lactic acid in the system (Figure 5A). The lactic acid content was 40 g / L at the start of fermentation, but decreased to 34.12 ± 1.07 g / L at the end of fermentation, indicating that 5.88 g / L was decomposed. Ethanol measurements revealed that Bayer conjugated yeast ZB can not only adapt to 40 g / L of lactic acid stress, but can also produce high concentrations of ethanol under lactic acid stress, with the ethanol content reaching 11.99 ± 0.13 g / L at the end of fermentation (Figure 5B).

[0062] As shown in Figure 6, Bayer conjugated yeast CCTCC NO:M2023729 can produce a variety of flavor substances under lactic acid stress. Among these, the flavor compounds with a content exceeding 0.1 g / L were ethyl acetate (1.02 g / L), isoamyl alcohol (1.52 g / L), phenethyl alcohol (0.72 g / L), phenylethyl acetate (0.50 g / L), isobutyl alcohol (0.13 g / L), isoamyl lactate (0.10 g / L), and isovaleric acid (0.10 g / L). Flavor compounds with a content of less than 0.1 g / L included isoamyl acetate, ethyl lactate, and 1-octanol.

[0063] From these results, it was revealed that the Bayer junctioned yeast CCTCC NO:M2023729 can adapt well to a lactic acid environment, not only reducing the lactic acid content in the system but also producing ethanol and flavor compounds.

[0064] Example 4: Application of yeast preparations in baijiu brewing From the initial raw material input to the final cycle of distillation and waste disposal, the production of soy sauce-flavored baijiu follows seasonal changes, with one production cycle lasting approximately one year. It involves two raw material input cycles (initial and final), nine distillation cycles, eight fermentation cycles, and seven distillation cycles. This production process is shown in Figure 9.

[0065] Bayer conjugation yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugation yeast ZB) was inoculated into a starter culture medium and cultured at 30°C and 200 rpm for 48 hours to obtain a starter culture of 1 × 10⁸ cells / mL. Next, 10 mL of this starter culture was centrifuged at 10000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding mycelial solution. This mycelial solution was applied to the fourth cycle production process of soy sauce-flavored baijiu fermentation.

[0066] The specific steps are as follows:

[0067] After distilling the sake lees, cooling it by spreading it out, and mixing it with koji, 30 kg of sake lees was taken out, and 1000 mL of microbial solution was uniformly sprayed onto the 30 kg of sake lees and mixed uniformly. The blank control was 30 kg of sake lees uniformly sprayed with 1000 mL of sterile water.

[0068] Each process is repeated three times, and finally, the corresponding liquor is placed in bamboo baskets and then placed in fermentation mounds and cellars for on-site fermentation. The procedure is shown in Figure 7.

[0069] Sampling was performed at the end of stack fermentation and at the end of cellar fermentation, and lactic acid, ethanol, and volatile flavor substances in the liquor were measured.

[0070] The results are as follows:

[0071] The measurement results for lactic acid and ethanol are shown in Figure 8. The initial lactic acid content in the fourth cycle of fermentation of soy sauce-flavored baijiu exceeded 60 g / kg, confirming that lactic acid accumulation is an important factor limiting the yield and quality of soy sauce-flavored baijiu.

[0072] The addition of the experimental fungal agent effectively reduced the lactic acid content during the fermentation process (see Figure 8A). At the end of stacking, the lactic acid content in the group without fungal agent (empty cellar) was 73.84 ± 3.65 g / kg, while the lactic acid content in the group with fungal agent (cellar ZB) was 35.45 ± 7.09 g / kg, showing a decrease of 51.99%.

[0073] At the end of fermentation in the cellar, the lactic acid content of the control group (empty cellar) was 85.07 ± 3.52 g / kg, while the lactic acid content of the group treated with fungicide (ZB cellar) was 54.51 ± 2.08 g / kg, indicating a 35.92% decrease.

[0074] Regarding ethanol, the addition of the experimental fungal agent significantly increased ethanol production during the stacking fermentation stage (P<0.05, see Figure 8B). At the end of stacking, the ethanol content of the control group was 6.37±1.45 g / kg, while the ethanol content of the fungal agent-added group was 12.30±1.01 g / kg, showing an increase of 92.93%. At the end of cellar fermentation, the ethanol content of the control group was 28.00±0.65 g / kg, while the ethanol content of the fungal agent-added group was 29.98±0.34 g / kg, confirming an increase of 7.07%.

[0075] Furthermore, the addition of experimental microbial agents significantly increased volatile flavor substances (mainly esters, alcohols, and aromatic compounds) in the liquor (Table 2). Esters that increased significantly included ethyl acetate, ethyl propionate, isoamyl acetate, ethyl isobutyrate, furfuryl acetate, ethyl phenylacetate, and phenethyl acetate. Alcohols that increased significantly included isobutanol, phenethyl alcohol, isoamyl alcohol, and 2-heptanol. Aromatic compounds that increased significantly included 2-ethyl-6-methylpyrazine, 2,6-diethylpyrazine, and tetramethylpyrazine.

[0076] Table 2 Effects of the addition of CCTCC NO:M2023729 fungicide on volatile flavor compounds in the fermentation process of soy sauce-flavored baijiu. JPEG0007900872000003.jpg208170

[0077] Example 5: Application of yeast preparations in baijiu brewing The specific steps are as follows:

[0078] Bayer conjugating yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugating yeast ZB) was inoculated into a seed culture medium and cultured at 30°C and 200 rpm for 48 hours to obtain a seed solution of 1 × 10⁸ cells / mL. Next, 10 mL of this seed solution was centrifuged at 10000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial solution.

[0079] (1) Omagari Kiyoka type baijiu JPEG0007900872000004.jpg42170

[0080] (2) Komagaki Kiyoka type baijiu JPEG0007900872000005.jpg44170

[0081] (3) Old Bai Dangxiang Baijiu: JPEG0007900872000006.jpg51170

[0082] These results demonstrate that the fungal agent of the present invention increases flavor substances in baijiu (Chinese liquor).

[0083] Example 6: Application of yeast preparations in yellow wine (cooking wine) (1) Bayer conjugating yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugating yeast ZB) was inoculated into a seed culture medium and cultured at 30°C and 200 rpm for 48 hours to obtain a seed solution of 1 × 10⁸ cells / mL. Next, 10 mL of this seed solution was centrifuged at 10000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial solution.

[0084] (2) Preparation of the yeast starter: First, the saccharification solution was prepared. Glutinous rice was placed in a rice soaking tank and soaked for 30 minutes. After discarding the rice water, it was steamed for 30-40 minutes. The steamed rice was cooled to 60°C and set aside. 2‰ of saccharifying enzyme and 1‰ of liquefying enzyme were added and mixed uniformly. Then, the mixture was placed in a water bath pot and saccharified at a constant temperature of 60°C for at least 4 hours, stirring every hour to ensure uniform saccharification. The saccharification solution was filtered through gauze to obtain a sugar solution, which was then diluted with water to 13±0.5°Bx (20°C). The solution was placed in a bottle, sealed with a sealing film, sterilized in a sterilization pot at 121°C for 20 minutes, removed, cooled, and ready for use. The cultured bacterial solution was inoculated into the rice koji saccharification solution at a 5% (v / v) inoculation ratio and further cultured at a constant temperature for 36-48 hours to make it suitable for use in the fermentation process.

[0085] (3) Fermentation of yellow wine: The ingredients were added according to the blending ratio for yellow wine brewing. 1000g glutinous rice, 1500g cooked rice, 1100mL brewing water, 110g oatmeal, 100mL yeast starter.

[0086] The total fermentation time was approximately 20 days. During the fermentation process, care was taken to avoid opening the bags, and the temperature for pre-fermentation was controlled to below 33°C, while the temperature for post-fermentation was controlled to approximately 15±2°C. The temperature for pre-fermentation was 28°C, the total fermentation period was 5 days, and the bags were opened twice daily. The temperature for post-fermentation was 15°C, and fermentation continued for 15 days, with the bags being opened once every 5 days.

[0087] These results demonstrate that the fungal agent of the present invention increases the flavor substances in yellow wine (cooking wine).

[0088] Example 7: Application of yeast preparations in soy sauce brewing (1) Bayer conjugating yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugating yeast ZB) was inoculated into a seed culture medium and cultured at 30°C and 200 rpm for 48 hours to obtain a seed solution of 1 × 10⁸ cells / mL. Next, 10 mL of this seed solution was centrifuged at 10000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial solution.

[0089] (2) Clean soybeans with full grains were selected, washed, and soaked in water for 5-10 hours. After the beans had absorbed enough water and swelled, the water was drained and the mixture was steamed under pressure. The hot beans were removed from the pot and cooled to 70-80°C. Then, 30-40% of the soybean mass in wheat flour was added and mixed uniformly, and the mixture was cooled further to 30-40°C. After that, 2-5% (v / v) of a fungicide solution was added and thoroughly mixed uniformly, and the mixture was fermented while maintaining the temperature at around 28-30°C. During fermentation, heat is generated due to the metabolism of microorganisms, so it is necessary to continuously stir the koji and ventilate to cool it down, and soy sauce koji was obtained after 72-96 hours. Saltwater was added to the manufactured soy sauce koji in a ratio of 1:1.5 (m / v) and thoroughly mixed uniformly to make baijiu (a technical term used in the baijiu manufacturing process, referring to the "process of preparing baijiu"). The process involved fermentation for 3 to 6 months, with the mixture being stirred once every 15 days. After fermentation was complete, the soy sauce syrup was pressed, filtered to remove impurities, and sterilized to obtain the finished soy sauce.

[0090] These results demonstrate that the fungal agent of the present invention increases flavor substances in soy sauce.

[0091] Example 8: Application of yeast preparations in vinegar brewing (1) Bayer conjugating yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugating yeast ZB) was inoculated into a seed culture medium and cultured at 30°C and 200 rpm for 48 hours to obtain a seed solution of 1 × 10⁸ cells / mL. Next, 10 mL of this seed solution was centrifuged at 10000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial solution.

[0092] (2) Selection process: Select 100 kg of high-quality glutinous rice, add 200 kg of water and soak for 15 to 24 hours, then wash the rice with water until no more cloudy starch liquid runs out, drain the water, steam, sprinkle with water and cool to 25 to 30°C.

[0093] Mixing ingredients into the container: Before putting the glutinous rice into the container, first add 1.5-2 kg of fermented rice liquor and mix, then saccharify at a low temperature for 72-96 hours.

[0094] Alcoholic fermentation: After saccharification was complete, 30 kg of water and 6 kg of mycelial solution were added, and fermentation was carried out at 28°C for 144 to 168 hours to obtain mature alcoholic beverage.

[0095] Pei production: 150 kg of wheat gluten was placed in the fermentation pond and spread evenly. The matured fermented rice liquor was then added to the pond and mixed uniformly. 5 kg of rice husks was taken and spread evenly on the top layer of the pond. Another 5 kg of matured vinegar was taken and mixed evenly, and then 5 kg of rice husks was placed over it and spread evenly to complete the pei production.

[0096] Acetic acid fermentation: The mixture was stirred every 24 hours during fermentation, and after each stirring, rice hulls were added to maintain temperature and humidity. From the 11th day of fermentation, the addition of rice hulls was stopped, and the mixture was stirred to cool the temperature. After 18-20 days, when it was confirmed that the rise in acidity had stopped, 4 kg of salt was added, and the mixture was sealed for 30-45 days.

[0097] Rinse and boiled vinegar: After the aging of the vinegar is complete, the color of roasted rice is added in proportion to the vinegar, and after soaking for several hours, a multi-stage spraying method is used to circulate and drain the vinegar, sugar is added to the resulting vinegar liquid and mixed, and after clarification, boiled vinegar is made, and once the temperature has cooled to 75-80°C it is canned and sealed for storage.

[0098] These results demonstrate that the fungicide of the present invention increases flavor substances in vinegar.

[0099] Example 9: Application of yeast preparations in sake brewing Bayer conjugating yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugating yeast ZB) was inoculated into a seed culture medium and cultured at 30°C and 200 rpm for 48 hours to obtain a seed solution of 1 × 10⁸ cells / mL. Next, 10 mL of this seed solution was centrifuged at 10000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1000 mL of sterile water to obtain the corresponding bacterial solution.

[0100] (1) Rice milling: Using a rice milling machine, 20-30% of the mass of the raw rice was removed. The number of broken rice grains was kept to a minimum, and the polished rice resembled the shape of brown rice, with the germ and grooves of the raw rice completely removed.

[0101] (2) Soaking the rice: The temperature of the soaking water was set to room temperature, the water absorption rate to be approximately 28-30%, and the soaking time was set to 9-12 hours.

[0102] (3) Steamed rice: The rice grains were distinct, hard on the outside and soft on the inside, with no white core inside, fluffy and not gelatinized, well-cooked, retaining the shape of the rice grains, uniform and consistent, and the cooking rate of the glutinous rice was generally around 140-150%.

[0103] (4) Raw material addition: When adding the raw materials, the rice was cooled to 25-30°C, and 100 kg of rice, 140 kg of cooked rice, rice koji, 20 kg of microbial solution, 3 kg of sake starter, and 120 L of water were added to it.

[0104] (5) Fermentation: The initial fermentation temperature was controlled to 28-30°C for 3 days, followed by fermentation at approximately 20°C for 22 days, for a total fermentation time of 25 days.

[0105] These results demonstrate that the microbial agent of the present invention increases flavor substances in sake.

[0106] Example 10: Application of yeast preparations in baijiu brewing Bayer conjugating yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugating yeast ZB) was inoculated into a starter culture medium and cultured for 48 hours under conditions of 30°C and 200 rpm, resulting in 1 × 10⁶ 8 A cell / mL inoculation solution was obtained. Next, 10 mL of this inoculation solution was centrifuged at 10,000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1,000 mL of sterile water to obtain the corresponding bacterial solution.

[0107] (1) Sorghum (40-50%), wheat (6-10%), corn (5-10%), glutinous rice (10-12%), and rice (15-20%) were weighed in the specified proportions and soaked in hot water (55-65°C) for 24-32 hours. Rice hulls (equivalent to 20-35% of the total weight of grains) were steamed at 90-100°C for 30-40 minutes, then spread out to cool and stored.

[0108] (2) Initial steaming: After draining the surface moisture from the soaked corn, sorghum, wheat, etc., the raw grains were steamed for 30 to 60 minutes.

[0109] (3) Soaking in hot water: After the initial steaming is complete, add 40-50°C warm water so that the water level is at least 15 cm higher than the surface of the grain, soak the grain for 10-30 minutes, and then drain the water.

[0110] (4) Second steaming: After the initial steaming is complete, the rice and glutinous rice moistened with water in step (1) are added to the raw grains, and after being thoroughly and uniformly mixed, the mixture is placed in a steaming pot and second steamed for 60 to 70 minutes.

[0111] (5) Cooling and koji addition: The re-steamed sake lees were cooled to 30-35°C, the rice husks processed in step (1) were added, 18-28% of baijiu daqu was added, and 5-10% of the fungal agent solution (by grain weight) was added, and the mixture was uniformly stirred and fermented in the cellar.

[0112] (6) Sealed cellar fermentation: After placing the sake lees in the cellar, a lid was placed over it, with the lid being at least 40 cm higher than the ground. The cellar was then sealed with cellar mud to a thickness of 15-20 cm, and fermentation was carried out for 45-70 days.

[0113] (7) Distillation of sake: The sake lees, after fermentation is complete, are removed from the cellar and distilled using a steaming pot to extract the sake. The rate of sake outflow is set to 1.5-2.5 kg / min, and the temperature of the sake outflow is set to 25-30°C.

[0114] JPEG0007900872000007.jpg44170

[0115] Example 11: Application of yeast preparations in amazake brewing Inoculate Saccharomyces bayanus CCTCC NO: M2023729 (hereinafter abbreviated as Saccharomyces bayanus ZB) into the seed culture medium and culture it at 30 °C and 200 rpm for 48 hours to obtain a seed solution of 1×10 8 cells / mL. Then, centrifuge 10 mL of this seed solution at 10000×g for 5 minutes. After discarding the supernatant, resuspend the precipitate in 1000 mL of sterile water to obtain the corresponding fungicide solution.

[0116] Weigh rice as the main raw material at a predetermined ratio, soak it at room temperature for about 8 - 12 hours to fully absorb water. Drain the water from the soaked rice grains, steam them under normal pressure for about 30 - 40 minutes until there is no white core in the center of the rice. After the rice is steamed and cooled, pour cold water over it to about 25 - 28 °C and transfer it to a fermentation tank. Add the fungicide solution at a predetermined ratio of 0.4 - 0.8% (mass ratio), inoculate 0.5 - 1.5% (mass ratio) of brewing yeast, stir evenly, add drinking water 1 - 1.5 times the mass of the raw rice, and carry out constant temperature fermentation at 28 - 30 °C for 48 - 72 hours. The obtained sake had an alcohol content of 2 - 4% (v / v), with a harmonious aroma, rich taste, uniform texture, and a sweet and sour taste in the mouth, being refreshing.

[0117] From these results, it was shown that the fungicide of the present invention increases flavor substances in sake.

[0118] Example 12: Application of Yeast Fungicide in Rice Wine Inoculate Saccharomyces bayanus CCTCC NO: M2023729 (hereinafter abbreviated as Saccharomyces bayanus ZB) into the seed culture medium and culture it at 30 °C and 200 rpm for 48 hours to obtain a seed solution of 1×10 ' 8 cells / mL. Then, centrifuge 10 mL of this seed solution at 10000×g for 5 minutes. After discarding the supernatant, resuspend the precipitate in 1000 mL of sterile water to obtain the corresponding fungicide solution.

[0119] After weighing the rice in the specified proportions, it was soaked at room temperature for 8 to 12 hours to allow it to absorb water thoroughly. After soaking, the rice should be able to be easily crushed by hand, with no hard core remaining. The water was drained from the soaked rice grains, and they were steamed under atmospheric pressure for 30 to 40 minutes to ensure there was no white core in the center of the rice. After the rice was steamed, it was heated in cooled water to about 25 to 28°C, and 5 to 10% (by mass) of a microbial solution was added in the specified proportions to carry out saccharification. After saccharification was complete, more rice was added, 5 to 10% (by mass) of brewing yeast was inoculated, and it was mixed uniformly. 1 to 1.5 times the mass of raw rice was added as drinking water, and fermentation was carried out for 3 to 5 days under conditions of 28 to 30°C. Then the temperature was lowered to a range of 10 to 15°C and fermentation was carried out for another 10 to 15 days. After fermentation was complete, the rice was pressed and filtered to obtain rice wine. The rice wine had an alcohol content of 5-10% (v / v), a rich aroma of rice and fruit, a harmonious blend of fragrances, a full-bodied flavor, and a uniform texture.

[0120] These results demonstrate that the microbial agent of the present invention increases flavor substances in rice wine.

[0121] Example 13: Application of yeast preparations in fermented dairy products Bayer conjugating yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugating yeast ZB) was inoculated into a starter culture medium and cultured for 48 hours under conditions of 30°C and 200 rpm, resulting in 1 × 10⁶ 8 A cell / mL inoculated culture was obtained. Next, 10 mL of this inoculated culture was centrifuged at 10,000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1,000 mL of sterile water to obtain the corresponding bacterial solution.

[0122] It can be used in the production of lactic acid bacteria milk beverages, and the specific preparation process is as follows.

[0123] The skim milk used as raw material is heat-sterilized at 95°C for 20 minutes, then cooled to 4°C, and a bacterial solution is added to increase the bacterial concentration to 10. 6 A milk beverage was obtained by increasing the CFU / mL level to above and storing it under refrigeration at 4°C.

[0124] These results demonstrate that the microbial agent of the present invention increases flavor substances in milk beverages.

[0125] Example 14: Application of yeast preparations in fermented dairy products Bayer conjugating yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugating yeast ZB) was inoculated into a starter culture medium and cultured for 48 hours under conditions of 30°C and 200 rpm, resulting in 1 × 10⁶ 8 A cell / mL inoculated culture was obtained. Next, 10 mL of this inoculated culture was centrifuged at 10,000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1,000 mL of sterile water to obtain the corresponding bacterial solution.

[0126] It can be used in the production of soy milk, and the specific preparation process is as follows:

[0127] Soybeans were soaked in soft water, with the water volume being three times the volume of the original soybeans. They were soaked at 80°C for 1-2 hours, and the soybean skins were removed. Next, the soaking water was drained, and boiling water was added for wet grinding. The mixture was then kept warm at a temperature of 80°C or higher for 10-15 minutes. The slurry was filtered through a 150-mesh filter membrane, then centrifuged. The resulting liquid was crude soy milk, which was then heated to 140-150°C. The hot crude soy milk was then quickly introduced into a vacuum cooling chamber and vacuumed, allowing off-flavor substances in the crude soy milk to be rapidly removed along with water vapor. After vacuum degassing, the temperature was lowered to around 37°C, and a bacterial solution was inoculated, with a concentration of 10. 6 Soy milk was obtained by increasing the CFU / mL level to above and storing it in a refrigerator at 4°C.

[0128] These results demonstrate that the microbial agent of the present invention increases flavor substances in soy milk.

[0129] Example 15: Application of yeast preparations in fermented fruit and vegetable beverages Bayer conjugating yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugating yeast ZB) was inoculated into a starter culture medium and cultured for 48 hours under conditions of 30°C and 200 rpm, resulting in 1 × 10⁶ 8A cell / mL inoculation solution was obtained. Next, 10 mL of this inoculation solution was centrifuged at 10,000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1,000 mL of sterile water to obtain the corresponding bacterial solution.

[0130] It can be used in the manufacture of fruit and vegetable beverages, and the specific preparation process is as follows:

[0131] Fresh vegetables (for example, one or more of the following: cucumber, carrot, sugar beet, celery, or cabbage) are selected, washed, and then juiced. Subsequently, high-temperature flash sterilization is performed, with the vegetables being heated at 140°C for 2 seconds, then immediately cooled to approximately 37°C. The bacterial solution of the present invention is then inoculated, and the concentration is set to 10. 6 A fruit and vegetable beverage was obtained by increasing the concentration to above CFU / mL and storing it under refrigeration at 4°C.

[0132] These results demonstrate that the fungal agent of the present invention increases flavor substances in fruit and vegetable beverages.

[0133] Example 16: Application of yeast preparations in whiskey It mainly includes the following steps.

[0134] Bayer conjugating yeast CCTCC NO:M2023729 (hereinafter abbreviated as Bayer conjugating yeast ZB) was inoculated into a starter culture medium and cultured for 48 hours under conditions of 30°C and 200 rpm, resulting in 1 × 10⁶ 8 A cell / mL inoculation solution was obtained. Next, 10 mL of this inoculation solution was centrifuged at 10,000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1,000 mL of sterile water to obtain the corresponding bacterial solution.

[0135] Crushing: The weighed malt was mechanically crushed to process it into a coarse powder.

[0136] Saccharification: Crushed malt and hot water were mixed at a water temperature of 63.5°C, and then saccharified to obtain malt juice.

[0137] Fermentation: After cooling the malt, it was pumped into a fermentation tank, and a 20% (v / v) microbial solution was added to initiate fermentation. The fermentation time was 56-60 hours.

[0138] Distillation: The fermented liquid was distilled twice in a copper Holstein still. First distillation: The distillate was added to the still, and once the temperature of the liquid in the still rose to 80°C and began to boil, the distillation stage began. The temperature of the cooling water was kept below 18°C. In the first distillation, the head and tail were not removed, the alcohol content of the initial distillate was controlled to 20-28% vol, and the distillation time for the first distillation was controlled to 8-9 hours. Second distillation: The initial distillate was distilled a second time and fractional distillation was performed to obtain the head, core, and tail. Only the core was distilled, and its alcohol content was reduced to 63.5% vol, and it was kept as component A. The head and tail were used for the next distillation, and the distillation time for the second distillation was approximately 10-12 hours.

[0139] Aging: The product was aged after distillation.

[0140] Blending and filtration: The alcohol content was reduced to 40% to obtain a semi-finished product, which was then filtered to obtain the final product.

[0141] These results demonstrate that the fungal agent of the present invention can increase the flavor substances in whiskey.

[0142] Although the present invention has already been disclosed as described above in preferred embodiments, this does not limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be defined by the claims.

Claims

1. A Bayerian zygomycetes bailii specimen, deposited on May 10, 2023, at the China Center for Typical Culture Depositary Studies, located at Wuhan University in Wuhan City, China, with deposit number CCTCC NO: M2023729.

2. A microbial agent characterized by containing the Bayer conjugating yeast CCTCC NO: M2023729 described in claim 1.

3. The microbial agent according to claim 2, characterized in that it uses Bayer conjugating yeast CCTCC NO: M2023729 as the main microorganism.

4. The number of viable Bayer zygote yeasts CCTCC NO: M2023729 in the aforementioned fungal agent is 10 ５ ~10 ８ The microbial agent according to claim 3, characterized in that it is CFU / mL.

5. The bacterial concentration range of Bayer zygomatic yeast CCTCC NO: M2023729 in the aforementioned bacterial agent is 10 ５ ~10 ８ The microbial agent according to claim 3, characterized in that it is CFU / mL.

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

7. The microbial agent according to claim 2, characterized in that it contains live cells of the Bayer junctioned yeast CCTCC NO: M2023729 strain of claim 1, dried cells obtained by freeze-drying, and immobilized cells.

8. The fermentation medium contains one or more selected from lactic acid decomposition, ethanol content improvement, and flavor substance content improvement. Specifically, a single colony of Bayer conjugated yeast CCTCC NO: M2023729 is taken and inoculated into sorghum medium, cultured for 24 hours at 200 rpm and 30°C to obtain a starter culture. Then, 1 mL of the starter culture of the Bayer conjugated yeast CCTCC NO: M2023729 is inoculated into 100 mL of fermentation medium to which 40 g / L of lactic acid has been added, and cultured for 72 hours at 30°C and 200 rpm; the sorghum medium is prepared by soaking sorghum and water in a ratio of 1:4 (M / V) overnight, adding amylase to liquefy by steaming, adding saccharifying enzyme at 60°C to saccharify, and finally filtering and centrifugation to obtain a sugar content of 5°Bx. The use of a microbial agent containing Bayer conjugated yeast CCTCC NO: M2023729 according to claim 1, characterized in that the fermentation medium is prepared by adjusting the pH of the sorghum medium to 3.2 using lactic acid, or Bayer conjugated yeast CCTCC NO: M2023729 according to claim 1.

9. The use of Bayer conjugated yeast CCTCC NO: M2023729 or a microbial agent containing Bayer conjugated yeast CCTCC NO: M2023729 in a fermented food, characterized in that the Bayer conjugated yeast CCTCC NO: M2023729 or a microbial agent containing Bayer conjugated yeast CCTCC NO: M2023729 as described in claim 1 is added during the manufacturing process of the fermented food.

10. A method for producing ethanol and flavor substances under lactic acid stress, characterized by taking a single colony of Bayer conjugated yeast CCTCC NO: M2023729, inoculating it into sorghum medium, culturing it to obtain a starter culture, and then inoculating the starter culture of Bayer conjugated yeast CCTCC NO: M2023729 into a fermentation medium to which lactic acid has been added and performing fermentation culture.

11. The method according to claim 10, characterized in that the sorghum juice medium is prepared by soaking sorghum and water in a ratio of 1:4 (M:V) overnight, adding amylase to liquefy by steaming, adding a saccharifying enzyme at 60°C to saccharify, and finally filtering and centrifuging to adjust the sugar content to 5°Bx, and the fermentation medium is prepared by adjusting the pH of the sorghum juice medium to 3.2 using lactic acid.

12. A method for producing ethanol and flavor substances under lactic acid stress, characterized by taking a single colony of Bayer conjugating yeast CCTCC NO: M2023729, inoculating it into sorghum medium, culturing it at 200 rpm and 30°C for 24 hours to obtain a starter culture, and then inoculating 1 mL of the starter culture of Bayer conjugating yeast CCTCC NO: M2023729 into 100 mL of fermentation medium to which 40 g / L of lactic acid has been added, and culturing it at 30°C and 200 rpm for 72 hours.

13. The method according to claim 12, characterized in that the sorghum juice medium is prepared by soaking sorghum and water in a ratio of 1:4 (M:V) overnight, adding amylase to liquefy by steaming, adding a saccharifying enzyme at 60°C to saccharify, and finally filtering and centrifuging to adjust the sugar content to 5°Bx, and the fermentation medium is prepared by adjusting the pH of the sorghum juice medium to 3.2 using lactic acid.

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