Application of a compound bacterium agent of Lactobacillus rhamnosus and Bacillus licheniformis for reducing acid and higher alcohols, Lactobacillus rhamnosus, and in Chinese liquor

Abstract

The present invention belongs to the technical field of microbial fermentation, and discloses the application of a compound bacterium agent of Lactobacillus rhamnosus and Bacillus licheniformis for reducing acids and higher alcohols, Lactobacillus rhamnosus, and in Baijiu. Lactobacillus rhamnosus L21 has a preservation number of: CGMCC NO. 32609, a preservation date of: November 13, 2024, and a preservation unit of: China General Microbiological Culture Collection Center. The use of the compound bacterium agent of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 can effectively reduce organic acids such as lactic acid and propionic acid in the Baijiu fermentation system, and at the same time can also reduce the content of n-propanol in the fermented grains, which is beneficial to the regulation of acidity and higher alcohols in the process of Baijiu production.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to the application of a compound bacterium agent of Lactobacillus rhamnosus and Bacillus licheniformis for reducing acid and higher alcohols, Lactobacillus rhamnosus, and in Baijiu. Background Art

[0002] High acid content is one of the characteristics of the brewing of Maotai-flavor Baijiu. The acid flavor substances mainly include organic acids such as acetic acid, lactic acid, butyric acid, caproic acid, and higher fatty acids. The multi-round fermentation process leads to the accumulation of non-volatile acids. The excessive accumulation of non-volatile acids will lead to abnormal later fermentation, further affecting the succession and growth metabolism of the microbial flora, and ultimately affecting the yield and quality of the base liquor. In addition, the high content of higher alcohols in the first-round base liquor is a common problem in the production of Maotai-flavor Baijiu. Appropriate higher alcohols can increase the aroma and sweetness of the liquor body, while excessive higher alcohols will reduce the comfort of drinking Baijiu. Therefore, controlling the content of higher alcohols in the first-round base liquor is of great significance for liquor blending.

[0003] At present, there have been studies on using functional microorganisms to control the increase in the acidity of fermented grains. For example, Pichia kudriavzevii can reduce the lactic acid concentration in fermented grains, thereby alleviating the impact of acid on Saccharomyces cerevisiae in the fermentation system (Journal of Agricultural and Food Chemistry, 2020, 68, 4903-4911, Cooperative Response of Pichia kudriavzevii and Saccharomyces cerevisiae to Lactic Acid Stress in Baijiu Fermentation). The reported patents mostly focus on regulating the problems of excessive acidity and higher alcohol content in fermented grains through microbial bacterium agents. For example, using Bacillus licheniformis to reduce the acidity and higher alcohols in the fermented grains of Maotai-flavor Baijiu (Chinese Patent CN117286064A), the acidity of the fermented grains decreased by 28%, and n-propanol decreased by 45.2%; using Candida parapsilosis to control the content of higher alcohols in Baijiu (Chinese Patent CN112322509A), the content of higher alcohols in the wine sample after fermentation with Candida parapsilosis was 69.5% of that of the wine sample fermented with the standard strain; adding Lactobacillus to reduce the content of higher alcohols in light-flavor Xiaoqu Baijiu (Chinese Patent CN115109674A), the content of higher alcohols in the fermented grains decreased by 52.2%, but the total acid increased by 2.5 times. However, so far, there are few reports on reducing the acidity and higher alcohols in the fermented grains of Maotai-flavor Baijiu simultaneously by adding a compound bacterium agent. Summary of the Invention

[0004] The object of the present invention is to overcome the deficiencies in the prior art and provide an application of a compound bacterium agent of Lactobacillus rhamnosus and Bacillus licheniformis for reducing acidity and higher alcohols, Lactobacillus rhamnosus, and in Baijiu.

[0005] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0006] A strain of Lactobacillus rhamnosus capable of reducing the acidity and higher alcohol content of fermented grains, named L21, with the taxonomic name: Lactobacillus rhamnosus, deposit number: CGMCC No. 32609, deposit date: November 13, 2024, deposit unit: General Microbiology Center of China Microbial Culture Collection Management Committee, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The application of the above-mentioned Lactobacillus rhamnosus in the brewing of Maotai-flavor Baijiu.

[0008] A compound bacterium agent of Lactobacillus rhamnosus and Bacillus licheniformis capable of reducing the acidity and higher alcohol content of fermented grains by using the above-mentioned Lactobacillus rhamnosus, the compound bacterium agent includes Lactobacillus rhamnosus L21 and Bacillus licheniformis;

[0009] Among them, the name of the Bacillus licheniformis is: XDNZ_Bl_27, selected from Chinese Patent, publication number CN117286064A, application number 202311257416.7, a strain of Bacillus licheniformis, method and application in Baijiu production capable of reducing the acidity and higher alcohol content of fermented grains.

[0010] Further, the viable cell number ratio of the above-mentioned Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 is 1:10.

[0011] Further, both the above-mentioned Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 are isolated and screened from Maotai-flavor production fermented grains.

[0012] The application of the above-mentioned compound bacterium agent in the brewing of Maotai-flavor Baijiu.

[0013] The application of the above-mentioned compound bacterium agent in the in-situ fermentation of Maotai-flavor Baijiu.

[0014] Furthermore, after the in-situ fermentation of Maotai-flavor Baijiu, the use of the composite bacterium agent of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 did not significantly change the moisture and starch concentration of the fermented grains at the two time points of the end of piling and the out of the pit, while the acidity of the fermented grains decreased significantly, from the original 1.16±0.01 mmol / 10 g of fermented grains to 0.79±0.07 mmol / 10 g of fermented grains, a decrease of 31.82%.

[0015] Acetic acid in the fermented grains decreased significantly from 9129.68±383.71 mg / kg to 7199.93±270.59 mg / kg, a decrease of 21.14%, and propionic acid decreased significantly from 119.82±1.06 mg / kg to 95.62±0.74 mg / kg, a decrease of 20.20%.

[0016] The content of n-propanol in the fermented grains decreased significantly from the original 2720.72±82.67 mg / kg to 2346.77±60.98 mg / kg, a decrease of 13.74%.

[0017] The method for in-situ fermentation of Maotai-flavor Baijiu using the above-mentioned composite bacterium agent includes the following steps:

[0018] Bacillus licheniformis XDNZ_Bl_27 was added to the fermented grains after steaming and cooling at an inoculation amount of 10 6 CFU / g, and Lactobacillus rhamnosus L21 was added at an inoculation amount of 10 5 CFU / g; after stirring evenly, it was divided into cloth bags and placed in the middle of the fermented grains pile. After 2-3 days of piling fermentation, the sample was stirred, simulating the operation of moving the pile. After sampling, it was put into a gauze bag again. As the fermented grains pile entered the cellar fermentation stage, the cellar fermentation lasted for 30 days to obtain Maotai-flavor Baijiu.

[0019] The advantages and positive effects achieved by the present invention are:

[0020] 1. The two strains of bacteria that can reduce the acidity and higher alcohol content in fermented grains in the present invention are: Lacticaseibacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27. Lacticaseibacillus rhamnosus L21 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 13, 2024. Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101, Deposit Number CGMCC NO. 32609. Bacillus licheniformis XDNZ_Bl_27 is selected from a Chinese patent, Publication Number CN117286064A, Application Number 202311257416.7, a Bacillus licheniformis strain, method and application in white liquor production that can reduce the acidity and higher alcohol content in fermented grains.

[0021] 2. The addition of a single Lacticaseibacillus rhamnosus L21 bacterial agent reduced the acidity of the fermented grains from the original 1.16 ± 0.01 mmol / 10 g to 0.87 ± 0.04 mmol / 10 g, a decrease of 25.00%. Acetic acid decreased from 9129.68 ± 383.71 mg / kg to 7633.16 ± 77.38 mg / kg, a decrease of 16.39%. Propionic acid decreased from 119.82 ± 1.06 mg / kg to 105.00 ± 2.69 mg / kg, a decrease of 12.37%. The content of n-propanol decreased significantly from the original 2720.72 ± 82.67 mg / kg to 2253.32 ± 3.07 mg / kg, a decrease of 17.18%. The use of a compound bacterial agent of Lacticaseibacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 can effectively reduce organic acids such as lactic acid and propionic acid in the white liquor fermentation system, and at the same time can also reduce the content of n-propanol in the fermented grains, which is beneficial to the regulation of acidity and higher alcohols in the white liquor production process.

[0022] 3. The use of the compound bacterium agent of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 significantly reduced the acidity of the fermented grains, from the original 1.16±0.01 mmol / 10 g to 0.79±0.07 mmol / 10 g, a decrease of 31.82%. Acetic acid in the fermented grains significantly decreased from 9129.68±383.71 mg / kg to 7199.93±270.59 mg / kg, a decrease of 21.14%, and propionic acid significantly decreased from 119.82±1.06 mg / kg to 95.62±0.74 mg / kg, a decrease of 20.20%. The compound bacterium agent showed a more excellent effect than the single bacterium agent in reducing the content of propionic acid. The content of n-propanol in the fermented grains significantly decreased from the original 2720.72±82.67 mg / kg to 2346.77±60.98 mg / kg, a decrease of 13.74%. Description of the Drawings

[0023] Figure 1 It is the colony morphology diagram of L21 in the present invention;

[0024] Figure 2 It is the cell morphology diagram of L21 in the present invention;

[0025] Figure 3 It is the diagram of the content of propionic acid in the fermented grains at the end of pit fermentation in the present invention, where * indicates that the significant difference is 0.01 < P < 0.05; ** indicates that the significant difference is 0.001 < P < 0.01.

[0026] A strain of Lactobacillus rhamnosus that can reduce the acidity and higher alcohol content of fermented grains, named L21, with the taxonomic name: Lactobacillus rhamnosus, deposit number: CGMCC No. 32609, deposit date: November 13, 2024, deposit unit: China General Microbiological Culture Collection Center, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Embodiments

[0027] The following further illustrates the present invention in combination with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0028] All kinds of experimental operations involved in the specific embodiments are conventional techniques in the art. For parts not specifically noted in this article, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literature, or relevant specifications, manuals, etc. before the application date of the present invention for implementation.

[0029] A Lactobacillus rhamnosus strain capable of reducing the acidity and higher alcohol content of fermented grains, named L21, with the taxonomic name: Lactobacillus rhamnosus, deposit number: CGMCC No. 32609, deposit date: November 13, 2024, depositary institution: China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0030] Application of the above-mentioned Lactobacillus rhamnosus in the brewing of Maotai-flavor Baijiu.

[0031] A compound bacterium agent of Lactobacillus rhamnosus and Bacillus licheniformis capable of reducing the acidity and higher alcohol content of fermented grains by using the above-mentioned Lactobacillus rhamnosus, the compound bacterium agent comprising Lactobacillus rhamnosus L21 and Bacillus licheniformis;

[0032] Among them, the name of the Bacillus licheniformis is: XDNZ_Bl_27, selected from Chinese Patent, publication number CN117286064A, application number 202311257416.7, a Bacillus licheniformis strain, method and application in Baijiu production capable of reducing the acidity and higher alcohol content of fermented grains.

[0033] Preferably, the viable cell number ratio of the Lactobacillus rhamnosus L21 and the Bacillus licheniformis XDNZ_Bl_27 is 1:10.

[0034] Preferably, both the Lactobacillus rhamnosus L21 and the Bacillus licheniformis XDNZ_Bl_27 are isolated and screened from the fermented grains in Maotai-flavor Baijiu production.

[0035] Application of the above-mentioned compound bacterium agent in the brewing of Maotai-flavor Baijiu.

[0036] Application of the above-mentioned compound bacterium agent in the in-situ fermentation of Maotai-flavor Baijiu.

[0037] Preferably, after the in-situ fermentation of Maotai-flavor Baijiu with the compound bacterium agent of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27, the use of the compound bacterium agent of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 did not significantly change the moisture and starch concentration of the fermented grains at the two time points of the end of piling and the out of cellar, while the acidity of the fermented grains decreased significantly, from the original 1.16 ± 0.01 mmol / 10 g significantly reduced to 0.79 ± 0.07 mmol / 10 g, a decrease of 31.82%.

[0038] The acetic acid in the fermented grains decreased significantly from 9129.68±383.71 mg / kg to 7199.93±270.59 mg / kg, a decrease of 21.14%. The propionic acid decreased significantly from 119.82±1.06 mg / kg to 95.62±0.74 mg / kg, a decrease of 20.20%.

[0039] The content of n-propanol in the fermented grains decreased significantly from the original 2720.72±82.67 mg / kg to 2346.77±60.98 mg / kg, a decrease of 13.74%.

[0040] A method for in-situ fermentation of Maotai-flavor Baijiu using the above-mentioned compound bacterial agent includes the following steps:

[0041] Bacillus licheniformis XDNZ_Bl_27 was added to the fermented grains after steaming and cooling at an inoculation amount of 10 6 CFU / g, and Lactobacillus rhamnosus L21 was added at an inoculation amount of 10 5 CFU / g. After thorough mixing, it was divided into cloth bags and placed in the middle of the fermented grains pile. After 2-3 days of stacking fermentation, the sample was stirred, simulating the operation of shifting the pile. After sampling, it was put into a gauze bag again and entered the cellar fermentation stage with the fermented grains pile. The cellar fermentation lasted for 30 days to obtain Maotai-flavor Baijiu.

[0042] Specifically, the related preparation and detection are as follows:

[0043] Lactobacillus rhamnosus L21 involved in the following examples was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms.

[0044] Example 1: Screening of Lactobacillus rhamnosus L21

[0045] Take 10.0 g of Daqu from a winery in Kuancheng Manchu Autonomous County, Chengde City, Hebei Province and put it into a 250 mL Erlenmeyer flask containing 100 mL of MRS liquid medium. Incubate at 50℃ for 24 h, then perform gradient dilution and spread it on an MRS plate containing 1.5% (w / v) CaCO3. Incubate statically at 37℃ for 48 h. Select the strains with clear zones and purify them 2-3 times by the streak plate method to obtain pure colonies. The pure colonies were preserved in a glycerol cryotube at -80℃. The tolerance of the strains was detected by the spot plate assay. The test strains were picked into 5 mL of MRS liquid medium and incubated at 37℃ for 12-16 h. The OD of the bacterial liquid 600Adjust to 1.0. Pipette 4 μL of the bacterial solution and drop it onto MRS plates containing different concentrations of ethanol (8%, 10%, 12% by volume), and incubate the plates upside down at 37 °C for 2 days. Pipette 4 μL of the bacterial solution and drop it onto ordinary plates, and place them in incubators at different temperatures (37, 45, 50 °C). Observe the growth of the strains every 12 h. After detection, the highest ethanol tolerance of the screened Lactobacillus rhamnosus L21 is 12%, and the highest temperature tolerance is 45 °C.

[0046] Colony morphology identification: Inoculate the purified single colony onto MRS medium by streaking, and incubate it at 37 °C for 2 - 3 days. Observe and record the colony morphology. The colonies on the MRS medium are round, white, convex, with a smooth and moist surface and regular edges. Among them Figure 1 is the colony morphology diagram of L21.

[0047] Microscopic examination: Stain by Gram staining method and observe under a 100× oil immersion lens. It is short rod-shaped, distributed singly or in pairs, non-motile, without spores, and Gram-positive. Among them, Figure 2 is the cell morphology of L21.

[0048] Molecular biology identification:

[0049] Identify L21 bacteria by 16S rDNA. The selected universal primers are:

[0050] 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′

[0051] 1492R: 5′-GGTTACCTTGTTACGACTT-3′

[0052] The 16S rRNA nucleotide sequence of the said L21 is as follows:

[0053]

[0054] Example 2: In-situ fermentation experiment of single Lactobacillus rhamnosus L21

[0055] Lactobacillus rhamnosus L21 was added to the fermented grains after steaming and cooling of liquor with an inoculation amount of 10 5 CFU / g. The control group was an equal volume of sterile normal saline with a mass concentration of 0.9%. After mixing evenly, it was divided into cloth bags and placed in the middle of the fermented grains pile. After 2 - 3 days of stacking fermentation, the samples were stirred to simulate the operation of shifting the pile. After sampling, they were put into gauze bags again. As the fermented grains pile entered the cellar fermentation stage, the cellar fermentation lasted for about 30 days. After the fermentation was completed, the samples were taken out and the contents of total acid, reducing sugar, moisture, starch, organic acid and flavor substances in the fermented grains were detected. The determination of total acid, reducing sugar, moisture and starch was carried out with reference to T / CBJ004-2018 "General Analytical Method for Solid-state Fermented Fermented Grains".

[0056] Sample pretreatment for organic acid detection: 10 g of fermented grains were placed in a 250 mL triangular flask, 50 mL of water was added, stirred evenly, and soaked at room temperature for 30 min. During the soaking time, it was stirred once every 5 min. The leaching solution was filtered through double-layer gauze or absorbent cotton into a 100 mL volumetric flask, and the residue was washed thoroughly with distilled water, made up to the mark and shaken well to obtain the test solution. Subsequently, the test solution was poured into a 50 mL centrifuge tube, centrifuged at 8000 r / min at 4℃ for 10 min, and the supernatant was taken and filtered through a 0.22 μL water-based filter membrane for detection.

[0057] The organic acids in the fermented grains were determined by liquid chromatography, mainly including lactic acid, acetic acid, butyric acid, caproic acid, formic acid and propionic acid. The chromatographic column was Bio-Rad organic acid analysis column Aminex HPX–87H (300 mm×7.8 mm, 9 μm); ultraviolet-visible absorption detector (VWD); the mobile phase was dilute sulfuric acid with a concentration of 5 mmol / L, the flow rate was 0.6 mL / min, the column temperature was 60℃, the ultraviolet wavelength was 210 nm, and the injection volume was 20 μL.

[0058] Gas chromatography flavor detection of flavor substances - three internal standard method, sample pretreatment: 10 g of fermented grains were dissolved in 20 mL of ethanol solution with a volume concentration of 60%, shaken for 30 min, centrifuged at 8000 r / min for 10 min, the supernatant was taken, 100 μL of three internal standard solution (2% tert-amyl alcohol, n-amyl acetate, 2-ethylbutyric acid) was added and made up to 10 ml in a volumetric flask, and filtered through a 0.22 μL organic filter membrane for detection.

[0059] The main volatile flavor substances in the wine samples, including ethyl hexanoate, ethyl lactate, ethyl butyrate, ethyl acetate, methanol, n-propanol, isobutanol, n-butanol, isoamyl alcohol, acetaldehyde, acetal, etc., were determined by gas chromatograph (GC). The chromatographic column was DB-WAX (60 m×250 μm×0.25 μm). The detector was a flame ionization detector. Chromatographic conditions: The carrier gas was high-purity nitrogen; the split ratio was 100:1; the inlet temperature was 300 °C; the detector temperature was 250 °C; the injection volume was 1 μL. Temperature programming: The initial temperature was 40 °C, held for 8 min; increased to 90 °C at 5 °C / min, and then increased to 180 °C at 6 °C / min, held for 10 min, and each sample was run for 43 min.

[0060] As shown in Table 1, the physicochemical analysis of the fermented grains showed that the use of Lactobacillus rhamnosus L21 inoculant did not significantly change the moisture and starch concentration of the fermented grains at the two time points of the end of piling and the end of cellar exit, while the acidity of the fermented grains decreased significantly, from the original 1.16±0.01 mmol / 10 g to 0.87±0.04 mmol / 10 g, a decrease of 25.00%.

[0061] Table 1 Physicochemical detection and analysis of fermented grains at the end of piling and the end of cellar

[0062]

[0063] The change in the content of organic acids made an important contribution to the change in the acidity of the fermented grains. We conducted a quantitative analysis of the flavor substances in the fermented grains, and the results are shown in Table 2. Compared with the control group, the addition of Lactobacillus rhamnosus L21 inoculant decreased the acidity instead. Acetic acid in the fermented grains decreased from 9129.68±383.71 mg / kg to 7633.16±77.38 mg / kg, a decrease of 16.39%, and propionic acid decreased from 119.82±1.06 mg / kg to 105.00±2.69 mg / kg, a decrease of 12.37%.

[0064] Flavor analysis found that the content of n-propanol in the fermented grains decreased significantly from the original 2720.72±82.67 mg / kg to 2253.32±3.07 mg / kg, a decrease of 17.18%, which helped to improve the comfort after drinking. Thus, it can be seen that the single Lactobacillus rhamnosus L21 inoculant can effectively reduce organic acids and higher alcohols in the liquor fermentation system. At present, there is no research on the reduction of higher alcohols by Lactobacillus rhamnosus. This strain of inoculant provides data support for reducing organic acids and higher alcohols in liquor fermented grains, and provides ideas for the application of Lactobacillus rhamnosus inoculant in liquor production.

[0065] Table 2 Content of flavor substances in fermented grains after cellar fermentation

[0066]

[0067] Example 3: In-situ fermentation experiment of the compound bacterium agent of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27

[0068] Add Bacillus licheniformis XDNZ_Bl_27 into the fermented grains after steaming and cooling of liquor-making at an inoculation amount of 10 6 CFU / g, and add Lactobacillus rhamnosus L21 into the fermented grains after steaming and cooling of liquor-making at an inoculation amount of 10 5 CFU / g. The control group is an equal volume of sterile normal saline with a mass concentration of 0.9%. After stirring evenly, divide it into cloth bags and place it in the middle position of the fermented grains pile. After 2 - 3 days of stacking fermentation, stir the sample to simulate the operation of moving the pile. After sampling, put it back into the gauze bag. As the fermented grains enter the cellar fermentation stage, the cellar fermentation lasts for about 30 days. After the fermentation is completed, take out the sample and detect the contents of total acid, reducing sugar, moisture, starch, organic acid and flavor substances in the fermented grains. The relevant detection methods are the same as those in Example 1.

[0069] As shown in Table 3, the physicochemical analysis of the fermented grains shows that the use of the compound bacterium agent of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 did not significantly change the moisture and starch concentrations of the fermented grains at the two time nodes of the end of stacking and the end of cellar fermentation. However, the acidity of the fermented grains decreased significantly, from the original 1.16 ± 0.01 mmol / 10 g to 0.79 ± 0.07 mmol / 10 g, a decrease of 31.82%. It is worth noting that the addition of the compound bacterium agent did not significantly reduce the reducing sugar and starch in the fermented grains, indicating that the addition of the compound bacterium agent did not affect the utilization rate of raw material starch and was conducive to controlling the liquor yield of each round.

[0070] Table 3 Physicochemical detection and analysis of fermented grains at the end of stacking and the end of cellar fermentation

[0071] The change in the content of organic acids makes an important contribution to the change in the acidity of the fermented grains. We conducted a quantitative analysis of the flavor substances in the fermented grains. The results are shown in Table 4. Compared with the control group, the acetic acid in the fermented grains decreased from 9129.68 ± 383.71 mg / kg to 7199.93 ± 270.59 mg / kg, a decrease of 21.14%, and the propionic acid decreased from 119.82 ± 1.06 mg / kg to 95.62 ± 0.74 mg / kg, a decrease of 20.20%.

[0072] Flavor analysis found that the content of n-propanol in the fermented grains decreased significantly from the original 2720.72 ± 82.67 mg / kg to 2346.77 ± 60.98 mg / kg, a decrease of 13.74%, which helps to improve the comfort after drinking. As Figure 3, the addition of a single Lactobacillus rhamnosus L21 bacterium agent can significantly reduce propionic acid in the fermented grains by 12.37%. Compared with the control group, the content of propionic acid decreased significantly after adding the compound bacterium agent, with a decrease of 20.20%; and compared with the single bacterium agent, the compound bacterium agent showed a more excellent effect in reducing the content of propionic acid. Applying the compound bacterium agent in liquor production is beneficial to the regulation of acidity and higher alcohols in the liquor production process. Generally, the addition of Lactobacillus will increase the total acid, but the compound bacterium agent in this experiment added Lactobacillus rhamnosus to achieve a reduction in acidity. And in previous studies, Lactobacillus rhamnosus does not produce other acids during fermentation, only producing L-lactic acid, and humans can only utilize L-lactic acid, so foods fermented with Lactobacillus rhamnosus will not cause adverse reactions in humans. At present, there is no research on discussing the effect of reducing acid and higher alcohols in liquor fermented grains by combining Lactobacillus rhamnosus and Bacillus licheniformis. This experiment fills this gap, provides new ideas for solving the problems of excessive acidity and higher alcohols in liquor brewing, provides a theoretical basis for the utilization of diversified compound microbial bacterium agents, and is beneficial to the application of compound bacterium agents in liquor production.

[0073] Table 4 Contents of flavor substances in fermented grains after pit fermentation

[0074]

[0075] Although embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. Lactobacillus rhamnosus capable of reducing the acidity and higher alcohol content of fermented grains, characterized in that: Its name is L21, and the classification name is: Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), the preservation number is: CGMCC No. 32609, the preservation date: November 13, 2024, the preservation unit: China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Lacticaseibacillus rhamnosus according to claim 1 in the brewing of Maotai-flavor Baijiu.

3. A compound bacterium agent of Lactobacillus rhamnosus and Bacillus licheniformis that can reduce the acidity and higher alcohol content of fermented grains by using Lactobacillus rhamnosus as described in claim 1, characterized in that: The composite bacterial agent includes Lacticaseibacillus rhamnosus L21 and Bacillus licheniformis; Among them, the name of the Bacillus licheniformis is: XDNZ_Bl_27, selected from the publication number CN117286064A.

4. The composite microbial agent according to claim 3, characterized in that: The viable bacteria number ratio of the Lacticaseibacillus rhamnosus L21 and the Bacillus licheniformis XDNZ_Bl_27 is 10:

1.

5. The composite bacterial agent according to claim 3 or 4, characterized in that: Both the Lacticaseibacillus rhamnosus L21 and the Bacillus licheniformis XDNZ_Bl_27 are isolated and screened from the fermented grains in the production of Maotai-flavor Baijiu.

6. Use of the composite bacterial agent according to any one of claims 3 to 5 in the brewing of Maotai-flavor Baijiu.

7. Use of the composite bacterial agent according to any one of claims 3 to 5 in the in-situ fermentation of Maotai-flavor Baijiu.

8. The application according to claim 7, characterized in that: After the in-situ fermentation of Maotai-flavor Baijiu with the composite bacterial agent of Lacticaseibacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27, the use of the composite bacterial agent of Lacticaseibacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 did not change the moisture and starch concentration of the fermented grains at the two time nodes of the end of piling and the out of the pit, while the acidity of the fermented grains decreased significantly, from the original 1.16±0 mmol / 10 g fermented grains to 0.79±0.07 mmol / 10 g fermented grains, a decrease of 31.82%; Acetic acid in the fermented grains decreased significantly from 9129.68±383.71 mg / kg to 7199.93±270.59 mg / kg, a decrease of 21.14%, and propionic acid decreased significantly from 119.82±1.06mg / kg to 95.62±0.74 mg / kg, a decrease of 20.20%; The content of n-propanol in the fermented grains decreased significantly from the original 2720.72±82.67 mg / kg to 2346.77±60.98mg / kg, a decrease of 13.74%.

9. A method for in-situ fermentation of Maotai-flavor Baijiu using the composite microbial agent according to any one of claims 3 to 5, characterized in that: Including the following steps: Bacillus licheniformis XDNZ_Bl_27 was added to the steamed and cooled fermented grains at an inoculation amount of 10 6 CFU / g, and Lactobacillus rhamnosus L21 was added at an inoculation amount of 10 5 CFU / g; after thorough mixing, it was divided into cloth bags and placed in the middle of the fermented grains pile. After 2-3 days of stacking fermentation, the samples were stirred, simulating the operation of moving the pile. After sampling, it was put back into the gauze bag, and as the fermented grains pile entered the cellar fermentation stage, the cellar fermentation continued for 30 days to obtain Maotai-flavor liquor.

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