Lactobacillus rhamnosus and bacillus licheniformis complex microbial inoculant for reducing acids and higher alcohols, lactobacillus rhamnosus and application of lactobacillus rhamnosus and bacillus licheniformis complex microbial inoculant in Baijiu

Abstract

The invention belongs to the technical field of microbial fermentation, and discloses a complex microbial inoculant of lactobacillus rhamnosus and bacillus licheniformis for reducing acid and higher alcohols, the lactobacillus rhamnosus and application of the complex microbial inoculant in baijiu, the lactobacillus rhamnosus L21 is preserved in CGMCC (China General Microbiological Culture Collection Center) NO.32609 on November 13, 2024, and the preservation number is CGMCC NO.32609. The preservation number of the lactobacillus rhamnosus L21 is CGMCC NO.32609. The preservation unit is China General Microbiological Culture Collection Center (CGMCC). By using the composite microbial agent of the rhamnosus lactobacillus L21 and the bacillus licheniformis XDNZBl27, organic acids such as lactic acid, propionic acid and the like in a liquor fermentation system can be effectively reduced, meanwhile, the content of n-propyl alcohol in fermented grains can also be reduced, and regulation and control of acidity and higher alcohols in the liquor production process are facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, in particular to an acid- and higher alcohol-reducing Lactobacillus rhamnosus and Bacillus licheniformis composite bacterial agent, Lactobacillus rhamnosus and application in liquor. Background Art

[0002] High acidity is one of the characteristics of Maotai-flavor liquor production, among which acidic flavor substances mainly include organic acids such as acetic acid, lactic acid, butyric acid, caproic acid and higher fatty acids. Multiple rounds of fermentation processes lead to the accumulation of non-volatile acids. Excessive accumulation of non-volatile acids will lead to abnormal fermentation in the later stage, which will in turn affect the succession and growth metabolism of microbial flora, and ultimately affect the base wine yield and quality. In addition, the high content of higher alcohols in the first round of base wine is a common problem in the production of Maotai-flavor liquor. The right amount of higher alcohols can increase the aroma and sweetness of the wine, while excessive higher alcohols will reduce the comfort of the liquor after drinking. Therefore, controlling the content of higher alcohols in the first round of base wine is of great significance for the blending of the wine.

[0003] At present, there have been studies on using functional microorganisms to control the increase in the acidity of the mash. For example, Pichia kudriavzevii can reduce the lactic acid concentration in the mash, thereby alleviating the effect of acid in the fermentation system on brewing yeast (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). Most of the reported patents focus on the problem of regulating the acidity and high content of higher alcohols in fermented grains by microbial agents, such as using Bacillus licheniformis to reduce the acidity and higher alcohols of fermented grains of Maotai-flavor liquor (Chinese patent CN117286064A), the acidity of fermented grains was reduced by 28%, and the n-propanol was reduced by 45.2%; using dwarf Candida to control the content of higher alcohols in liquor (Chinese patent CN112322509A), the higher alcohol content of the liquor sample after fermentation with dwarf Candida was 69.5% of the liquor sample after fermentation with standard strains; adding Lactobacillus to reduce the higher alcohol content of light-flavor Xiaoqu liquor (Chinese patent CN115109674A), the higher alcohol content in the fermented grains was reduced by 52.2%, but the total acid would increase by 2.5 times. However, so far, there are few reports on the research on reducing the acidity and higher alcohols of Maotai-flavor liquor by adding composite bacterial agents. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a composite bacterial agent of Lactobacillus rhamnosus and Bacillus licheniformis for reducing acid and higher alcohol, Lactobacillus rhamnosus and application in liquor.

[0005] The technical solution adopted by the present invention to solve the technical problem is:

[0006] A strain of Lactobacillus rhamnosus that can reduce the acidity and higher alcohol content of mash is named L21, classified as Lactobacillus rhamnosus, with a preservation number of CGMCC No.32609, a preservation date of November 13, 2024, and a preservation unit at the General Microbiology Center of China National Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The application of Lactobacillus rhamnosus in the brewing of Maotai-flavor liquor as described above.

[0008] A composite bacterial agent of Lactobacillus rhamnosus and Bacillus licheniformis that utilizes the above-mentioned Lactobacillus rhamnosus and can reduce the acidity and higher alcohol content of fermented grains, wherein the composite bacterial agent comprises 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 capable of reducing the acidity and higher alcohol content of mash, method and application in liquor production.

[0010] Furthermore, the ratio of the number of live bacteria of Lactobacillus rhamnosus L21 to Bacillus licheniformis XDNZ_Bl_27 is 1:10.

[0011] Furthermore, the Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 are both obtained by separation and screening from fermented grains produced in the sauce-flavor liquor.

[0012] The application of the composite bacterial agent as described above in the brewing of Maotai-flavor liquor.

[0013] The application of the composite bacterial agent as described above in the in-situ fermentation of Maotai-flavor liquor.

[0014] Furthermore, after the in-situ fermentation of Maotai-flavor liquor was completed, the use of the composite bacterial agent of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 did not significantly change the moisture and starch concentrations of the mash at the end of stacking and out of the cellar, while the acidity of the mash decreased significantly from the original 1.16±0.01 mmol / 10 g mash to 0.79±0.07 mmol / 10 g mash, a decrease of 31.82%.

[0015] The acetic acid in the mash was significantly reduced from 9129.68±383.71 mg / kg to 7199.93±270.59 mg / kg, a decrease of 21.14%, and the propionic acid was significantly reduced 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 mash was significantly reduced from 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 liquor using the composite bacterial agent as described above comprises the following steps:

[0018] Bacillus licheniformis XDNZ_Bl_27 with 10 6 CFU / g inoculation amount, Lactobacillus rhamnosus L21 at 10 5 The inoculum amount of CFU / g is added to the mash after the distilled liquor is cooled; after being stirred evenly, it is divided into cloth bags and placed in the middle of the mash pile. After 2-3 days of stacking and fermentation, the sample is stirred to simulate the pile transfer operation. After sampling, it is put into the gauze bag again. As the mash pile enters the cellar fermentation stage, the cellar fermentation lasts for 30 days to obtain the sauce-flavored liquor.

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

[0020] 1. The two bacteria capable of reducing the acidity and higher alcohol content of fermented grains in the present invention are Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27, Lactobacillus rhamnosus L21 was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on November 13, 2024, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, with a deposit number of CGMCC NO.32609, and Bacillus licheniformis XDNZ_Bl_27 ... licheniformis)XDNZ_Bl_27 is selected from Chinese patent, publication number CN117286064A, application number 202311257416.7, a licheniformis strain capable of reducing the acidity and higher alcohol content of mash, method and application in liquor production.

[0021] 2. The addition of a single Lactobacillus rhamnosus L21 bacterial agent reduced the acidity of the mash 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%, and 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 the composite bacterial 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 liquor fermentation system, and can also reduce the content of n-propanol in the mash, which is beneficial to the regulation of acidity and higher alcohols in the liquor production process.

[0022] 3. The use of the composite bacterial agent of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 significantly decreased the acidity of the mash from the original 1.16±0.01 mmol / 10 g to 0.79±0.07 mmol / 10 g, a decrease of 31.82%; the acetic acid in the mash was significantly reduced from 9129.68±383.71 mg / kg to 7199.93±270.59 mg / kg, a decrease of 21.14%, and the propionic acid was significantly reduced from 119.82±1.06 mg / kg to 95.62±0.74 mg / kg, a decrease of 20.20%. The composite bacterial agent showed better effect in reducing the propionic acid content than the single bacterial agent. The content of n-propanol in the mash was significantly reduced from 2720.72±82.67 mg / kg to 2346.77±60.98 mg / kg, a decrease of 13.74%. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 This is a bacterial morphology diagram of L21 in the present invention;

[0025] Figure 3 Graph showing the propionic acid content in the fermented grains after the cellar fermentation in the present invention, wherein * indicates a significant difference of 0.01< P < 0.05; ** indicates a significant difference of 0.001< P < 0.01.

[0026] A strain of Lactobacillus rhamnosus that can reduce the acidity and higher alcohol content of mash is named L21, classified as Lactobacillus rhamnosus, with a preservation number of CGMCC No.32609, a preservation date of November 13, 2024, and a preservation unit at the General Microbiology Center of China National Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0028] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0029] A strain of Lactobacillus rhamnosus that can reduce the acidity and higher alcohol content of mash is named L21, classified as Lactobacillus rhamnosus, with a preservation number of CGMCC No.32609, a preservation date of November 13, 2024, and a preservation unit at the General Microbiology Center of China National Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0030] The application of Lactobacillus rhamnosus in the brewing of Maotai-flavor liquor as described above.

[0031] A composite bacterial agent of Lactobacillus rhamnosus and Bacillus licheniformis that utilizes the above-mentioned Lactobacillus rhamnosus and can reduce the acidity and higher alcohol content of fermented grains, wherein the composite bacterial agent comprises 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 strain of Bacillus licheniformis capable of reducing the acidity and higher alcohol content of mash, method and application in liquor production.

[0033] Preferably, the ratio of the number of live bacteria of Lactobacillus rhamnosus L21 to that of Bacillus licheniformis XDNZ_Bl_27 is 1:10.

[0034] Preferably, the Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 are both obtained by separation and screening from fermented grains produced in sauce-flavor liquor.

[0035] The application of the composite bacterial agent as described above in the brewing of Maotai-flavor liquor.

[0036] The application of the composite bacterial agent as described above in the in-situ fermentation of Maotai-flavor liquor.

[0037] Preferably, after the in-situ fermentation of Maotai-flavor liquor is completed, the use of the composite bacterial agent of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 does not significantly change the moisture and starch concentration of the mash at the end of stacking and out of the cellar, while the acidity of the mash decreases significantly from the original 1.16±0.01 mmol / 10 g to 0.79±0.07 mmol / 10 g, a decrease of 31.82%.

[0038] The acetic acid in the mash was significantly reduced from 9129.68±383.71 mg / kg to 7199.93±270.59 mg / kg, a decrease of 21.14%, and the propionic acid was significantly reduced 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 mash was significantly reduced from 2720.72±82.67 mg / kg to 2346.77±60.98 mg / kg, a decrease of 13.74%.

[0040] The method for in-situ fermentation of Maotai-flavor liquor using the composite bacterial agent as described above comprises the following steps:

[0041] Bacillus licheniformis XDNZ_Bl_27 with 10 6 CFU / g inoculation amount, Lactobacillus rhamnosus L21 at 10 5 The inoculum amount of CFU / g is added to the mash after the distilled liquor is cooled; after being stirred evenly, it is divided into cloth bags and placed in the middle of the mash pile. After 2-3 days of stacking and fermentation, the sample is stirred to simulate the pile transfer operation. After sampling, it is put into the gauze bag again. As the mash pile enters the cellar fermentation stage, the cellar fermentation lasts for 30 days to obtain the sauce-flavored liquor.

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

[0043] The Lactobacillus rhamnosus L21 involved in the following examples is deposited in the General Microbiological Center of China National Committee for the Preservation and Administration of Microbiological Cultures.

[0044] Example 1: Screening of Lactobacillus rhamnosus L21

[0045] Take 10.0 g of Daqu from the 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 culture medium. Culture it at 50°C for 24 h, then perform gradient dilutions, spread it on an MRS plate containing 1.5% CaCO3, and culture it at 37°C for 48 h. Select strains with transparent circles and purify them 2 to 3 times by plate streaking to obtain pure colonies. Pure colonies were stored in glycerol cryovials at -80°C. The tolerance of the strains was detected by the spot plate test method. The test strains were picked into 5 mL of MRS liquid culture medium and cultured at 37°C for 12 to 16 h. The OD value of the bacterial solution was 0.04. 600Adjust to 1.0, and pipette 4 μL of bacterial solution onto MRS plates containing different ethanol (volume concentrations of 8%, 10%, and 12%), and invert and culture at 37°C for 2 days. Pipette 4 μL of bacterial solution onto ordinary plates and place them in incubators at different temperatures (37, 45, and 50°C), and observe the growth of the strain every 12 hours. According to the test, the maximum ethanol tolerance of the currently screened Lactobacillus rhamnosus L21 is 12%, and the maximum temperature tolerance is 45°C.

[0046] Colony morphology identification: The purified single colony was inoculated into MRS medium by streaking, cultured at 37℃ for 2-3 days, and the colony morphology was observed and recorded. The colony on MRS medium was round, white, convex, with a smooth and moist surface and neat edges. Figure 1 This is the colony morphology of L21.

[0047] Microscopic examination: Gram staining, 100× oil immersion lens. Short rod-shaped, single or in pairs, non-motile, no spores, Gram staining positive. Figure 2 This is the bacterial morphology of L21.

[0048] Molecular Biology Identification:

[0049] 16S rDNA was used to identify L21 bacteria. The selected universal primers were:

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

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

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

[0053]

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

[0055] Lactobacillus rhamnosus L21 was added to 10 5 The inoculation amount of CFU / g is added to the fermented grains after the distilled wine is cooled, and the control group is an equal volume of sterile saline with a mass concentration of 0.9%; after stirring evenly, it is divided into cloth bags and placed in the middle of the fermented grains pile. After 2-3 days of stacking and fermentation, the sample is stirred to simulate the pile transfer operation. After sampling, it is put into the gauze bag again. As the fermented grains pile enters the cellar fermentation stage, the cellar fermentation lasts about 30 days. After the fermentation is completed, the sample is taken out to detect the content of total acid, reducing sugar, water, starch, organic acid and flavor substances in the fermented grains. The determination of total acid, reducing sugar, water and starch refers to T / CBJ004-2018 "General Analysis Method for Solid Fermentation Fermented Grains".

[0056] Sample pretreatment for organic acid detection: 10 g of fermented grains were placed in a 250 mL conical flask, 50 mL of water was added, stirred, and soaked at room temperature for 30 min. During the soaking time, stir once every 5 min. The extract was filtered into a 100 mL volumetric flask with double-layer gauze or absorbent cotton, and the residue was fully washed with distilled water, fixed to volume, and shaken to obtain the test solution. The test solution was then poured into a 50 mL centrifuge tube, centrifuged at 8000 r / min at 4°C for 10 min, and the supernatant was taken and tested through a 0.22 μL water filter membrane.

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

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

[0059] Gas chromatography (GC) was used to determine the main volatile flavor substances in the wine samples, including ethyl caproate, ethyl lactate, ethyl butyrate, ethyl acetate, methanol, n-propanol, isobutanol, n-butanol, isopentanol, acetaldehyde, acetal, etc. The chromatographic column was DB-WAX (60 m×250 μm×0.25 μm). The detector was a hydrogen flame ionization detector. Chromatographic conditions: carrier gas was high-purity nitrogen; split ratio was 100:1; injection port temperature was 300℃; detector temperature was 250℃; injection volume was 1 μL. Heating program: initial temperature was 40℃, maintained for 8 min; increased to 90℃ at 5℃ / min, increased to 180℃ at 6℃ / min, maintained for 10 min, and each sample was run for 43 min.

[0060] As shown in Table 1, the physical and chemical analysis of the mash showed that the use of Lactobacillus rhamnosus L21 did not significantly change the moisture and starch concentrations of the mash at the end of stacking and out of the cellar, while the acidity of the mash 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 Physical and chemical analysis of the fermented grains at the end of the pile and the end of the cellar

[0062]

[0063] The change of organic acid content has an important contribution to the change of acidity of fermented mash. We conducted a quantitative analysis of the flavor substances in the fermented mash. The results are shown in Table 2. Compared with the control group, the addition of Lactobacillus rhamnosus L21 inoculant reduced the acidity. The acetic acid in the fermented mash decreased from 9129.68±383.71 mg / kg to 7633.16±77.38 mg / kg, a decrease of 16.39%, and the 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 n-propanol content in the mash was significantly reduced from the original 2720.72±82.67 mg / kg to 2253.32±3.07 mg / kg, a decrease of 17.18%, which helps to improve the comfort after drinking. It can be seen that a single Lactobacillus rhamnosus L21 inoculant can effectively reduce organic acids and higher alcohols in the liquor fermentation system, and there is no research on the reduction of higher alcohols by Lactobacillus rhamnosus. This strain of bacteria provides data support for reducing organic acids and higher alcohols in liquor mash, and provides ideas for the application of Lactobacillus rhamnosus inoculants in liquor production.

[0065] Table 2 Content of flavor substances in the mash after cellar fermentation

[0066]

[0067] Example 3: In situ fermentation experiment of Lactobacillus rhamnosus L21 and Bacillus licheniformis XDNZ_Bl_27 composite bacterial agent

[0068] Bacillus licheniformis XDNZ_Bl_27 was added at 10 6 CFU / g inoculation amount, Lactobacillus rhamnosus L21 with 10 5 An inoculum of 1000 CFU / g is added to the fermented grains after the distilled wine is cooled, and the control group is an equal volume of sterile saline with a mass concentration of 0.9%; after stirring evenly, it is divided into cloth bags and placed in the middle of the fermented grains pile. After 2-3 days of stacking and fermentation, the sample is stirred to simulate the pile transfer operation. After sampling, it is put into the gauze bag again. As the fermented grains enter the cellar fermentation stage, the cellar fermentation lasts about 30 days. After the fermentation is completed, the sample is taken out to detect the content of total acid, reducing sugar, water, starch, organic acid and flavor substances in the fermented grains. The relevant detection method is the same as Example 1.

[0069] As shown in Table 3, the physical and chemical analysis of the fermented grains showed that the use of the composite bacterial 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 end of the pile and the time of leaving the cellar, while 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 composite bacterial agent did not significantly reduce the reducing sugar and starch in the fermented grains, indicating that the addition of the composite bacterial agent did not affect the utilization rate of the raw material starch, which was conducive to controlling the wine yield of each round.

[0070] Table 3 Physical and chemical analysis of the fermented grains at the end of the pile and the end of the cellar

[0071] The change of organic acid content has an important contribution to the change of acidity of the mash. We conducted a quantitative analysis of the flavor substances in the mash. The results are shown in Table 4. Compared with the control group, the acetic acid in the mash 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 n-propanol content in the mash was significantly reduced 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. Figure 3, the addition of a single Lactobacillus rhamnosus L21 bacterial agent can significantly reduce the propionic acid in the mash by 12.37%. Compared with the control group, the propionic acid content was significantly reduced after adding the composite bacterial agent, with a decrease of 20.20%; and compared with the single bacterial agent, the composite bacterial agent showed a better effect in reducing the propionic acid content. The application of composite bacterial agents in liquor production is conducive 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 addition of Lactobacillus rhamnosus to the composite bacterial agent in this experiment has achieved a reduction in acidity. In addition, existing studies have shown that Lactobacillus rhamnosus will not produce other acids during the fermentation process, only L-lactic acid, and humans can only use L-lactic acid, so foods fermented with Lactobacillus rhamnosus will not cause adverse reactions in people. At present, there has been no research on the effect of combining Lactobacillus rhamnosus and Bacillus licheniformis to reduce the acidity and higher alcohols in liquor mash. This experiment fills this gap, provides new ideas for solving the problems of excessive acidity and higher alcohols in the liquor brewing process, and provides a theoretical basis for the use of diversified composite microbial agents, which is beneficial to the application of composite agents in liquor production.

[0073] Table 4 Content of flavor substances in the mash after cellar fermentation

[0074]

[0075] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate 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 contents disclosed in the embodiments.

Claims

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

2. Application of Lactobacillus rhamnosus as claimed in claim 1 in the brewing of Maotai-flavor liquor.

3. A composite bacterial agent of Lactobacillus rhamnosus and Bacillus licheniformis that can reduce the acidity and higher alcohol content of fermented grains using the Lactobacillus rhamnosus as claimed in claim 1, characterized in that: The composite bacterial agent includes Lactobacillus rhamnosus L21 and Bacillus licheniformis; Wherein, the name of the Bacillus licheniformis is: XDNZ_Bl_27.

4. The composite bacterial agent according to claim 3, characterized in that: The ratio of the number of live bacteria of Lactobacillus rhamnosus L21 and Bacillus chlamydosus XDNZ_Bl_27 is 10:

1.

5. The composite bacterial agent according to claim 3 or 4, characterized in that: The Lactobacillus rhamnosus L21 and Bacillus chlamydosus XDNZ_Bl_27 are both obtained by separation and screening from fermented grains produced in sauce-flavor liquor.

6. Use of the composite bacterial agent as described in any one of claims 3 to 5 in the brewing of Maotai-flavor liquor.

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

8. The use according to claim 7, characterized in that: After the in-situ fermentation of Maotai-flavor liquor was completed, the use of the composite bacterial agent of Lactobacillus rhamnosus L21 and Bacillus chlamydione XDNZ_Bl_27 did not change the moisture and starch concentration of the fermented grains at the end of stacking and out of the cellar, 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%; The acetic acid in the mash was significantly reduced from 9129.68±383.71 mg / kg to 7199.93±270.59 mg / kg, a decrease of 21.14%, and the propionic acid was significantly reduced from 119.82±1.06 mg / kg to 95.62±0.74 mg / kg, a decrease of 20.20%; The content of n-propanol in the mash was significantly reduced from 2720.72±82.67 mg / kg to 2346.77±60.98 mg / kg, a decrease of 13.74%.

9. A method for in-situ fermentation of Maotai-flavor liquor using the composite bacterial agent according to any one of claims 3 to 5, characterized in that: The steps include: Bacillus licheniformis XDNZ_Bl_27 with 10 6 CFU / g inoculation amount, Lactobacillus rhamnosus L21 at 10 5 The inoculum amount of CFU / g is added to the mash after the distilled liquor is cooled; after being stirred evenly, it is divided into cloth bags and placed in the middle of the mash pile. After 2-3 days of stacking and fermentation, the sample is stirred to simulate the pile transfer operation. After sampling, it is put into the gauze bag again. As the mash pile enters the cellar fermentation stage, the cellar fermentation lasts for 30 days to obtain the sauce-flavored liquor.

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