Method for activating yeast by mixed bacteria to strengthen esterification capacity of high-temperature daqu

Abstract

The present application relates to a kind of mixed bacteria activated yeast reinforced high-temperature Daqu esterification capacity method.The present application is inoculated in inorganic nitrogen culture with Rhizopus arrhizus and Hansenula polymorpha, and incubation activation is 1-3 days, then inoculates Saccharomyces cerevisiae, incubation activation is 3-5 days, then inoculates Lactobacillus plantarum, and continue to activate 5-7 days, separate bacterial body;The obtained isolated bacterial body is inoculated again in inorganic nitrogen culture medium, and repeated delay acid co-activation until activation is completed, and bacterial body is separated with fermentation liquor;Then, activation seed liquid preparation, second-order koji making and Daqu fermentation are carried out, and the koji is obtained.The total bacteria amount in the koji obtained by the present application can be increased by 4.12 × 10 7 cfu / g;Hexanoic acid ethyl ester, ethyl lactate, ethyl acetate is respectively compared with traditional block koji to increase 38.21 μg / g, 39.95 μg / g and 48.06 μg / g, and ester diversity is increased by 6.

Description

Technical Field

[0001] This invention belongs to the field of brewing koji technology, specifically involving a method for enhancing the esterification ability of high-temperature koji by activating mixed-strain yeast. Background Technology

[0002] Yeast (or koji) is an essential starting fermentation agent in the brewing process, providing the necessary enzyme system for fermentation, which is crucial for improving the quality of the liquor. High-temperature koji accounts for a large proportion of the production input in Maotai-flavor liquor, which is known for its ester aroma. This requires high-temperature koji to impart efficient esterification capabilities to the liquor brewing process.

[0003] Yeasts typically possess superior esterase activity and substrate esterification potential, but the esterification capacity of most yeasts decreases due to the acid, alcohol, and high-temperature stress in the high-temperature starter culture. Furthermore, other microorganisms are similarly affected by these stresses, leading to reduced metabolic efficiency and an inability to accumulate the substrates required for esterification reactions. This further deteriorates the starter culture and can even directly impact the quality of the liquor.

[0004] Therefore, there is an urgent need in this field to provide a method for enhancing the esterification ability of high-temperature koji by activating mixed yeast. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for enhancing the esterification ability of high-temperature koji by activating mixed-culture yeast.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a method for enhancing the esterification ability of high-temperature koji (a type of starter culture) by activating mixed-culture yeast, characterized by comprising the following steps:

[0008] (1) In an inorganic nitrogen culture, inoculate *Zygosacchariformis* and *Hansøe du Barry*, keep warm and activate for 1 to 3 days, then inoculate *Sacchariformis*, keep warm and activate for 3 to 5 days, then inoculate *Lactobacillus plantarum*, continue activation for 5 to 7 days, and isolate the cells.

[0009] (2) Re-inoculate the isolated bacterial cells obtained in step (1) into the inorganic nitrogen culture medium, and repeat step (1) until activation is complete, and separate the bacterial cells from the fermentation broth; each time the activation temperature is increased by 5℃~10℃;

[0010] (3) Mix the bacterial suspension containing the bacterial cells obtained in step (2) with soybean oil containing polyglycerol ricinoleate and shear until the oil droplets are uniform. Then add the fermentation liquid obtained in step (2) and shear until uniform to obtain activated seed liquid.

[0011] (4) After the crushed wheat is moistened and steamed, it is mixed with 0.5wt% to 5wt% of the activated seed liquid obtained in step (3), and loosened until the material thickness is 20cm to 40cm. After 1 to 3 days of ventilation and loosening, the koji is produced; 0.5wt% to 5wt% of the mother koji and the fermentation liquid obtained in step (2) are mixed in to make koji blocks;

[0012] (5) Place the koji blocks obtained in step (4) in a sealed koji room for initial fermentation at a low temperature for 1 to 3 days, then ventilate for 2 to 3 days and turn the koji once a day; reseal and raise the temperature by 3°C to 5°C every day for 5 to 7 days, then lower the temperature by 3°C to 5°C every day for 6 to 8 days to obtain the finished koji; the finished koji is used as the mother koji or directly for fermentation.

[0013] In one embodiment of the present invention, the inorganic nitrogen culture medium comprises, by weight, the following components: 10 to 20 parts glucose, 10 to 20 parts ammonium sulfate, 0.01 to 0.05 parts vitamins, 10 to 15 parts inorganic salts, and 1000 parts water.

[0014] In one embodiment of the present invention, in step (1), the inoculation amount of *Zygosacchariformis* and *Hansøe d'Bary* is 10. 6 cfu / mL ~10 7 cfu / mL.

[0015] In one embodiment of the present invention, in step (1), the inoculation amount of the brewing yeast is 10. 6 cfu / mL ~10 7 cfu / mL.

[0016] In one embodiment of the present invention, in step (1), the inoculum amount of *Lactobacillus plantarum* is 10. 5 cfu / mL ~10 6 cfu / mL.

[0017] In one embodiment of the present invention, in step (3), the bacterial suspension is obtained by mixing the bacterial cells with an equal mass of PBS buffer containing 1 wt% NaCl.

[0018] In one embodiment of the present invention, in step (3), by weight, the activated seed liquid contains 30 to 50 parts of bacterial suspension, 50 to 70 parts of soybean oil containing polyglycerol ricinoleate, and 100 to 200 parts of fermentation broth.

[0019] In one embodiment of the present invention, in step (3), the soybean oil containing polyglycerol ricinoleate is soybean oil containing 2 wt% t polyglycerol ricinoleate; the fermentation broth contains 1 wt% Tween 80.

[0020] In one embodiment of the present invention, in step (4), the moisture content of the shaped block is 35% to 40%.

[0021] In one embodiment of the present invention, in step (5), the initial temperature is 35°C to 40°C.

[0022] A second objective of this invention is to provide the application of the method in the preparation of Maotai-flavor liquor.

[0023] The technical solution of the present invention has the following advantages compared with the prior art:

[0024] (1) The microbial strain activation of this invention uses ammonium sulfate nitrogen source medium. Ammonium sulfate is a readily available nitrogen source that accelerates microbial growth, and the total microbial count after activation is 1.8 times higher than that of organic nitrogen sources. Simultaneously, sequential inoculation extends the activation period to enrich esterification metabolites. Non-Saccharomyces cerevisiae and Lactobacillus plantarum are co-activated, forming a cross-feeding process to enhance strain activity (compared to co-inoculation, acids and alcohols are enriched by 1.68 mmol / 10g and 0.229% vol, respectively). This enhanced strain improves tolerance to the acid, alcohol, and high-temperature stresses unique to the mixed-culture metabolism of this invention, increasing the total microbial count of mature yeast by 4.12 × 10⁻⁶. 7 cfu / g.

[0025] (2) The acid-producing yeast and lactic acid bacteria selected in this invention are combined with alcohol-producing brewing yeast to form a cross-feeding and mutually beneficial effect. The combination of the microbial emulsion and the segmented fermentation method promotes the orderly succession of metabolites. The resulting koji contains 38.21 μg / g, 39.95 μg / g and 48.06 μg / g more ethyl hexanoate, lactate and ethyl acetate than traditional koji blocks, respectively, and the diversity of esters is increased by 6 types. Attached Figure Description

[0026] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0027] Figure 1 This is a comparison of the total bacterial count of the activated fermentation broth and the mature yeast in this invention;

[0028] Figure 2 This is a comparison of the content of kojic acid and alcohol metabolites in this invention;

[0029] Figure 3 This is a comparison of the esters in the yeast used in this invention. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] This invention provides a method for enhancing the esterification ability of high-temperature koji (a type of starter culture) by activating mixed-culture yeast, comprising the following steps:

[0032] (1) Inorganic nitrogen culture medium: by weight, 10-20 parts glucose, 10-20 parts ammonium sulfate, 0.01-0.05 parts vitamins, 10-15 parts inorganic salts, and 1000 parts water are used to prepare the culture medium. Sterilize at 121℃ and 0.1MP for 20 min, then cool to 40℃ and keep warm.

[0033] (2) Delayed acid co-activation: Inorganic nitrogen culture was first inoculated with 10 6 ~10 7 After activation by incubation for 1–3 days, *Zygosaccharomyces rouxii* and *Hansøe d'Baryia* were inoculated with 10 cfu / mL *Zygosaccharomyces rouxii*. 6 ~10 7 CFU / mL Saccharomyces cerevisiae was activated by incubation for 3–5 days before inoculation with Lactobacillus plantarum (10). 6 Continue activation for 5–7 days (cfu / mL) and then isolate the bacterial cells.

[0034] (3) Delayed high temperature co-activation: The isolated cells are re-inoculated into fresh inorganic nitrogen medium and step (2) is repeated. Each time the activation temperature is increased by 5℃~10℃ until activation is completed at 60℃. The cells and fermentation broth are then separated for later use.

[0035] (4) Preparation of activated seed culture: By weight, the prepared bacterial cells are mixed with an equal mass of PBS containing 1% wt NaCl to form a bacterial suspension. Take 30-50 parts of the bacterial suspension and mix it with 50-70 parts of soybean oil (containing 2% wt polyglycerol ricinoleate) and shear until the oil droplets are uniform. Then add 100-200 parts of the prepared fermentation broth (containing 1% wt Tween 80) and shear until uniform.

[0036] (5) Second-stage koji making: Crushed wheat is moistened with 30wt%–35wt% water and steamed at 0.2MPa for 30min. Transfer to a sterilized disc koji making machine, and evenly mix in 0.5wt%–5wt% activated seed liquid. Loosen the koji until the material thickness is 20cm–40cm. Ventilate and loosen the koji every 8h and cultivate for 2 days to produce koji. Evenly mix 0.5wt%–5wt% mother koji into the koji material, and adjust the moisture content of the reserved fermentation liquid to 35%–40% before making koji blocks.

[0037] (6) Fermentation of Daqu: Place the Daqu blocks in a sealed Daqu room and ferment at an initial temperature of 35℃~40℃ for 2 days. Then ventilate for 2~3 days and turn the Daqu once a day. Reseal the room and raise the temperature by 3℃ every day for 7 days. Then lower the temperature by 5℃ every day for 6 days to obtain Daqu. Daqu can be used as mother Daqu or directly for fermentation.

[0038] Further, the inorganic nitrogen culture medium, by weight, comprises 15 parts glucose, 15 parts ammonium sulfate, 0.006 parts biotin, 0.0012 parts calcium pantothenate, 0.00056 parts folic acid, 0.002 parts inositol, 0.000056 parts para-aminobenzoic acid, 0.0048 parts pyridoxine hydrochloride, 0.0009 parts riboflavin, 0.00056 parts thiamine hydrochloride, 3.12 parts potassium dihydrogen phosphate, 6.48 parts dipotassium hydrogen phosphate, 1 part sodium chloride, 0.39 parts magnesium chloride, 0.1 parts manganese sulfate, 0.005 parts ferrous sulfate heptahydrate, and 1000 parts water.

[0039] Furthermore, the order of the retardant acid co-activation strains is as follows: inorganic nitrogen culture is first inoculated with 10... 6 *Zygosaccharomyces rouxii* and *Hansøe d'Baryia* were activated by incubation for 2 days and then inoculated with 10 cfu / mL *Zygosaccharomyces rouxii*. 6 CFU / mL Saccharomyces cerevisiae was activated by incubation for 3 days before inoculation with Lactobacillus plantarum (10). 6 Continue activation for 6 days (cfu / mL).

[0040] Furthermore, the delayed high-temperature co-activation step involves re-inoculating the bacterial cells isolated in step (2) into a fresh inorganic nitrogen culture medium and supplementing it with 10... 6 *Zygosaccharomyces rouxii* and *Hansøe d'Baryia* were activated at 45°C for 2 days and then inoculated with 10... 6 CFU / mL Saccharomyces cerevisiae was activated at 45℃ for 3 days before inoculation with Lactobacillus plantarum (10). 6 Continue activation for 6 days (cfu / mL). Repeat this process, isolating the cells again and incubating at 50℃, 55℃, and 60℃ sequentially. Stop activation after reaching 60℃, then separate the cells and fermentation broth for later use.

[0041] Further, the step of preparing the activated seed liquid is as follows: by weight, the prepared bacterial cells are mixed with an equal mass of PBS containing 1 wt% NaCl to form a bacterial suspension. 30 parts of the bacterial suspension are mixed with 70 parts of soybean oil (containing 2 wt% polyglycerol ricinoleate) and sheared until the oil droplets are uniform. Then, 200 parts of the prepared fermentation broth (containing 1 wt% Tween 80) are added and sheared until uniform.

[0042] Furthermore, the first stage of the second-stage koji-making process is disc koji-making, where crushed wheat is moistened with 35wt% water and steamed at 0.2MPa for 30 minutes. The mixture is then transferred to a sterilized disc koji-making machine, where 5wt% activated seed liquid is evenly mixed in, and the mixture is loosened until the material thickness reaches 30cm. Ventilation and loosening are performed every 8 hours, and the koji is cultured for 2 days to produce the final product.

[0043] Furthermore, the second stage of the second-stage koji making process is the preparation of koji blocks, in which 3% wt of mother koji is evenly mixed into the koji material, and the moisture content of the fermentation liquid is adjusted to 40% before koji blocks are made.

[0044] Furthermore, the fermentation method of the Daqu (a type of starter culture) is as follows: the starter culture blocks are placed in a sealed fermentation room and fermented at an initial temperature of 40°C for 2 days, followed by ventilation for 2 days and turning the starter culture once a day. The room is then sealed again and the temperature is increased by 3°C each day for 7 days of fermentation, and then the temperature is decreased by 5°C each day for 6 days of fermentation to obtain the finished starter culture. The finished starter culture can be used as the mother starter culture or directly for fermentation.

[0045] Materials used in this invention:

[0046] Zygosacchariformis CICC 1380, Hansenula d'Barry yeast CICC 1714, Saccharomyces cerevisiae CICC 1406, Lactobacillus plantarum CICC 23115: China Industrial Microbial Culture Collection Center;

[0047] Ammonium sulfate, biotin, calcium pantothenate, folic acid, inositol, para-aminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, magnesium chloride, manganese sulfate, ferrous sulfate heptahydrate, polyglycerol ricinoleate, Tween 80, and commercial MRS culture medium were all purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.

[0048] Unless otherwise specified, all other raw materials and equipment are commercially available.

[0049] Example 1

[0050] This embodiment provides a method for enhancing the esterification ability of high-temperature koji (a type of starter culture) by mixed-culture activated yeast, specifically including the following steps:

[0051] (1) Inorganic nitrogen culture medium: By weight, the culture medium was prepared by combining 15 parts glucose, 15 parts ammonium sulfate, 0.006 parts biotin, 0.0012 parts calcium pantothenate, 0.00056 parts folic acid, 0.002 parts inositol, 0.000056 parts para-aminobenzoic acid, 0.0048 parts pyridoxine hydrochloride, 0.0009 parts riboflavin, 0.00056 parts thiamine hydrochloride, 3.12 parts potassium dihydrogen phosphate, 6.48 parts dipotassium hydrogen phosphate, 1 part sodium chloride, 0.39 parts magnesium chloride, 0.1 parts manganese sulfate, 0.005 parts ferrous sulfate heptahydrate, and 1000 parts water. After sterilization at 121℃ and 0.1 MPa for 20 min, the temperature was lowered to 40℃ and kept warm to obtain the inorganic nitrogen culture medium.

[0052] (2) Delayed acid co-activation: Inorganic nitrogen medium was first inoculated with 10 6 *Zygosaccharomyces rouxii* and *Hansøe d'Baryia* were activated by incubation for 2 days and then inoculated with 10 cfu / mL *Zygosaccharomyces rouxii*. 6 CFU / mL Saccharomyces cerevisiae was activated by incubation for 3 days before inoculation with Lactobacillus plantarum (10). 6 Continue activation for 6 days (cfu / mL) and then isolate the bacterial cells.

[0053] (3) Delayed high temperature co-activation: The bacterial cells separated in step (2) are re-inoculated into fresh inorganic nitrogen medium and step (2) is repeated. Each time the activation temperature is increased by 5°C until activation is completed at 60°C. The bacterial cells and fermentation broth are then separated for later use.

[0054] (4) Preparation of activated seed solution: Take the bacterial cells separated in step (3) and mix them with an equal mass of PBS containing 1% wt NaCl to form a bacterial suspension. Take 30 parts of the bacterial suspension and mix it with 70 parts of soybean oil (containing 2 wt% polyglycerol ricinoleate) and shear until the oil droplets are uniform. Then add 200 parts of the fermentation broth separated in step (3) (containing 1 wt% t Tween 80) and shear it evenly to obtain the activated seed solution.

[0055] (5) Second-stage koji making: Crushed wheat is moistened with 35wt% water and steamed at 0.2MPa for 30min. Transfer to a sterilized disc koji making machine, and evenly mix in 5wt% activated seed liquid. Loosen the koji until the material thickness is 30cm. Ventilate and loosen the koji every 8h and cultivate for 2 days to produce koji. Evenly mix 3wt% mother koji (purchased from Liangshan Changjiu Biological Koji Making Co., Ltd., model: high-temperature koji) into the koji material, and adjust the moisture content to 40% with the reserved fermentation liquid before making koji blocks.

[0056] (6) Fermentation of Daqu: Place the Daqu blocks in a sealed Daqu room and ferment at an initial temperature of 40℃ for 2 days. Then ventilate for 2 days and turn the Daqu once a day. Reseal the room and raise the temperature by 3℃ every day for 7 days. Then lower the temperature by 5℃ every day for 6 days to obtain Daqu. Daqu can be used as mother Daqu or directly for fermentation.

[0057] Comparative Example 1

[0058] This comparative example provides a method for enhancing the esterification ability of high-temperature Daqu (a type of starter culture) by activating mixed-culture yeast. It is similar to Example 1, except that 15 parts of ammonium sulfate, the only nitrogen source in the inorganic nitrogen culture medium, are replaced with organic nitrogen sources, namely 7.5 parts of beef extract and 7.5 parts of peptone. All other process conditions are the same as in Example 1.

[0059] Comparative Example 2

[0060] This comparative example provides a method for enhancing the esterification ability of high-temperature Daqu (a type of starter culture) by activating mixed-culture yeast, similar to Example 1, except that steps (2) and (3) are as follows:

[0061] (2) The inorganic nitrogen medium was inoculated with 10g each of Zygosacchari rouxae, Sacchari hanshen d'Bary, Sacchari sacchari, and Lactobacillus plantarum. 6 After activating the cells with CFU / mL for 11 days, the bacterial cells were isolated.

[0062] (3) The isolated cells are re-inoculated into fresh inorganic nitrogen medium and step (2) is repeated. Each time the activation temperature is increased by 5-10°C until activation is completed at 60°C. The cells and fermentation broth are then separated for later use.

[0063] Test Example 1

[0064] Collect the activated fermentation broth from step (3) of each embodiment, as well as the mature yeast sample, and dilute with sterile water at a concentration of 10 g / L. 5 10 6 10 7 The samples were spread onto MRS solid medium and incubated for 48 hours. Colony counting was performed to assess the biomass of the activated strain. The acidity of the koji was determined according to the light industry standard QB / T 4257—2011, and the total alcohol was determined according to the national standard GB 5009.225-2016 (samples were mixed before testing, with 10 samples, and the results are presented as average values).

[0065] The total bacterial count of the activated fermentation broth and the mature yeast is as follows: Figure 1 As shown in the comparison between Examples 1 and 2, the total number of bacteria in the activated fermentation broth increased by about 1.8 times under inorganic nitrogen treatment. Ammonium sulfate, as a fast-acting nitrogen source and a potential stimulating factor, can promote the rapid reproduction of bacteria, while avoiding the exclusionary inhibition effect caused by the absolute dominance of Lactobacillus plantarum under organic nitrogen environment.

[0066] Comparing Examples 1 and 3, it can be seen that ( Figure 1 In Example 1, the total bacterial count of the mature yeast after strengthening stress tolerance increased by an order of magnitude, approximately 4.12 × 10⁻⁶. 7 The cfu / g concentration showed significant activation effects. Simultaneous sequential inoculation prolonged the activation period to enrich esterification metabolic substrates (CFU / g). Figure 2 Compared with co-inoculation, the acid and alcohol were enriched by 1.68 mmol / 10g and 0.229% vol, respectively.

[0067] Comparative Example 3

[0068] This comparative example provides a method for preparing high-temperature Daqu (a type of starter culture) without mixed-culture activation for control verification, including:

[0069] (1) Activation of the strain: Prepare a culture medium by mixing 50 parts by weight of commercial MRS medium with 1000 parts by weight of water. Sterilize at 121℃ and 0.1 MPa for 20 min, then cool to 40℃ and incubate. *Zygosacchariformis*, *Hansophila d'Barry*, *Sacchariformis*, and *Lactobacillus plantarum* were incubated at 10... 6 CFU / mL was inoculated alone and activated for 11 days.

[0070] (2) Koji making: Crushed wheat is moistened with 35wt% water and steamed at 0.2MPa for 30min. Transfer to a sterilized disc koji making machine, and evenly mix in 5wt% activated seed liquid (1.25% of each of the four activated bacteria). Loosen the koji until the material is 30cm thick. Ventilate and loosen the koji every 8h and cultivate for 2 days to produce koji. Evenly mix 3wt% mother koji into the koji material, and adjust the moisture content of the reserved fermentation liquid to 40% before making koji blocks.

[0071] (3) Fermentation of Daqu: Place the Daqu blocks in a sealed Daqu room and ferment at an initial temperature of 40℃ for 2 days. Then ventilate for 2 days and turn the Daqu once a day. Reseal the room and raise the temperature by 3℃ every day for 7 days. Then lower the temperature by 5℃ every day for 6 days to obtain Daqu. Daqu can be used as mother Daqu or directly for fermentation.

[0072] Test Example 2

[0073] Based on the paper "Study and Analysis of Volatile Flavor Compounds of Sweet Wine Starter from Different Regions by Combining HS-SPME and GC-MS" published by Xiang Ling et al., the esterification capacity of Daqu was determined (samples were mixed and tested, with 10 samples, and the results are presented as average values).

[0074] like Figure 3 As shown, the esterification ability of the koji in this invention is significantly improved. The characteristic flavors representing the aroma of wine, such as ethyl hexanoate, ethyl lactate, and ethyl acetate, are increased by 38.21 μg / g, 39.95 μg / g, and 48.06 μg / g, respectively, compared with traditional koji blocks. Furthermore, the diversity of esters is increased by 6 types.

[0075] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for enhancing the esterification ability of high-temperature koji (a type of starter culture) by mixed-culture activated yeast, characterized in that, Includes the following steps: (1) In an inorganic nitrogen culture, inoculate *Zygosacchariformis* and *Hansøe du Barry*, keep warm and activate for 1 to 3 days, then inoculate *Sacchariformis*, keep warm and activate for 3 to 5 days, then inoculate *Lactobacillus plantarum*, continue activation for 5 to 7 days, and then isolate the cells. (2) Re-inoculate the isolated bacterial cells obtained in step (1) into the inorganic nitrogen culture medium, and repeat step (1) until activation is complete, and separate the bacterial cells from the fermentation broth; each time the activation temperature is increased by 5℃~10℃; (3) Mix the bacterial suspension containing the bacterial cells obtained in step (2) with soybean oil containing polyglycerol ricinoleate and shear until the oil droplets are uniform. Then add the fermentation broth obtained in step (2) and shear until uniform to obtain activated seed liquid. (4) After the crushed wheat is moistened and steamed, it is mixed with 0.5 wt%~5 wt% of the activated seed liquid obtained in step (3), and loosened until the material thickness is 20 cm~40 cm. After 1 to 3 days of ventilation and loosening, the koji is produced; 0.5 wt%~5 wt% of the mother koji and the fermentation liquid obtained in step (2) are mixed in to make koji blocks; (5) Place the koji blocks obtained in step (4) in a sealed koji room for initial fermentation at a low temperature for 1 to 3 days, then ventilate for 2 to 3 days and turn the koji once a day; reseal and raise the temperature by 3℃ to 5℃ every day for 5 to 7 days, then lower the temperature by 3℃ to 5℃ every day for 6 to 8 days to obtain the finished koji; the finished koji can be used as the mother koji or directly for fermentation. The inorganic nitrogen culture medium, by weight, comprises the following components: 10-20 parts glucose, 10-20 parts ammonium sulfate, 0.01-0.05 parts vitamins, 10-15 parts inorganic salts, and 1000 parts water.

2. The method according to claim 1, characterized in that, In step (1), the inoculum size of *Zygosacchariformis* and *Hansøe dulcis* is 10. 6 cfu / mL ~10 7 cfu / mL.

3. The method according to claim 1, characterized in that, In step (1), the inoculation amount of the brewing yeast is 10. 6 cfu / mL ~10 7 cfu / mL.

4. The method according to claim 1, characterized in that, In step (1), the inoculum size of *Lactobacillus plantarum* is 10. 5 cfu / mL ~10 6 cfu / mL.

5. The method according to claim 1, characterized in that, In step (3), the bacterial suspension is obtained by mixing the bacterial cells with an equal mass of PBS buffer containing 1 wt% NaCl.

6. The method according to claim 1, characterized in that, In step (3), by weight, the activated seed liquid contains 30 to 50 parts of bacterial suspension, 50 to 70 parts of soybean oil containing polyglycerol ricinoleate, and 100 to 200 parts of fermentation liquid.

7. The method according to claim 1, characterized in that, In step (4), the moisture content of the block is 35%~40%.

8. The method according to claim 1, characterized in that, In step (5), the initial temperature is 35℃~40℃.

9. The application of the method according to any one of claims 1-8 in the preparation of Maotai-flavor liquor.

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