A method for increasing the thickness of mellow liquor

Abstract

The invention discloses a method for improving the thickness of mellow liquor, and belongs to the technical field of winemaking. The invention improves the thickness of mellow liquor by synergistically strengthening aerobic microorganisms and anaerobic microorganisms. Among them, aerobic microorganisms refer to the screened pyrazine-producing Bacillus subtilis CCTCC No: M2023090 and guaiacol-producing abnormal Wickham yeast CCTCC No: M2023089, and anaerobic microorganisms are Clostridium cohnii CICC 24509, Sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 24376. The anaerobic functional bacteria promote the production of complex acids, and the aerobic functional microorganisms enrich the trace flavor and health function active ingredients. The synergistic effect of the two greatly improves the thickness of mellow liquor, and the produced raw liquor has an elegant and coordinated cellar aroma, a mellow and full taste, a long and round aftertaste, and a refreshing and clean tail.

Description

Technical Field

[0001] The invention relates to a method for improving the thickness of mellow and soft liquor, belonging to the technical field of winemaking. Background Art

[0002] Baijiu, also known as Shaojiu, Laobaigan, Shaodaozi, etc., has a complex aroma with esters as the main component. It is made from starch (sugar) raw materials through cooking, saccharification, fermentation, distillation, aging and blending, using koji and yeast as saccharification and fermentation agents. Baijiu is divided into the following flavor types: sauce-flavored, light-flavored, strong-flavored, Laobaigan-flavored, rice-flavored, phoenix-flavored, mixed-flavored, Dong-flavored, and other flavor types.

[0003] A mellow and soft type of liquor refers to a liquor that uses grains as raw materials, uses medium- and high-temperature Daqu, high-temperature Daqu, etc. as saccharification and fermentation agents, and is steamed, hung, mixed with ingredients, layered into cellars for solid-state fermentation, solid-state distilled, and staged wine collection. It is a combination of multiple flavors and has a unique style of mellow, sweet, soft, thick, coordinated, and long-lasting.

[0004] The thickness of mellow liquor, or the thickness of mellowness, is a sensory feature used to characterize and evaluate the quality and style differences of mellow liquor. It refers to the comprehensive sensory experience of mellow liquor being soft, gentle, full and heavy in the mouth. It is the feeling of thickness felt by the taster through the taste buds in the mouth. The thickness of mellow liquor is an evaluation system that supports the qualitative characteristics of the sensory system through the quantitative expression of the content of flavor substances. Flavor substances mainly refer to trace components such as heterocyclic substances and organic acids with high contents of macromolecules represented by pyrazine and guaiacol. The targeted improvement from microorganisms to metabolic components to sensory thickness through modern biotechnology is an important means to promote the dual improvement of the sensory quality and health functions of mellow liquor.

[0005] The prior art has also made some attempts to improve the flavor of liquor. For example, CN102766576A discloses a combination of bacterial agents, which includes yeast, Bacillus, and mold. When introduced into the fermented grains or fermented glutinous rice in the brewing process, the alcohol, volatile acid, and ester in the liquor can be increased, and the characteristic aroma, fullness, fineness, sweetness, and comfort of the liquor can also be improved. For another example, CN116064270A discloses a strain of Bacillus licheniformis that produces protease and multiple flavor substances at the same time. The strain can produce protease, 2,5-dimethylpyrazine, trimethylpyrazine, tetramethylpyrazine, phenylethanol, etc. at the same time, which is beneficial to the decomposition of protein in the raw materials and the improvement of the flavor of the liquor in the brewing of liquor.

[0006] However, these methods of improving the flavor of liquor by using microbial compositions cannot achieve the effect of increasing the thickness of mellow liquor. The main reason is that there is no clear concept of the characteristic indicators of the sensory quality dimension supported by the flavor substance dimension of thickness, nor is it clear that improving thickness requires the comprehensive improvement of multiple flavor substances, and there is no disclosure of the metabolomics regulatory mechanism of the interaction between the flavor substance system of thickness, the microecological system, and multiple microbial flora. Summary of the invention

[0007] In order to improve the thickness of mellow liquor, the present invention provides a method for improving the thickness of mellow liquor, which improves the thickness of mellow liquor by synergistically strengthening aerobic microorganisms and anaerobic microorganisms. The aerobic microorganisms are the screened pyrazine-producing Bacillus subtilis CCTCC No: M2023090 and the guaiacol-producing Wickerhamomyces anomalus CCTCC No: M2023089, which have strong advantages in metabolizing pyrazine and guaiacol compounds, respectively. They are now preserved in the China Collection Center for Typical Objects. The aerobic microorganisms are prepared into solid special flavor targeted songs using specific culture media and culture conditions to enhance the sensory flavor and the content of health active substances, including pyrazine, terpenes, guaiacol and other heterocyclic compounds; the anaerobic microorganisms are Clostridium kluyveri CICC 24509, Sporosarcina sp. CICC24685, Clostridium tyrobutyricum CICC 24376, which can efficiently metabolize and produce a variety of complex acids in liquor, wherein the complex acids refer to monobasic or polybasic organic acids such as caproic acid, lactic acid, butyric acid, propionic acid, acetic acid, 1,2-dipropionic acid, and succinic acid. The anaerobic microorganisms are prepared into liquid functional microbial enhanced liquid under specific culture conditions, which is used for regulating the cellar microbial system to increase the number of target bacteria, inhibit the content of anaerobic bacteria producing single acids, and ultimately increase the complex organic acids and other flavor components.

[0008] The method for increasing the thickness of mellow liquor mainly comprises the following steps:

[0009] (1) A variety of acid-producing anaerobic bacteria were inoculated into a selective medium for producing complex acids using lactic acid as the sole carbon source, and strains of Clostridium cohnii CICC 24509, Sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 24376 with strong ability to produce complex acids using lactic acid as the sole carbon source were screened out, and the strains were inoculated into a primary expansion culture medium, and statically cultured at 37°C for 8-10 days to obtain a primary seed solution;

[0010] (2) The first-level seed liquid of Clostridium cohnii CICC 24509, Sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 24376 were mixed at a ratio of 1:1:1 and inoculated into the second-level expansion culture medium, and the second-level expansion culture was carried out at 37°C for 8-10 days to obtain the composite culture liquid of anaerobic bacteria producing composite acid;

[0011] (3) using a common bacterial nutrient medium and a fungal YPD medium to carry out liquid primary expansion culture on Bacillus subtilis CCTCC No: M2023090 and abnormal Wickham yeast CCTCC No: M2023089, respectively, wherein the liquid primary expansion culture is to culture Bacillus subtilis CCTCC No: M2023090 and abnormal Wickham yeast CCTCC No: M2023089 at 30° C. and 37° C. with ventilation and stirring for 1 day, respectively, to prepare liquid seeds for preparing solid special flavor targeted koji;

[0012] (4) preparing a solid culture medium with wheat flour, barley flour and pea flour in a mass ratio of 89:10:1, wherein the moisture content of the solid culture medium is 35%, and inoculating liquid seeds of Bacillus subtilis CCTCC No: M2023090 and abnormal Wickham yeast CCTCC No: M2023089 at 0.6-1% (w / w), respectively, and carrying out a ventilation solid culture at 37° C. for 36 hours in a solid disc, and then gradually increasing the temperature to 55° C. for further culturing for 36 hours, and carrying out a ventilation solid culture at 30° C. for 72 hours for abnormal Wickham yeast CCTCC No: M2023089, and drying the Bacillus subtilis and abnormal Wickham yeast at 40° C. after the cultivation is completed, and then mixing them to prepare a special flavor targeted koji for use;

[0013] (5) Before the mash is put into the cellar, the temperature should be 0.8-1.2kg / m 3 / The amount added to the cellar is to evenly spray the composite culture solution of anaerobic bacteria producing composite acids on the bottom of the cellar and the surrounding cellar walls; the solid special flavor targeted qu is evenly mixed with the daqu at a ratio of 1-5% of the daqu dosage and then added to the mash, and the mixture is layered into the cellar, fermented, distilled, and the wine is taken.

[0014] In one embodiment, the formula of the selective culture medium for producing complex acid prepared with lactic acid as the sole carbon source in step (1) is (w / w): 2% lactic acid, 0.5% cellar mud extract (prepared by soaking and filtering mature cellar mud), 0.5% yeast extract, pH 7.0, sterilized at 121°C for 20 minutes. The culture temperature of the anaerobic complex bacteria is 37°C.

[0015] In one embodiment, the formula of the primary expansion culture medium in step (1) and the secondary expansion culture medium in step (2) is (w / w): 1% peptone, 0.5% yeast extract, 0.5% sodium chloride, pH 7.0. The liquid volume is ≥90%, and sterilized at 121°C for 20 minutes.

[0016] In one embodiment, the formula of the common bacterial nutrient medium used in step (3) is (w / w): 1% beef extract, 2% yeast extract, 0.5% peptone, 0.5% NaCl, pH 7.0. Sterilize at 121°C for 20 minutes. The bacterial culture temperature is 37°C.

[0017] In one embodiment, the fungal YPD medium used in step (3) has a formula (w / w): 1% yeast extract, 2% peptone, pH 7.0, sterilized at 121°C for 20 min, and the fungal culture temperature is 37°C.

[0018] In one embodiment, the solid culture medium used in step (4) is formulated as follows: wheat flour, barley flour, and pea flour are mixed in a mass ratio of 89:10:1, with a moisture content of 35%, and sterilized at 121° C. for 20 min.

[0019] In one embodiment, the inoculation amount of Bacillus subtilis and yeast liquid seeds in step (4) is 1% (w / w).

[0020] In one embodiment, in step (4), Bacillus subtilis is cultured in a solid disc at 37°C for 36 hours in a ventilated solid state, then the temperature is gradually increased to 55°C for further culturing for 36 hours, and abnormal Wickham yeast is cultured in a ventilated solid state at 30°C for 72 hours. After the cultivation of both, they are dried at 40°C to prepare special flavor targeted koji for use.

[0021] Beneficial effects of the present invention:

[0022] The inventors have found that the thickness of liquor has a significant positive correlation with the complex acids, flavors and health functional active substances in the liquor, and "enriching the taste" is the key to improving the thickness of liquor. Complex acids are mainly generated by the metabolism of anaerobic microorganisms provided by the cellar mud in the brewing system. Screening, expanding and applying anaerobic microorganisms that produce complex acids are the key to improving the content of complex acids in mellow liquor; Clostridium cohnii, Sarcina, Clostridium tyrobutyricum and other microorganisms that produce complex acids can not only ensure the main style of mellow liquor, but also increase the types and content of complex acids. For complex flavors and health functional active substances, such as pyrazine, terpenes, guaiacol and other heterocyclic compounds, they mainly come from aerobic microorganisms in the koji making and stacking processes. Screening, expanding and applying key functional microorganisms that produce key flavors and health functional active substances are the key to improving the functional trace components of liquor.

[0023] The invention selects, purifies and then expands the culture of functional bacteria that can use lactic acid as the sole carbon source to produce complex acids in the mud of old thick-producing pits, thereby obtaining anaerobic functional strains that can grow and metabolize complex acids, including Clostridium cohnii CICC 24509, Sporosarcina CICC 24685 and Clostridium tyrobutyricum CICC 243776. On the one hand, using lactic acid as the only carbon source can deeply convert lactic acid that exists in large quantities in the mash and is not easily distilled into the wine. On the other hand, anaerobic bacteria producing compound acids can inhibit the abundance of caproic acid bacteria producing single caproic acid, thereby achieving the purpose of reducing single lactic acid and single caproic acid and increasing compound acids in the mash; anaerobic bacteria producing compound acids can utilize lactic acid to increase the types and contents of compound acids, laying the foundation for increasing the thickness of mellow-type liquor, and promoting the diversity of bacterial species and metabolic diversity of cellar mud functional flora. Anaerobic microorganisms producing compound acids are made into bacterial liquid and sprayed on the bottom of the cellar and the surrounding walls of the cellar, which can increase the relative abundance of anaerobic bacteria in the cellar mud by more than 2 times and increase the content of organic acids in high-grade liquor by more than 8.88%, thereby promoting the realization of the purpose of "enriching microorganisms and increasing flavor".

[0024] The present invention screens out Bacillus subtilis with high tetramethylpyrazine production and yeast with high 4-ethylguaiacol production, and cultivates them under the most suitable composite culture medium conditions by using modern disc koji making technology combined with temperature gradient cultivation to obtain special flavor targeted koji that has both high-yield functional active substance bacteria and rich flavor and aroma active substances. These two strains are made into special flavor targeted koji and then mixed with Daqu, which can achieve targeted enhancement of functional microorganisms in the cellar and make up for the lack of microorganisms producing key flavor components in Daqu.

[0025] The present invention sprays the expanded culture of anaerobic functional bacteria liquid with high production of composite acid into the cellar for brewing mellow and soft liquor, and combines the application of special flavor targeted koji, and ferments them together in the cellar. The anaerobic functional bacteria promote the production of composite acid, and the aerobic functional microorganisms enrich the trace flavor and health functional active ingredients to achieve the purpose of "enriching the trace flavor and enhancing the taste". The synergistic effect of the two greatly improves the thickness of the mellow and soft liquor. Production tests show that the above technology can make the original liquor have an elegant and coordinated cellar aroma, a mellow and full taste, a long and round aftertaste, a refreshing and clean tail, and a 30-40% increase in the total amount of acids compared with the control, and a 3-5 times increase in functional active substances represented by tetramethylpyrazine and 4-ethylguaiacol.

[0026] The present invention proposes for the first time a new targeted regulation technology of aerobic and anaerobic microorganisms multi-mode preparation, multi-strain application of synergistic fermentation, and multi-flavor substance complementation; multi-strains lay the foundation for multi-metabolism of solid-state fermentation, multi-mode preparation can increase the number of microbial cultures under optimal conditions, avoiding competitive inhibition in early culture, and the gradient temperature rise culture mode simulates the traditional Daqu natural fermentation process, which is conducive to stimulating the microorganisms' own metabolism to produce flavor and heat, and improving the adaptability of high-temperature microbial cells and the synthesis of flavor substances under high temperature conditions.

[0027] Biomaterial Deposit

[0028] Wickerhamomyces anomalus YHY0216 was deposited in the China Center for Type Culture Collection on February 3, 2023, with the deposit address being Wuhan University, China, and the deposit number being CCTCC No: M2023089.

[0029] Bacillus subtilis YHB0304 (Bacillus subtilis) was deposited in the China Center for Type Culture Collection on February 3, 2023, with the deposit address being Wuhan University, China, and the deposit number being CCTCC No: M2023090. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 :Soft and mellow process flow chart. DETAILED DESCRIPTION

[0031] The strains used in the following examples are:

[0032] Bacillus subtilis CCTCC No: M2023090 and Wickerhamomyces anomalus CCTCC No: M2023089 were deposited in China Center for Type Culture Collection. Caproicibacterium sp. CICC 24509, Sporosarcina sp. CICC 24685, and Clostridium tyrobutyricum CICC 24376 were purchased from China Center of Industrial Culture Collection (CICC).

[0033] Example 1 (Acidification + Pyrazine and Guaiacol)

[0034] The formula of the complex acid production selective culture medium made with lactic acid as the sole carbon source is (w / w): lactic acid 2%, pit mud extract 0.5%, yeast extract 0.5%, pH 7.0. Sterilize at 121°C for 20 minutes. Cultivate the bacteria at 37°C.

[0035] The formula of the primary expansion culture medium and the secondary expansion culture medium for the production of composite acid from high-quality cellar mud of thick liquor is (w / w): 1% peptone, 0.5% yeast extract, 0.5% sodium chloride, liquid volume ≥ 90%, pH 7.0, sterilized at 121℃ for 20min. The cellar mud functional bacteria were deep cultured at 37℃.

[0036] The liquid high-yield tetramethylpyrazine medium is (w / w): corn saccharification liquid (9BX), peptone 1%, beef extract 0.5%, ammonium sulfate 0.09%, magnesium sulfate 0.03%, pH 7.0. Sterilize at 121°C for 20 minutes.

[0037] The liquid high-yield 4-ethylguaiacol culture medium is (w / w): 25% bran, 25% corn flour, 50% water, pH 7.0, and sterilized at 121°C for 20 minutes.

[0038] The formula of the common nutrient medium for bacteria (Bacillus subtilis) is (w / w): 1% beef extract, 2% yeast extract, 0.5% peptone, 0.5% NaCL, pH 7.0. Sterilize at 121°C for 20 minutes. Cultivate Bacillus subtilis at 37°C.

[0039] The formula of fungus (yeast) YPD medium is (w / w): 1% yeast extract, 2% peptone, pH 7.0. Sterilize at 121°C for 20 min. Cultivate yeast at 37°C.

[0040] The solid culture medium formula used for preparing special flavor targeted koji is: wheat flour, barley flour, pea flour at a ratio of 89:10:1, with a moisture content of 35%, and sterilized at 121°C for 20 minutes.

[0041] Use a sterilized sampler to take about 10g of fresh pit mud around and at the bottom of a typical thick-style high-quality old pit, immediately put it into a sampling bag, seal it and store it in a refrigerator at 0-4℃ for later use; inoculate the above pit mud at 1% (w / w) into the sterilized pit mud functional bacteria selective culture medium for producing complex acids with lactic acid as the only carbon source, and culture it at 37℃ for 10 days in deep culture. Inoculate the above obtained anaerobic functional microorganisms for producing complex acids at 10% (w / w) into the sterilized pit mud functional bacteria expansion culture medium for producing complex acids with lactic acid as the only carbon source, and culture it at 37℃ for 3 days in deep culture. Purify and identify the following strains of pit mud functional microorganisms for producing complex acids with lactic acid as the only carbon source: Clostridium cohnii CICC 24509, Sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 243776.

[0042] Use a sterilized sampler to select high-quality Daqu to separate high-yield tetramethylpyrazine and 4-ethylguaiacol functional microorganisms. Specifically, weigh 1g of crushed Daqu and add it to a test tube containing 10mL of sterilized physiological saline. After shaking, treat it in an 80℃ water bath for 10min. Take 1mL of liquid and add it to 10mL of liquid high-yield tetramethylpyrazine culture medium, culture it at 37℃ for 96h, and perform GC-MS analysis after fermentation. After determining the high-yield tetramethylpyrazine Bacillus, use the above solid culture medium for separation and purification to obtain a pure strain of high-yield tetramethylpyrazine Bacillus subtilis CCTCC No: M2023090.

[0043] Weigh 1 g of crushed Daqu and add it to a test tube containing 10 mL of sterile physiological saline. After shaking, take 1 mL of the liquid and add it to 10 mL of liquid high-yield 4-ethylguaiacol culture medium. Cultivate at 37° C. for 96 h. After the fermentation is completed, perform GC-MS analysis to determine the yeast with high 4-ethylguaiacol production, and then use the above solid culture medium for separation and purification to obtain a pure strain of high-yield 4-ethylguaiacol yeast CCTCC No: M2023089.

[0044] (1) Inoculating caproic acid bacteria CICC 24509, sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 243776 into a primary expansion culture medium, respectively, and culturing them in a closed static culture at 37° C. for 8-10 days to obtain a primary seed solution;

[0045] (2) The first-level seed liquids of caproic acid bacteria CICC 24509, sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 243776 were mixed at a ratio of 1:1:1, and then inoculated into a 1000 L fermentation tank filled with a second-level expansion culture medium at a ratio of 5% (w / w), and cultured in a closed environment at 37°C for 8-10 days to obtain a composite culture liquid of anaerobic bacteria producing composite acid;

[0046] (3) using a common bacterial nutrient medium and a fungal YPD medium to carry out liquid primary expansion culture on Bacillus subtilis CCTCC No: M2023090 and abnormal Wickham yeast CCTCC No: M2023089, respectively, wherein the liquid primary expansion culture is to culture Bacillus subtilis CCTCC No: M2023090 and abnormal Wickham yeast CCTCC No: M2023089 at 30° C. and 37° C. with ventilation and stirring for 1 day, respectively, to prepare liquid seeds for preparing solid special flavor targeted koji;

[0047] (4) The solid culture medium is inoculated with liquid seeds of Bacillus subtilis CCTCC No: M2023090 and abnormal Wickham yeast CCTCC No: M2023089 at 1% (w / w), and then the Bacillus subtilis is cultured in a solid disc at 37° C. for 36 h in a ventilation solid state, and then the temperature is gradually increased to 55° C. for further culture for 36 h, and the abnormal Wickham yeast is cultured in a ventilation solid state at 30° C. for 72 h. After the two are cultured separately, they are dried at 40° C. and mixed to prepare a special flavor targeted koji for use.

[0048] (5) Before the mash is put into the cellar, the temperature should be 0.8-1.2kg / m 3 The cellar evenly sprays the culture solution of anaerobic functional microorganisms producing complex acid on the bottom of the cellar and the surrounding cellar walls. At the same time, special flavor targeted qu accounting for 1-5% (w / w) of the qu dosage is mixed with the qu and added to the fermented grains, and then layered into the cellar, fermented, distilled, and wine is taken. The fermentation and distillation methods are consistent with the control group, with low temperature (17℃-19℃) and slow fermentation.

[0049] Example 2 (acidification only)

[0050] The culture medium formulation and strain screening used refer to Example 1.

[0051] (1) Inoculating caproic acid bacteria CICC 24509, sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 243776 into a primary expansion culture medium, respectively, and culturing them in a closed static culture at 37° C. for 8-10 days to obtain a primary seed solution;

[0052] (2) The first-level seed liquids of caproic acid bacteria CICC 24509, sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 243776 were mixed at a ratio of 1:1:1, and then inoculated into a 1000 L fermentation tank filled with a second-level expansion culture medium at a ratio of 5% (w / w), and cultured in a closed environment at 37°C for 8-10 days to obtain a composite culture liquid of anaerobic bacteria producing composite acid;

[0053] (3) Before the mash is put into the cellar, the temperature should be 0.8-1.2kg / m 3 The cellar will evenly spray the culture solution of anaerobic microorganisms producing complex acid on the bottom of the cellar and the surrounding cellar walls, and then put it into the cellar in layers, ferment, distill and take the wine. The fermentation and distillation methods are the same as those of the control group, with low temperature (17℃-19℃) and slow fermentation.

[0054] Embodiment 3 (acidification + pyrazine-rich)

[0055] The culture medium formulation and strain screening used refer to Example 1.

[0056] (1) Inoculating caproic acid bacteria CICC 24509, sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 243776 into a primary expansion culture medium, respectively, and culturing them in a closed static culture at 37° C. for 8-10 days to obtain a primary seed solution;

[0057] (2) The first-level seed liquids of caproic acid bacteria CICC 24509, sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 243776 were mixed at a ratio of 1:1:1, and then inoculated into a 1000 L fermentation tank filled with a second-level expansion culture medium at a ratio of 5% (w / w), and cultured in a closed environment at 37°C for 8-10 days to obtain a composite culture liquid of anaerobic bacteria producing composite acid;

[0058] (3) using a common bacterial nutrient medium to carry out a liquid primary expansion culture of Bacillus subtilis CCTCC No: M2023090, wherein the liquid primary expansion culture is to culture Bacillus subtilis CCTCC No: M2023090 at 30° C. and 37° C. with ventilation and stirring for 1 day, and prepare liquid seeds respectively, in preparation for preparing a solid special flavor targeted koji;

[0059] (4) Inoculating liquid seeds of Bacillus subtilis CCTCC No: M2023090 into a solid culture medium at 1% (w / w), then, the Bacillus subtilis is cultured on a disk at 37° C. for 36 h in a ventilated solid state, then gradually raised to 55° C. for further 36 h, and dried at 40° C. to prepare a special flavor targeted koji for later use.

[0060] (5) Before the mash is put into the cellar, the temperature should be 0.8-1.2kg / m 3 The cellar evenly sprays the culture solution of anaerobic functional bacteria producing complex acid on the bottom of the cellar and the surrounding cellar walls. At the same time, special flavor targeted qu accounting for 1-5% (w / w) of the qu dosage is mixed with the qu and added to the fermented grains, and then layered into the cellar, fermented, distilled, and wine is taken. The fermentation and distillation methods are consistent with the control group, with low temperature (17℃-19℃) and slow fermentation.

[0061] Example 4 (pyrazine + guaiacol only)

[0062] The culture medium formulation and strain screening used refer to Example 1.

[0063] (1) using a common bacterial nutrient medium and a fungal YPD medium to carry out liquid primary expansion culture on Bacillus subtilis CCTCC No: M2023090 and abnormal Wickham yeast CCTCC No: M2023089, respectively, wherein the liquid primary expansion culture is to culture Bacillus subtilis CCTCC No: M2023090 and abnormal Wickham yeast CCTCC No: M2023089 at 30° C. and 37° C. for 1 day with ventilation and stirring, respectively, to prepare liquid seeds for preparing solid special flavor targeted koji;

[0064] (2) Inoculate the solid culture medium with 1% (w / w) liquid seeds of Bacillus subtilis CCTCC No: M2023090 and abnormal Wickham yeast CCTCC No: M2023089, respectively. Then, the Bacillus is cultured in a solid disc at 37° C. for 36 h in a ventilation solid state, and then the temperature is gradually increased to 55° C. for further 36 h. The abnormal Wickham yeast is cultured in a solid state at 30° C. for 72 h in a ventilation solid state. After the two are cultured separately, they are dried at 40° C. and mixed to prepare a special flavor targeted koji for later use.

[0065] (3) Before the mash is put into the cellar, the special flavor targeted qu is mixed with the daqu at a rate of 1-5% (w / w) of the daqu, and then added to the mash, and the mash is put into the cellar in layers, fermented, distilled, and the wine is taken. The fermentation and distillation methods are the same as those of the control group, with low temperature (17°C-19°C) and slow fermentation.

[0066] Comparative Example 1 (Current Situation)

[0067] Compared with Example 1, a conventional mellow liquor process is adopted, and the step of strengthening aerobic microorganisms and anaerobic microorganisms is omitted (i.e., no composite culture solution of anaerobic bacteria producing composite acid is sprayed, and no solid special flavor targeted koji is used). The specific process route is as follows: Figure 1 shown.

[0068] Comparison of the mellow and soft liquor bases obtained according to the methods of Examples 1-4 and Comparative Example 1, the results are shown in Table 1.

[0069] The evaluation method of the cotton thickness level is as follows: 10 national judges are randomly selected from the national judge talent pool, and samples are taken in the same round and three groups of parallel methods for each embodiment and control, and then mixed evenly to reduce the alcohol content to 55 degrees. The "five cups method" is used to randomly encode and then evaluate and score, and the results are summarized and analyzed. Based on the evaluation conclusions of national wine judges and combined with market consumer feedback information, the cotton thickness is divided into the following three levels:

[0070] High Mian Thickness (8-10 points): Elegant and harmonious cellar aroma, mellow and full-bodied taste, long and mellow aftertaste, refreshing and clean aftertaste

[0071] Medium thickness (6-8 points): elegant and harmonious cellar aroma, round and full taste, long aftertaste, refreshing and clean aftertaste

[0072] Low cotton thickness (4-6 points): typical pure cellar aroma, mellow taste, lack of lingering aftertaste, and lack of clean aftertaste

[0073] Table 1 Comparison of sensory and main flavor substances in wine produced by the examples and control examples

[0074]

[0075]

[0076] Analysis of the data in Table 1 shows that caproic acid, acetic acid, lactic acid, butyric acid, and butyric acid are representative organic acids. The increase in cotton thickness is the increase in the types and contents of multiple organic acids, which is directly manifested by the increase in total acid, not the increase in a single acid. The increase in composite acid increases the cotton thickness, and a single acid is prone to irritation; ethyl caproate, ethyl acetate, ethyl lactate, ethyl butyrate, and ethyl butyrate are representative esters, and a single ester is prone to irritation. The increase or decrease of monomeric esters has not been directly associated with the thickness of cotton. Clostridium cohnii, Sarcina, and Clostridium tyrobutyricum in the microorganisms producing composite acid substances used in Example 1 can inhibit the abundance of caproic acid bacteria producing single caproic acid, thereby achieving the purpose of reducing single lactic acid and single caproic acid, and increasing the composite acid of fermented grains. Bacillus subtilis CCTCC No: M2023090 and abnormal Wickham yeast CCTCC No: M2023089 are made into special flavor targeted koji and then mixed with the Daqu, which can achieve targeted enhancement of functional microorganisms in the cellar and make up for the lack of microorganisms producing key flavor components in the Daqu.

[0077] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for increasing the thickness of mellow liquor, characterized in that: The main steps include: (1) Clostridium cohnii CICC 24509, Sporosarcina sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 24376 strains were inoculated into a primary expansion culture medium, and cultured in a closed manner to obtain a primary seed solution; (2) The first-level seed liquid of Clostridium cohnii CICC 24509, Bacillus sporosarcina CICC 24685, and Clostridium tyrobutyricum CICC 24376 were mixed at a ratio of 1:1:1 and inoculated into the second-level expansion culture medium, and the culture was closed to obtain the composite culture liquid of complex acid-producing anaerobic bacteria; (3) Bacillus subtilis ( Bacillus subtilis ) YHB0304 and Wickham's yeast ( Wickerhamomyces anomalus ) YHY0216 were respectively subjected to liquid primary expansion culture and made into liquid seeds in preparation for the production of solid special flavor targeted koji; (4) preparing a solid culture medium with wheat flour, barley flour and pea flour in a mass ratio of 89:10:1, wherein the moisture content of the solid culture medium is 35%, and inoculating liquid seeds of Bacillus subtilis YHB0304 and abnormal Wickham yeast YHY0216 at 0.6-1% (w / w), respectively; after Bacillus subtilis YHB0304 is cultured in a solid disc at 37°C for 36 hours, the temperature is gradually increased to 55°C for further culture for 36 hours, and abnormal Wickham yeast YHY0216 is cultured in a solid disc at 30°C for 72 hours; after the culture of Bacillus subtilis and abnormal Wickham yeast is completed, they are dried at 40°C, and then mixed to prepare a special flavor targeted koji for use; (5) Before the mash is put into the cellar, the temperature should be 0.8-1.2kg / m 3 / Amount added to the cellar: Spray the composite culture solution of anaerobic bacteria producing composite acid evenly on the bottom of the cellar and the surrounding cellar walls; Mix the solid special flavor targeted qu with the daqu at a ratio of 1-5% of the daqu dosage and add it to the fermented grains, put them into the cellar in layers, ferment, distill and take out the wine; Among them, Bacillus subtilis ( Bacillus subtilis ) YHB0304, deposited on February 3, 2023 in China Center for Type Culture Collection, Wuhan University, China, with the deposit number CCTCC No: M2023090; Abnormal Wickham yeast ( Wickerhamomyces anomalus )YHY0216, deposited in the China Center for Type Culture Collection on February 3, 2023, with the deposit address at Wuhan University, China, and the deposit number is CCTCC No: M2023089.

2. The method for increasing the thickness of mellow and soft liquor according to claim 1, characterized in that: The formula of the primary expansion culture medium in step (1) and the secondary expansion culture medium in step (2) is (w / w): 1% peptone, 0.5% yeast extract, 0.5% sodium chloride, pH 7.

0.

3. The method for increasing the thickness of mellow and soft liquor according to claim 1, characterized in that: The formula of the bacterial common nutrient medium used in step (3) is (w / w): 1% beef extract, 2% yeast extract, 0.5% peptone, 0.5% NaCl, pH 7.0, sterilized at 121°C for 20 min.

4. The method for increasing the thickness of mellow and soft liquor according to claim 1, characterized in that: The fungal YPD medium used in step (3) has the following formula (w / w): 1% yeast extract, 2% peptone, pH 7.0, sterilized at 121°C for 20 min, and the fungal culture temperature is 37°C.

5. The method for increasing the thickness of mellow liquor according to claim 1, characterized in that: The inoculation amount of Bacillus subtilis and yeast liquid seeds in step (4) is 1% (w / w).

6. The method for increasing the thickness of mellow liquor according to claim 1, characterized in that: The closed culture in step (1) and step (2) is static closed culture at 37° C. for 8-10 days.

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