Production method for increasing content of pyrazine substances in wine by adopting out-cellar fermentation and fragrance mixing process

Abstract

The invention discloses a production method for increasing the content of pyrazine substances in liquor by adopting an out-cellar fermentation and fragrance mixing process, and belongs to the technical field of liquor brewing. The method comprises the following steps: by utilizing the metabolic characteristic of high-yield pyrazine substances of bacillus subtilis YHB0304 screened from medium-high temperature Daqu, taking wheat and peas as raw materials, crushing, cooking, spreading for cooling, inoculating seed fermentation liquor, and carrying out two-stage temperature-controlled fermentation outside a cellar, so as to prepare a fermentation material rich in pyrazine substances. And mixing aroma with strong-flavor fermented grains which are maturely fermented, distilling and receiving the wine, so that pyrazine substances in the raw wine reach more than 12mg / L. The strain, the raw materials and the production method all follow the characteristics of traditional brewing processes such as solid fermentation and distillation of baijiu, the independence and integrity of the brewing process of the strong-flavor baijiu are reserved, food safety risks caused by exogenous addition do not exist, and the method has good application and popularization value.

Description

Technical Field

[0001] The present invention relates to a production method for increasing the content of pyrazine substances in liquor by means of out-cellar fermentation and flavor-strengthening process, belonging to the technical field of liquor brewing. Background Art

[0002] Pyrazine substances play an important role in the flavor of liquor due to their flavor characteristics such as roasted aroma, nutty aroma, and coffee aroma. In addition, they also have certain benefits to human health, such as preventing cardiovascular diseases and regulating physiological functions. Especially tetramethylpyrazine (chuanxiongzine) has pharmacological effects such as dilating blood vessels, slightly reducing blood pressure, improving tissue circulation, inhibiting platelet adhesion aggregation and thrombosis formation.

[0003] There are significant differences in the content of pyrazine substances in different flavor-type liquors. For example, the content of pyrazine compounds in sauce-flavor liquor is the highest and the variety is also the most, with the content range roughly being 3 - 6 mg / L, while the content and variety of pyrazine compounds in strong-flavor liquor are less, below 1 mg / L. This difference is related to various factors such as the brewing process, raw material selection, and geographical environment of liquor. The formation mechanism of pyrazine substances is generally that pyruvate is produced by glycolysis, pyruvate generates α-acetolactate under the action of acetolactate synthase, then decarboxylates to form acetoin. Acetoin, as an intermediate of aroma substances, reacts with ammonia converted from amino acids in the fermentation system through a non-enzymatic reaction to finally generate tetramethylpyrazine. It is the characteristics of high-temperature koji-making, high-temperature stacking, and high-temperature fermentation in the sauce-flavor process that promote the formation of tetramethylpyrazine.

[0004] Aiming at the problem that the brewing process of "low-temperature entering the pit and low-temperature fermentation" in strong-flavor liquor is not conducive to the production of pyrazine substances, it is urgent to develop a new brewing process in order to increase the content of pyrazine substances in base liquor. Summary of the Invention

[0005] Aiming at the deficiencies of the above-mentioned existing technologies, the present invention provides a production method for increasing the content of pyrazine substances in liquor by means of out-cellar fermentation and flavor-strengthening process, aiming to solve the technical problem that the existing brewing process of "low-temperature entering the pit and low-temperature fermentation" in strong-flavor liquor is not conducive to the production of pyrazine substances.

[0006] The first technical method provided by the present invention is a brewing method for strong-flavor liquor, including the following steps:

[0007] (1) Expansion culture of high-yield pyrazine strains: Inoculate Bacillus subtilis YHB0304 into a liquid medium to prepare a seed solution;

[0008] (2) Preparation and steaming-cooling of fermentation substrate: Mix crushed wheat and peas, then steam and cool them to obtain a fermentation substrate;

[0009] (3) fermentation outside the cellar: inoculating the seed solution prepared in step (1) into the fermentation matrix prepared in step (2), performing medium-temperature and high-temperature two-stage temperature-controlled fermentation, and drying after completion to obtain a fermentation material rich in pyrazines;

[0010] (4) soaking the material: soaking the fermented material prepared in step (3) in advance with water or original wine as a soaking liquid to obtain a soaked material for standby use;

[0011] (5) Aroma concatenation process: the soaking material prepared in step (4) and the fermented grains of Luzhou-flavor liquor are loaded into the retort in any of the following ways: (a) the soaking material and the fermented grains of Luzhou-flavor liquor are stirred evenly and put into the retort together; (b) after the fermented grains of Luzhou-flavor liquor are loaded into the retort, an appropriate amount of the soaked material is spread on the upper layer;

[0012] (6) After the filling of the steamer is completed, the original liquor is distilled and selected.

[0013] In certain embodiments, in step (1), the liquid culture medium comprises the following components by mass concentration: 0.8-1.5% peptone, 0.3-0.5% beef extract, 0.3-0.5% sodium chloride, 0.01-0.05% magnesium sulfate, 0.001-0.005% manganese sulfate, and the solvent is pure water with a pH of 6.8-7.4.

[0014] In certain embodiments, in step (1), the deposit number of Bacillus subtilis YHB0304 is CCTCC NO: M 2023090.

[0015] In certain embodiments, in step (2), the proportion of wheat in the total material is 20-80%, and the cooling temperature is 35-45°C.

[0016] In certain embodiments, in step (3), the medium temperature fermentation condition is 35-45° C. for 1-3 days, and the high temperature fermentation condition is 45-55° C. for 2-4 days.

[0017] In certain embodiments, in step (4), the mass ratio of the soaking liquid to the fermentation material is 1:1-3:1.

[0018] In certain embodiments, in step (5), the ratio of the soaking material to the fermented grains of the Luzhou-flavor liquor out of the tank is 0.5-5%.

[0019] The second technical solution provided by the present invention is a Luzhou-flavor liquor prepared by the brewing method described in the first technical solution, wherein the mixed alcohol content of the Luzhou-flavor liquor is 70-73% vol.

[0020] The third technical solution provided by the present invention is a method for increasing the content of pyrazines in wine. The method uses wheat and peas as fermentation mashes, utilizes Bacillus subtilis YHB0304 seed liquid to carry out fermentation outside the cellar, and then uses the aroma-binding process to load the fermented grains of Luzhou-flavor liquor into the steamer together.

[0021] In some embodiments, the steps include:

[0022] (1) inoculating Bacillus subtilis YHB0304 into a liquid culture medium to prepare a seed solution;

[0023] (2) mixing the crushed wheat and peas, steaming, and cooling to obtain a fermentation matrix;

[0024] (3) inoculating the seed solution prepared in step (1) into the fermentation matrix prepared in step (2), performing medium-temperature and high-temperature two-stage temperature-controlled fermentation, and drying after completion to obtain a fermentation material rich in pyrazines;

[0025] (4) soaking the fermented material prepared in step (3) in advance using water or original wine as a soaking liquid to obtain a soaked material;

[0026] (5) The soaking material prepared in step (4) and the fermented grains of Luzhou-flavor liquor are loaded into a retort in any of the following ways: (a) the soaking material and the fermented grains of Luzhou-flavor liquor are stirred evenly and loaded into a retort together; (b) after the fermented grains of Luzhou-flavor liquor are loaded into the retort, an appropriate amount of the soaked material is spread on the upper layer;

[0027] (6) After the filling of the steamer is completed, the original liquor is distilled and selected.

[0028] In certain embodiments, in step (1), the liquid culture medium comprises the following components by mass concentration: 0.8-1.5% peptone, 0.3-0.5% beef extract, 0.3-0.5% sodium chloride, 0.01-0.05% magnesium sulfate, 0.001-0.005% manganese sulfate, and the solvent is pure water with a pH of 6.8-7.4.

[0029] In certain embodiments, in step (1), the deposit number of Bacillus subtilis YHB0304 is CCTCC NO: M 2023090.

[0030] In certain embodiments, in step (2), the proportion of wheat in the total material is 20-80%, and the cooling temperature is 35-45°C.

[0031] In some embodiments, in step (3), the medium-temperature fermentation conditions are fermentation at 35 - 45°C for 1 - 3 days, and the high-temperature fermentation conditions are fermentation at 45 - 55°C for 2 - 4 days.

[0032] In some embodiments, in step (4), the mass ratio of the soaking solution to the fermented material is 1:1 - 3:1.

[0033] In some embodiments, in step (5), the proportion of the soaked material in the fermented grains for Luzhou-flavor liquor entering the pit is 0.5 - 5%.

[0034] The fourth technical solution provided by the present invention is the application of the method described in the first technical solution, or the Luzhou-flavor liquor described in the second technical solution, or the method described in the third technical solution in the brewing field.

[0035] The technical effects of the present invention are as follows:

[0036] 1. The present invention combines out-of-pit fermentation, flavor-strengthening process with traditional Luzhou-flavor liquor brewing techniques, compensates for the deficiency of pyrazine substances in Luzhou-flavor liquor, and makes the content of pyrazine substances in the liquor reach more than 12 mg / L.

[0037] 2. The high-pyrazine-producing strain used in the present invention is Bacillus subtilis YHB0304, screened from the medium-high temperature Daqu of our company, and this strain is safe, efficient and stable.

[0038] 3. The fermented material rich in pyrazine substances prepared by the present invention uses raw materials such as wheat and peas, which are all commonly used raw materials for traditional liquor brewing, avoiding the food safety risks brought by exogenous addition and meeting the requirements of relevant national laws and regulations.

[0039] 4. The out-of-pit fermentation process of the fermented material rich in pyrazine substances prepared by the present invention is independent of the in-pit fermentation of Luzhou-flavor liquor, retaining the independence and integrity of the Luzhou-flavor liquor brewing process. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a technical roadmap for the present invention to improve the content of pyrazine substances in liquor by using out-of-pit fermentation and flavor-strengthening process. DETAILED DESCRIPTION OF THE INVENTION

[0041] Referring to the attached Figure 1 , the preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0042] Testing method:

[0043] For the same method of testing pyrazine substances, the steps are as follows:

[0044] Using gas chromatography-mass spectrometry (GC-MS) method, the sample treatment method is as follows: Shake the wine sample well, take 20 mL of the wine sample diluted to 14% vol and transfer it to a 100 mL sealed sample bottle. Add NaCl to saturation (about 9 g), accurately add 50 μL of the internal standard mixture (2-ethylbutyric acid 2000 mg / L, 2-octanol 1600 mg / L, and the final concentrations of the internal standards in the 20 mL wine sample system are 2-ethylbutyric acid 5 mg / L and 2-octanol 4 mg / L respectively). Then transfer 5 mL of diethyl ether, shake and extract at 350 r / min for 30 min, and extract 3 times in total. Combine the three organic phases into a 20 mL sealed reagent bottle, add 5 g of anhydrous sodium sulfate and dry overnight. The next day, transfer the extract to a 25 mL KD concentrator tube, concentrate to 0.5 mL by nitrogen blowing, and then transfer it to an injection vial containing a 300 or 500 μL liner for GC-MS analysis.

[0045] Analysis conditions: The carrier gas is nitrogen, the flow rate is 1.0 mL / min, the inlet temperature is 230 °C, and the injection volume is 1 μL. Programmed temperature rise: The initial temperature is 40 °C, hold for 5 min, then rise to 100 °C at a rate of 3.5 °C / min, hold for 2 min, then rise to 200 °C at a rate of 5 °C / min, hold for 4 min, and finally rise to 220 °C at a rate of 20 °C / min, hold for 15 min. After the program runs, run at 40 °C for 1 min.

[0046] Raw materials used in the examples:

[0047] 1. Bacillus subtilis YHB0304 was deposited at the China Center for Type Culture Collection on February 3, 2023. The deposit address is Wuhan University, China, and the deposit number is CCTCC No: M2023090, which has been disclosed in the patent document CN117210358A.

[0048] 2. The thick-flavor Chinese liquor fermented grains taken out of the pit involved in the following examples are from Jiangsu Yanghe Brewery Co., Ltd.

[0049] Example 1 High content of pyrazine substances in liquor

[0050] The specific steps are as follows:

[0051] (1) Expansion culture of high-yield pyrazine strains: Inoculate a strain of Bacillus subtilis YHB0304 with high pyrazine-producing substances into a liquid medium. The liquid medium includes the following components by mass concentration: peptone 0.8%, beef extract 0.3%, sodium chloride 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.004%, the solvent is pure water, and the pH is 6.8, and a seed solution is prepared.

[0052] (2) Preparation of fermentation substrate and steaming and cooling: Mix the crushed wheat and peas, with the proportion of wheat in the total material being 20%; then steam and cool the mixture, with the cooling temperature being 38°C.

[0053] (3) Fermentation outside the pit: The seed liquid prepared in step (1) is inoculated into the fermentation matrix prepared in step (2), and medium-temperature and high-temperature two-stage temperature-controlled fermentation is performed. The medium-temperature fermentation condition is 35° C. for 2 days, and the high-temperature fermentation condition is 45° C. for 4 days. After the fermentation is completed, the fermented material rich in pyrazines is dried.

[0054] (4) Material soaking: The fermentation material prepared in step (3) is soaked with 60% vol of original wine to obtain a soaking material, and the mass ratio of the original wine to the fermentation material is 1:1.

[0055] (5) Aroma-mixing process: The soaking material prepared in step (4) is mixed with the fermented grains of Luzhou-flavor liquor and stirred evenly, wherein the soaking material accounts for 1% of the fermented grains. The mixture is then put into a retort for distillation, and a high-quality raw liquor with a mixed alcohol content of 72% vol is selected.

[0056] Example 2: Increase the content of pyrazines in wine according to the following steps

[0057] The specific steps are as follows

[0058] (1) Propagation of high-pyrazine-producing strains: A strain of Bacillus subtilis YHB0304 that produces high pyrazines was inoculated into a liquid culture medium, which contained the following components by mass concentration: 1.0% peptone, 0.5% beef extract, 0.3% sodium chloride, 0.05% magnesium sulfate, 0.003% manganese sulfate, pure water as the solvent, pH 7.4, to prepare a seed solution.

[0059] (2) Preparation of fermentation substrate and steaming and cooling: Mix the crushed wheat and peas, with the wheat accounting for 60% of the total material; then steam and cool the mixture, with the cooling temperature being 40°C.

[0060] (3) Fermentation outside the pit: The seed liquid prepared in step (1) is inoculated into the fermentation matrix prepared in step (2), and medium-temperature and high-temperature two-stage temperature-controlled fermentation is performed. The medium-temperature fermentation condition is 45° C. for 1 day, and the high-temperature fermentation condition is 50° C. for 3 days. After the fermentation is completed, the fermented material rich in pyrazines is dried.

[0061] (4) Material soaking: The fermentation material prepared in step (3) is soaked with 35% vol of original wine to obtain a soaking material, and the mass ratio of the original wine to the fermentation material is 3:1.

[0062] (5) Aroma-mixing process: The soaking material prepared in step (4) is mixed with the fermented grains of Luzhou-flavor liquor and stirred evenly, wherein the soaking material accounts for 3% of the fermented grains. The mixture is then put into a retort for distillation, and a high-quality raw liquor with a mixed alcohol content of 72% vol is selected.

[0063] Example 3: Increase the content of pyrazines in wine according to the following steps

[0064] The details are as follows:

[0065] (1) Propagation of high-pyrazine-producing strains: A strain of Bacillus subtilis YHB0304 that produces high pyrazines was inoculated into a liquid culture medium, wherein the liquid culture medium comprises the following components by mass concentration: 1.2% peptone, 0.4% beef extract, 0.3% sodium chloride, 0.01% magnesium sulfate, 0.002% manganese sulfate, the solvent is pure water, and the pH is 7.2, to prepare a seed solution.

[0066] (2) Preparation of fermentation substrate and steaming and cooling: Mix the crushed wheat and peas, with the proportion of wheat in the total material being 40%; then steam and cool the mixture, with the cooling temperature being 45°C.

[0067] (3) Fermentation outside the pit: The seed liquid prepared in step (1) is inoculated into the fermentation matrix prepared in step (2), and medium-temperature and high-temperature two-stage temperature-controlled fermentation is performed. The medium-temperature fermentation condition is 30° C. for 3 days, and the high-temperature fermentation condition is 55° C. for 2 days. After the fermentation is completed, the fermented material rich in pyrazines is dried.

[0068] (4) Material soaking: The fermentation material prepared in step (3) is soaked in water to obtain a soaking material, and the mass ratio of water to the fermentation material is 2:1.

[0069] (5) Aroma-mixing process: After the fermented grains of the Luzhou-flavor liquor are taken out of the tank and loaded into the steamer, the soaking material prepared in step (4) is spread on the upper layer, wherein the soaking material accounts for 5% of the fermented grains taken out of the tank, and then they are put into the steamer for distillation, and the high-quality original liquor with a mixed alcohol content of 72% vol is selected.

[0070] Example 4: Increase the content of pyrazines in wine according to the following steps

[0071] The specific steps are as follows:

[0072] (1) Propagation of high-pyrazine-producing strains: A strain of Bacillus subtilis YHB0304 that produces high pyrazines was inoculated into a liquid culture medium, which contained the following components by mass concentration: 1.5% peptone, 0.3% beef extract, 0.4% sodium chloride, 0.03% magnesium sulfate, 0.005% manganese sulfate, pure water as the solvent, pH 7.0, to prepare a seed solution.

[0073] (2) Preparation of fermentation substrate and steaming and cooling: Mix the crushed wheat and peas, with the wheat accounting for 80% of the total material; then steam and cool the mixture, with the cooling temperature being 35°C.

[0074] (3) Fermentation outside the pit: The seed liquid prepared in step (1) is inoculated into the fermentation matrix prepared in step (2), and medium-temperature and high-temperature two-stage temperature-controlled fermentation is performed. The medium-temperature fermentation condition is 35° C. for 2 days, and the high-temperature fermentation condition is 48° C. for 4 days. After the fermentation is completed, the fermented material rich in pyrazines is dried.

[0075] (4) Material soaking: The fermentation material prepared in step (3) is soaked with 72% vol of original wine to obtain a soaking material, and the mass ratio of the original wine to the fermentation material is 1:1.

[0076] (5) Aroma-mixing process: After the fermented grains of the Luzhou-flavor liquor are taken out of the tank and loaded into the retort, the soaking material prepared in step (4) is spread on the upper layer, wherein the soaking material accounts for 2% of the fermented grains taken out of the tank, and then they are put into the retort for distillation, and the high-quality original liquor with a mixed alcohol content of 72% vol is selected.

[0077] Comparative Example

[0078] The traditional Luzhou-flavor liquor production process is adopted, and the selected mixed alcohol content is 72% vol. of high-quality raw liquor.

[0079] Test Case

[0080] The pyrazine substances in the liquor obtained in Examples 1-4 and the comparative example were detected, and the results are shown in Table 1.

[0081] Table 1: Comparison of pyrazine content in liquor of Example and Comparative Example

[0082]

[0083] As can be seen from Table 1, the pyrazine content of the original liquor produced by Examples 1-4 is above 12 mg / L, while the pyrazine content of the liquor produced by the traditional Luzhou-flavor process in the comparative example is 0.83 mg / L, which is increased by 15.01 to 26.91 times. This proves that the process adopted by the present invention can greatly increase the content of pyrazines in the liquor and achieve the purpose of upgrading the quality of the liquor.

[0084] 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 brewing Luzhou-flavor liquor, characterized in that: The steps include: (1) Propagation of high-yield pyrazine strains: Bacillus subtilis YHB0304 was inoculated into a liquid culture medium to prepare a seed solution; (2) Preparation of fermentation matrix and steaming and cooling: mixing the crushed wheat and peas, steaming and cooling to obtain a fermentation matrix; (3) fermentation outside the cellar: inoculating the seed liquid prepared in step (1) into the fermentation matrix prepared in step (2), performing medium temperature and high temperature two-stage temperature controlled fermentation, and drying after completion to obtain the fermented material; (4) soaking the material: soaking the fermented material prepared in step (3) with water or original wine as a soaking liquid to obtain a soaked material; (5) Aroma concatenation process: the soaking material prepared in step (4) and the fermented grains of Luzhou-flavor liquor are loaded into the retort in any of the following ways: (a) the soaking material and the fermented grains of Luzhou-flavor liquor are stirred evenly and put into the retort together; (b) after the fermented grains of Luzhou-flavor liquor are loaded into the retort, an appropriate amount of the soaked material is spread on the upper layer; (6) After the filling of the steamer is completed, the original liquor is distilled and selected.

2. The method according to claim 1, characterized in that In step (1), the liquid culture medium comprises the following components by mass concentration: 0.8-1.5% peptone, 0.3-0.5% beef extract, 0.3-0.5% sodium chloride, 0.01-0.05% magnesium sulfate, and 0.001-0.005% manganese sulfate; The deposit number of Bacillus subtilis YHB0304 is CCTCC NO: M2023090.

3. The method according to claim 1, characterized in that In step (2), the proportion of wheat in the total material is 20-80%, and the cooling temperature is 35-45°C.

4. The method according to claim 1, characterized in that: In step (3), the medium temperature fermentation condition is 35-45°C for 1-3 days, and the high temperature fermentation condition is 45-55°C for 2-4 days.

5. The method according to claim 1, characterized in that In step (4), the mass ratio of the soaking liquid to the fermentation material is 1:1-3:

1.

6. The method according to claim 1, characterized in that In step (5), the ratio of the soaking material to the fermented grains of the Luzhou-flavor liquor out of the pond is 0.5-5%.

7. A Luzhou-flavor liquor prepared by the brewing method according to any one of claims 1 to 6, characterized in that: The mixed alcohol content of the Luzhou-flavor liquor is 70-73% vol.

8. A method for increasing the content of pyrazines in wine, characterized in that: The method uses wheat and peas as fermentation substrates, utilizes Bacillus subtilis YHB0304 seed liquid to carry out fermentation outside the cellar, and then uses the aroma-binding process to load the fermented grains of Luzhou-flavor liquor into a steamer together.

9. The method according to claim 8, characterized in that The steps include: (1) inoculating Bacillus subtilis YHB0304 into a liquid culture medium to prepare a seed solution; (2) mixing the crushed wheat and peas, steaming, and cooling to obtain a fermentation matrix; (3) inoculating the seed solution prepared in step (1) into the fermentation matrix prepared in step (2), performing medium-temperature and high-temperature two-stage temperature-controlled fermentation, and drying to obtain the fermented material after completion; (4) soaking the fermented material prepared in step (3) with water or original wine as a soaking liquid to obtain a soaked material; (5) The soaking material prepared in step (4) and the fermented grains of Luzhou-flavor liquor are loaded into a retort in any of the following ways: (a) the soaking material and the fermented grains of Luzhou-flavor liquor are stirred evenly and loaded into a retort together; (b) after the fermented grains of Luzhou-flavor liquor are loaded into the retort, an appropriate amount of the soaked material is spread on the upper layer; (6) Distillation and selection of raw liquor.

10. Use of the method according to claims 1 to 6, or the Luzhou-flavor liquor according to claim 7, or the method according to claim 8 or 9 in the brewing field.

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