Preparation and application of pichia kudriavzevii active dry yeast

Abstract

The invention provides a preparation method and application of pichia kudriavzevii active dry yeast. The total number of cells of the pichia kudriavzevii in each gram of the active dry yeast is greater than or equal to 230 and less than or equal to 4 billion, the living cell rate of the pichia kudriavzevii in the active dry yeast is greater than or equal to 74% and less than or equal to 95%, and the moisture content is 4.5-5wt%. According to the preparation method of the pichia kudriavzevii active dry yeast, provided by the invention, sufficient flavor substances can be generated while alcohol is generated in a fermentation process, so that the quality of fen-flavor liquor is improved.

Description

Technical Field

[0001] The present invention belongs to the field of fermentation, and particularly relates to the preparation and application of Pichia kudriavzevii active dry yeast. Background Art

[0002] Currently, light-flavor Baijiu is mainly divided into Daqu light-flavor, Xiaoqu light-flavor, and bran Qu light-flavor. The characteristic flavor of light-flavor Baijiu has been clarified, and the main component is ethyl acetate.

[0003] The fermentation processes of the three types of light-flavor Baijiu are different. In terms of the main fermentation strains, non-Saccharomyces cerevisiae is mainly used in Daqu light-flavor, and Saccharomyces cerevisiae is mainly used in Xiaoqu light-flavor and bran Qu light-flavor. It is more difficult to enhance the main aroma of Xiaoqu light-flavor and bran Qu light-flavor. Some distilleries use Hansenula anomala, Pichia anomala, etc. for aroma-producing fermentation, and improve the content of ethyl acetate through processes such as tandem steaming. Saccharomyces cerevisiae is mostly still used for the main fermentation of alcohol.

[0004] In the existing Baijiu fermentation process, there are mainly the following several fermentation pathways for the production of ethyl acetate. Pathway 1: Medium and low-temperature Daqu are used. There is a complex microbial system in Daqu that can produce ethyl acetate and complete the fermentation of light-flavor. Generally, in Daqu Baijiu, 10%-20% of Daqu is used for fermentation. In Xiaoqu and bran Qu fermentation, the fermentation ability of ethyl acetate is improved by adding Daqu. Pathway 2: The tandem steaming process of fragrant fermented grains is adopted. The fragrant fermented grains can be produced in the following ways: (1) Add 1%-1.5% of Daqu or bran Qu, and ferment in the cellar for 20-30 days to obtain fragrant fermented grains; (2) Use the discarded grains of Daqu or Xiaoqu Baijiu, add Rhizopus koji, Daqu or Xiaoqu, and ferment in the pool for 20-30 days to obtain fragrant fermented grains; (3) Add about 10% of sorghum powder or starchy raw materials based on the amount of fermented grains, mix evenly, load into the wine still, steam until cooked, take out of the still, cool to 18-20 degrees, sprinkle 20% of Daqu and bran Qu based on the weight of the new raw materials, mix well, and ferment in the pool for 15-20 days to obtain fragrant fermented grains; (4) Directly use the fermented grains of Daqu Baijiu or Xiaoqu Baijiu as fragrant fermented grains. (5) Mix the fermented grains of Daqu Baijiu and yeast to make fragrant fermented grains. Summary of the Invention

[0005] However, the existing technology has the following problems:

[0006] Problem 1: Poor stability

[0007] During the Daqu-making process, the quantity and quality of microorganisms are easily affected by seasonal changes. Whether it is natural inoculation or the addition of mother Qu for enhancement, there are problems of volatility, resulting in poor stability of the Daqu quality.

[0008] Problem 2: The process of making fragrant fermented grains is cumbersome, and the aroma-producing fermentation effect is poor

[0009] In the production of fragrant fermented grains, the microorganisms used in the prior art, such as Hansenula anomala and Pichia anomala, need to produce better flavor substances under aerobic conditions. Moreover, both the fermentation alcohol and fermentation flavor systems need to be fermented separately. This leads to the cumbersome process, low production efficiency, and poor flavor production effect in the prior art for the production of fragrant fermented grains.

[0010] Pichia kudriavzevii is a yeast that utilizes monosaccharides and is directly produced using molasses. Since the sucrose content in molasses is usually above 70%, there is a problem that this yeast cannot be well cultured and the production environment of this yeast cannot be achieved. Therefore, in the process of preparing active dry yeast of the present invention, sucrose in molasses is hydrolyzed in advance by sucrase (the sucrose degradation rate reaches more than 95%), and it is used as a carbon source to ferment Pichia kudriavzevii, solving the problem that this yeast cannot be well cultured and the production environment of this yeast cannot be achieved.

[0011] In view of the problems existing in the above prior art, the present invention provides a preparation and application of Pichia kudriavzevii active dry yeast.

[0012] Specifically, the following technical solutions are provided:

[0013] Technical solution 1: A Pichia kudriavzevii active dry yeast, characterized in that the total number of Pichia kudriavzevii cells per gram of the active dry yeast is greater than or equal to 23 billion and less than or equal to 40 billion, the viable cell rate of Pichia kudriavzevii in the active dry yeast is greater than or equal to 74% and less than or equal to 95%, and the water content is 4.5 - 5 wt%.

[0014] Technical solution 2: The active dry yeast according to Technical solution 1, characterized in that the total number of Pichia kudriavzevii cells per gram of the active dry yeast is greater than or equal to 26 billion and less than or equal to 35 billion, and / or the viable cell rate of Pichia kudriavzevii in the active dry yeast is greater than or equal to 74% and less than or equal to 90%.

[0015] Technical solution 3: The active dry yeast according to Technical solution 1 or 2, characterized in that the Pichia kudriavzevii is Pichia kudriavzeii C4.12.

[0016] Technical solution 4: The active dry yeast according to any one of technical solutions 1-3, characterized in that it is prepared by a method comprising the following steps: culturing a seed culture solution of Pichia kudriavzevii in a mixture of a carbon source and a nutrient source to obtain a culture solution, then feeding the carbon source into the culture solution for culturing to obtain a fermentation culture solution, emulsifying the fermentation culture solution to obtain fermented milk, granulating and drying the fermented milk to obtain Pichia kudriavzevii active dry yeast.

[0017] Wherein, the carbon source is one or a combination of two of molasses and starch hydrolysate treated with invertase.

[0018] Wherein, the nutrient source comprises one or more substances selected from the group consisting of ammonium sulfate, ammonium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, nicotinic acid, inositol, biotin, ammonia water, diammonium hydrogen phosphate and ammonium hydrogen phosphate.

[0019] Technical solution 5: The active dry yeast according to any one of technical solutions 1-4, characterized in that the sugar content of the carbon source is 10-35 wt%, preferably 10-20 wt%.

[0020] Technical solution 6: The active dry yeast according to any one of technical solutions 1-5, characterized in that the molasses is one or a combination of two of cane molasses and beet molasses.

[0021] Technical solution 7: The active dry yeast according to technical solution 6, characterized in that the molasses treated with invertase is prepared by a method comprising the following steps: mixing molasses with invertase and reacting at 40-80 °C for 2-5 h to obtain molasses treated with invertase.

[0022] Preferably, the addition amount of invertase is 100-1000 U per g of sucrose in the molasses.

[0023] Technical solution 8: The active dry yeast according to any one of technical solutions 1-7, characterized in that the nutrient source is one or more substances selected from the group consisting of ammonium sulfate, ammonium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, nicotinic acid, inositol, biotin, ammonia water, diammonium hydrogen phosphate and ammonium hydrogen phosphate.

[0024] Technical solution 9: The active dry yeast according to technical solution 8, characterized in that, by weight, relative to 5500 - 6500 parts of the carbon source, the added amounts of the nutrient sources include 12 - 325 parts of ammonium sulfate, and / or 5 - 115 parts of ammonium phosphate, and / or 5 - 30 parts of potassium chloride, and / or 1 - 20 parts of magnesium sulfate, and / or 0.015 - 0.125 parts of calcium pantothenate, and / or 0.001 - 0.065 parts of nicotinic acid, and / or 0.005 - 0.125 parts of inositol, and / or 0.02 - 0.13 parts of biotin, and / or 135 - 260 parts of ammonia water, and / or 45 - 55 parts of diammonium hydrogen phosphate, and / or 58 - 59 parts of ammonium hydrogen phosphate.

[0025] Technical solution 10: The active dry yeast according to any one of technical solutions 1 - 9, characterized in that the emulsification is to mix the yeast milk and the emulsifier according to a weight ratio of 20000 - 60000:0.1 - 245 to emulsify the yeast milk, wherein the emulsifier includes one or more substances selected from the group consisting of vegetable oil, sorbitan monostearate, sorbitan laurate, sorbitan palmitate, sorbitan stearate, sorbitan stearoyl glycerol, sorbitan oleate, and sorbitan trioleate.

[0026] Technical solution 11: The active dry yeast according to technical solution 10, characterized in that the emulsifier is one of sorbitan monostearate, sorbitan laurate, sorbitan palmitate, or sorbitan stearate.

[0027] Technical solution 12: The active dry yeast according to technical solution 10 or 11, characterized in that, by weight, the emulsifier includes: 0.1 - 2 parts of sorbitan monostearate, and / or 235 - 245 parts of sorbitan laurate, and / or 125 - 135 parts of sorbitan palmitate, and / or 185 - 195 parts of sorbitan stearate.

[0028] Technical solution 13: A method for preparing the active dry yeast of Pichia kudriavzevii according to any one of technical solutions 1 - 12, characterized by comprising the following steps: Culturing the seed culture solution of Pichia kudriavzevii in a mixture of a carbon source and a nutrient source, then feeding the carbon source into the culture solution for culturing to obtain a fermentation culture solution, emulsifying the fermentation culture solution to obtain yeast milk, granulating and drying the yeast milk to obtain the active dry yeast of Pichia kudriavzevii, wherein the carbon source is one or a combination of molasses treated with invertase and starch hydrolysate, and the nutrient source includes one or more substances selected from the group consisting of ammonium sulfate, ammonium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, nicotinic acid, inositol, biotin, ammonia water, diammonium hydrogen phosphate, and ammonium hydrogen phosphate.

[0029] Technical solution 14: The preparation method according to technical solution 13, characterized by comprising the following steps:

[0030] (1) Inoculate the activated Pichia kudriavzevii strain into a liquid medium for cultivation to obtain a seed culture solution. Preferably, the liquid medium is a wort liquid medium;

[0031] (2) Inoculate the seed culture solution obtained in step (1) into the mixture of the carbon source and the nutrient source for primary cultivation, and then add the carbon source to the culture solution and introduce sterile air for secondary cultivation to obtain a fermentation culture solution;

[0032] (3) Separate the fermentation culture solution obtained in step (2) to obtain yeast cream, and then mix the yeast cream and an emulsifier, granulate and dry to obtain active dry Pichia kudriavzevii. Preferably, the drying temperature is: the inlet air temperature is 80 - 115 °C, the outlet air temperature is 40 - 80 °C, and / or the drying time is 25 - 65 min.

[0033] Technical solution 15: The preparation method according to technical solution 13 or 14, characterized in that in step (1), the sugar content of the liquid medium is 5 - 25 wt%, preferably 8 - 15 wt%.

[0034] Technical solution 16: The preparation method according to any one of technical solutions 13 - 15, characterized in that the cultivation temperature in step (1) is: 25 - 35 °C, and / or the primary cultivation temperature in step (2) is: 20 - 35 °C, and / or the secondary cultivation temperature in step (2) is: 20 - 35 °C.

[0035] Technical solution 17: The preparation method according to any one of technical solutions 13 - 15, characterized in that the cultivation time in step (1) is: 20 - 50 h, and / or the primary cultivation time in step (2) is: 20 - 50 h, and / or the secondary cultivation time in step (2) is: 15 - 30 h.

[0036] Technical solution 18: The preparation method according to any one of technical solutions 13 - 15, characterized in that in step (2), the flow rate of the carbon source is 200 - 2500 L / hr, and / or the air volume of the sterile air introduced is 28 - 32 m 3 air / fermentation broth m 3 / hr, and / or during the secondary cultivation, the ethanol content in the fermentation culture solution is below 0.4%.

[0037] Technical solution 19: The preparation method according to any one of technical solutions 13-15, characterized in that in step (3), the separation is to separate the fermentation culture solution at 3500-4500 rpm and retain the precipitate to obtain the yeast milk.

[0038] Technical solution 20: A Kluyveromyces pichia active dry yeast prepared by the preparation method according to any one of technical solutions 13-19.

[0039] Technical solution 21: Application of the Kluyveromyces pichia active dry yeast according to any one of technical solutions 1-12 or technical solution 20 in the preparation of koji.

[0040] Technical solution 22: A koji, characterized by containing the Kluyveromyces pichia active dry yeast according to any one of technical solutions 1-12 or technical solution 20.

[0041] Technical solution 23: The koji according to technical solution 22, characterized in that the koji is prepared by a method comprising the following steps: mixing the Kluyveromyces pichia active dry yeast and the strain to obtain koji, preferably, the strain is a Rhizopus strain or an Aspergillus niger strain.

[0042] Technical solution 24: The koji according to technical solution 22 or 23, characterized in that when the strain is a Rhizopus strain, the koji is prepared by a method comprising the following steps:

[0043] (1) Based on the weight of bran, inoculate 5-15 wt% of the Rhizopus strain and ferment and culture to obtain a fermented product, preferably, the temperature of the fermentation and culture is 28-33 °C and / or the time of the fermentation and culture is 70-73 h;

[0044] (2) Based on the weight of the fermented product, add 0.1-5 wt% of the Kluyveromyces pichia active dry yeast to the fermented product and mix to obtain koji.

[0045] Technical solution 25: The koji according to any one of technical solutions 22-24, characterized in that when the strain is an Aspergillus niger strain, the koji is prepared by a method comprising the following steps:

[0046] (1) Based on the weight of bran, inoculate 5-15 wt% of the Aspergillus niger strain and ferment and culture to obtain a fermented product, preferably, the temperature of the fermentation and culture is 28-33 °C and / or the time of the fermentation and culture is 4-5 d;

[0047] (2) Based on the weight of the fermented product, add 1-2 wt% of the Kluyveromyces pichia active dry yeast to the fermented product and mix to obtain koji.

[0048] Technical solution 26: The koji according to technical solution 22, characterized in that the koji is prepared by a method comprising the following steps: mixing the Pichia kudriavzevii active dry yeast and the daqu, to obtain the koji, wherein the daqu is prepared by a method comprising the following steps: mixing barley and peas in a weight ratio of 6:3-5, crushing, forming into a mass, fermenting and culturing, and drying, to obtain the daqu. Preferably, based on the mass of the daqu, the addition amount of the Pichia kudriavzevii active dry yeast is 0.1-5 wt%, and more preferably, based on the weight of the daqu, the addition amount of the Pichia kudriavzevii active dry yeast is 0.1-2 wt%.

[0049] Technical solution 27: The application of the koji according to any one of technical solutions 22-26 in the preparation of wine, preferably, the wine is white liquor.

[0050] Technical solution 28: A white liquor, characterized in that it contains the koji according to any one of claims 22-26.

[0051] Technical solution 29: The white liquor according to technical solution 28, characterized in that it is obtained by the following steps: mixing the koji according to any one of technical solutions 22-25 with the grain mash, saccharifying, fermenting and distilling to obtain the white liquor.

[0052] Technical solution 30: The white liquor according to technical solution 28 or 29, characterized in that the volatile substances contained in the white liquor include ester compounds. Preferably, based on each liter of the white liquor, the content of the ester compounds is 1000-3500 mg. Preferably, the ester compounds include one or more substances selected from the group consisting of ethyl acetate, ethyl lactate and ethyl butyrate.

[0053] Technical solution 31: The white liquor according to any one of technical solutions 28-30, characterized in that the volatile substances contained in the white liquor further include one or more substances selected from the group consisting of n-propanol, isobutanol, acetal, isoamyl alcohol and phenethyl alcohol,

[0054] Preferably, based on each liter of white liquor, the volatile substances contained in the white liquor include 1000-2500 mg of ethyl acetate, and / or 700-1300 mg of ethyl lactate, and / or 5-15 mg of ethyl butyrate, and / or 85-400 mg of n-propanol, and / or 100-800 mg of isobutanol, and / or 50-380 mg of acetal, and / or 150-950 mg of isoamyl alcohol, and / or 10-50 mg of phenethyl alcohol,

[0055] More preferably, 1000 - 2350 mg of ethyl acetate, and / or 90 - 390 mg of n-propanol, and / or 150 - 800 mg of isobutanol, and / or 50 - 380 mg of acetal, and / or 180 - 950 mg of isoamyl alcohol, and / or 10 - 45 mg of phenethyl alcohol.

[0056] Technical solution 32: The method for brewing Chinese liquor according to any one of technical solutions 28 - 31, characterized by comprising the following steps: mixing the Pichia kudriavzevii active dry yeast, the strain and the grain mash, saccharifying, fermenting and distilling to obtain Chinese liquor.

[0057] Advantages of the present invention

[0058] (1) A method for preparing Pichia kudriavzevii active dry yeast provided by the present invention. In the process of preparing Pichia kudriavzevii active dry yeast, sucrose in molasses is hydrolyzed in advance by invertase (the sucrose degradation rate reaches more than 95%), and it is used as a carbon source to ferment Pichia kudriavzevii yeast, solving the problem that this yeast cannot be well cultured and cannot reach its production environment, which is beneficial to the formation of highly active Pichia kudriavzevii dry yeast.

[0059] (2) The present invention mixes Pichia kudriavzevii active dry yeast and a strain to obtain a kind of koji. The liquor fermented by this koji has the characteristic of better aroma production effect. Combining with the method of solid-state light-aroma xiaoqu brewing, the method of bran koji light-aroma brewing or the method of daqu light-aroma brewing, the obtained Chinese liquor has the content of ester compounds in the Chinese liquor above 1 g / L.

[0060] Proof of strain material

[0061] The Pichia kudriavzevii (Pichia kudriavzeii) C4.12 used in the present invention was deposited at the China Center for Type Culture Collection (CCTCC) on February 5, 2021, with the deposit number CCTCC NO: 2021124, and the deposit address: Wuhan University, Wuhan, China, Postcode: 430072; Tel: 027 - 68754052, and has been recorded in the Chinese patent application with the publication number CN114107077B. Specific embodiments

[0062] In order to better understand the above technical solutions, the technical solutions of the present invention are clearly and completely explained below in conjunction with specific embodiments. It should be noted that the content in the specific embodiments is only a specific implementation and explanation of the technical solutions of the present invention, and should not be construed as a limitation of the protection scope of the present invention.

[0063] As described in the background art, in the production of distiller's yeast, microorganisms such as Hansenula anomala and Pichia anomala used in the prior art need to produce better flavor substances under aerobic conditions. Moreover, both the fermentation alcohol and fermentation flavor systems need to be fermented separately. This results in a cumbersome production process, low production efficiency, and poor flavor-producing effects in the prior art for making distiller's yeast.

[0064] To solve the above technical problems, the present invention provides a method for preparing Pichia kudriavzevii active dry yeast, which can produce sufficient flavor substances while fermenting alcohol, so as to achieve the quality of light-flavor Baijiu. Moreover, it can make up for the deficiencies of using Hansenula anomala and Pichia anomala for making distiller's yeast.

[0065] In some specific embodiments, the present invention provides a Pichia kudriavzevii active dry yeast, wherein the total cell number of Pichia kudriavzevii in each gram of the active dry yeast is greater than or equal to 23 billion and less than or equal to 40 billion, the viable cell rate of Pichia kudriavzevii in the active dry yeast is greater than or equal to 74% and less than or equal to 95%, and the moisture content is 4.5 - 5 wt%.

[0066] Preferably, in some specific embodiments, the total number of cells of Pichia kudriavzevii in each gram of the active dry yeast may be 23 billion, 23.1 billion, 23.2 billion, 23.3 billion, 23.4 billion, 23.5 billion, 23.6 billion, 23.7 billion, 23.8 billion, 23.9 billion, 24 billion, 24.1 billion, 24.2 billion, 24.3 billion, 24.4 billion, 24.5 billion, 24.6 billion, 24.7 billion, 24.8 billion, 24.9 billion, 25 billion, 25.1 billion, 25.2 billion, 25.3 billion, 25.4 billion, 25.5 billion, 25.6 billion, 25.7 billion, 25.8 billion, 25.9 billion, 26 billion, 26.1 billion, 26.2 billion, 26.3 billion, 26.4 billion, 26.5 billion, 26.6 billion, 26.7 billion, 26.8 billion, 26.9 billion, 27 billion, 27.1 billion, 27.2 billion, 27.3 billion, 27.4 billion, 27.5 billion, 27.6 billion, 27.7 billion, 27.8 billion, 27.9 billion, 28 billion, 28.1 billion, 28.2 billion, 28.3 billion, 28.4 billion, 28.5 billion, 28.6 billion, 28.7 billion, 28.8 billion, 28.9 billion, 29 billion, 29.1 billion, 29.2 billion, 29.3 billion, 29.4 billion, 29.5 billion, 29.6 billion, 29.7 billion, 29.8 billion, 29.9 billion, 30 billion, 30.1 billion, 30.2 billion, 30.3 billion, 30.4 billion, 30.5 billion, 30.6 billion, 30.7 billion, 30.8 billion, 30.9 billion, 31 billion, 31.1 billion, 31.2 billion, 31.3 billion, 31.4 billion, 31.5 billion, 31.6 billion, 31.7 billion, 31.8 billion, 31.9 billion, 32 billion, 32.1 billion, 32.2 billion, 32.3 billion, 32.4 billion, 32.5 billion, 32.6 billion, 32.7 billion, 32.8 billion, 32.9 billion, 33 billion, 33.1 billion, 33.2 billion, 33.3 billion, 33.4 billion, 33.5 billion, 33.6 billion, 33.7 billion, 33.8 billion, 33.9 billion, 34 billion, 34.1 billion, 34.2 billion, 34.3 billion, 34.4 billion, 34.5 billion, 34.6 billion, 34.7 billion, 34.8 billion, 34.9 billion, 35 billion, 35.1 billion, 35.2 billion, 35.3 billion, 35.4 billion, 35.5 billion, 35.6 billion, 35.7 billion, 35.8 billion, 35.9 billion, 36 billion, 36.1 billion, 36.2 billion, 36.3 billion, 36.4 billion, 36.5 billion, 36.6 billion, 36.7 billion, 36.8 billion, 36.9 billion, 37 billion, 37.1 billion, 37.2 billion, 37.3 billion, 37.4 billion, 37.5 billion, 37.6 billion, 37.7 billion, 37.8 billion, 37.9 billion, 38 billion, 38.1 billion, 38.2 billion, 38.3 billion, 38.4 billion, 38.5 billion, 38.6 billion, 38.7 billion, 38.8 billion, 38.9 billion, 39 billion, 39.1 billion, 39.2 billion, 39.3 billion, 39.4 billion, 39.5 billion, 39.6 billion, 39.7 billion, 39.8 billion, 39.9 billion or 40 billion, or the total number of cells of Pichia kudriavzevii within the numerical range formed by any two of the above specific numerical values as endpoints.

[0067] Preferably, in some specific embodiments, the viable cell rate of Pichia kudriavzevii in the active dry yeast can be 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%, or the viable cell rate of Pichia kudriavzevii within the numerical range formed by any two of the above specific values as endpoints.

[0068] In some specific embodiments, the present invention provides a technical solution for a method of brewing Chinese liquor (using the method of brewing Chinese liquor with solid-state light-flavor small starter), which is specifically as follows:

[0069] (1.1) A method of brewing Chinese liquor, characterized by comprising the following steps: mixing the Pichia kudriavzevii active dry yeast, rhizopus species and grain mash according to any one of Technical Solutions 1-12 or Technical Solution 20, saccharifying, fermenting and distilling to obtain Chinese liquor.

[0070] (1.2) The method of brewing Chinese liquor according to Technical Solution (1.1), characterized by comprising the following steps:

[0071] (1) Based on the weight of bran, inoculate 5-15 wt% of rhizopus species and ferment and culture to obtain a fermented product. Preferably, the temperature of the fermentation and culture is 28-33 °C and / or the time of the fermentation and culture is 70-73 h;

[0072] (2) Based on the weight of the fermented product, add 0.1-5 wt% of Pichia kudriavzevii active dry yeast to the fermented product and mix to obtain a koji. Preferably, the addition amount of Pichia kudriavzevii active dry yeast is 0.5-2 wt%;

[0073] (3) Soak, steam and cool the grains. Then, based on the weight of the grains before soaking, inoculate 0.3 wt%-0.8 wt% of the koji obtained in step (2) and mix to obtain the grain mash before saccharification, and then perform saccharification to obtain the saccharified grain mash.

[0074] Among them, the grains include one or more of sorghum, corn, wheat and rice; preferably, the grains are one or more of sorghum, corn and wheat;

[0075] (4) Ferment and distill by mixing the saccharified grain mash and the grain mash before saccharification at a weight ratio of 1:2-3 to obtain Chinese liquor.

[0076] (1.3) The method of brewing Chinese liquor according to Technical Solution (1.1) or (1.2), characterized in that the rhizopus species is Rhizopus sp. Q303 strain.

[0077] It should be noted that the source of the Rhizopus species used in this application can be any commercially available one or can be obtained through conventional screening. For example, the Rhizopus Q303 strain sold by the Guizhou Provincial Light Industry Research Institute can be used.

[0078] (1.4) The method for brewing Chinese liquor according to any one of the technical solutions (1.1)-(1.3), characterized in that the steaming time of the grains is 15-30 min.

[0079] (1.5) The method for brewing Chinese liquor according to any one of the technical solutions (1.1)-(1.4), characterized in that when soaking the grains, water is used for soaking, wherein the soaking temperature is: 70-75 °C and / or the soaking time is: 5-10 h.

[0080] (1.6) The method for brewing Chinese liquor according to any one of the technical solutions (1.1)-(1.4), characterized in that it is cooled to 30-35 °C.

[0081] (1.7) The method for brewing Chinese liquor according to any one of the technical solutions (1.1)-(1.6), characterized in that the saccharification temperature is 25-30 °C and / or the saccharification time is 18-32 h.

[0082] (1.8) The method for brewing Chinese liquor according to any one of the technical solutions (1.1)-(1.7), characterized in that the fermentation temperature is: 22-24 °C and / or the fermentation time is: 7-21 days.

[0083] (1.9) The method for brewing Chinese liquor according to any one of the technical solutions (1.1)-(1.8), characterized in that the volatile substances contained in the Chinese liquor include one or more substances selected from the group consisting of n-propanol, ethyl acetate, isobutanol, acetal, isoamyl alcohol, phenethyl alcohol, ethyl lactate, and ethyl butyrate.

[0084] (1.10) The method for brewing Chinese liquor according to any one of the technical solutions (1.1)-(1.9), characterized in that the volatile substances contained in the Chinese liquor include ester compounds. Preferably, based on each liter of the Chinese liquor, the content of the ester compounds is 1000-3500 mg, and preferably, the content of the ester compounds is 1000-1600 mg.

[0085] (1.11) The method for brewing Chinese liquor according to any one of the technical solutions (1.1)-(1.10), characterized in that the ester compounds include one or more substances selected from the group consisting of ethyl acetate, ethyl lactate, and ethyl butyrate, and preferably, the ester compound is ethyl acetate.

[0086] (1.12) The brewing method of Chinese liquor according to any one of technical solutions (1.1)-(1.11), characterized in that the volatile substances contained in the Chinese liquor further include one or more substances selected from the group consisting of n-propanol, isobutanol, acetal, isoamyl alcohol, and phenethyl alcohol.

[0087] (1.13) The brewing method of Chinese liquor according to any one of technical solutions (1.1)-(1.12), characterized in that, based on each liter of the Chinese liquor, the volatile substances contained in the Chinese liquor include 85-400 mg of n-propanol, and / or 100-800 mg of isobutanol, and / or 50-380 of acetal, and / or 150-950 of isoamyl alcohol, and / or 10-50 mg of phenethyl alcohol.

[0088] Preferably, 90-350 mg of n-propanol, and / or 250-800 mg of isobutanol, and / or 50-100 of acetal, and / or 400-950 of isoamyl alcohol, and / or 15-50 mg of phenethyl alcohol.

[0089] In some specific embodiments, the present invention provides a technical solution of a brewing method of Chinese liquor (using the bran koji Qingxiang brewing method), which is specifically as follows:

[0090] (2.1) A brewing method of Chinese liquor, characterized in that it includes the following steps: Mix the Pichia kudriavzevii active dry yeast, Aspergillus niger strain, and grain fermented grains described in any one of technical solutions 1-12 or technical solution 20, saccharify, ferment, and distill to obtain Chinese liquor.

[0091] (2.2) The brewing method of Chinese liquor according to technical solution (2.1), characterized in that it includes the following steps:

[0092] (1) Based on the weight of bran, inoculate 5-15 wt% of Aspergillus niger strain and ferment and culture to obtain a fermented product. Preferably, the temperature of the fermentation and culture is 28-33 °C, and / or the time of the fermentation and culture is 4-5 d. Preferably, the Aspergillus niger is Aspergillus niger UV-11.

[0093] It should be noted that the source of the Aspergillus niger strain used in this application can be any commercially available one, or can be obtained by screening through conventional methods. For example, the Aspergillus niger UV-11 strain sold by Beijing BioWin Biotechnology Co., Ltd. can be used.

[0094] (2) Based on the weight of the fermented product, add 0.1-5 wt% of Pichia kudriavzevii active dry yeast to the fermented product and mix to obtain koji. Preferably, the addition amount of Pichia kudriavzevii active dry yeast is 0.5-2 wt%.

[0095] (3) Soak the grains, steam them until cooked, let them cool, and then, based on the weight of the grains before soaking, add 10 wt% - 15 wt% of the koji obtained in step (2) and mix to obtain the grain mash before saccharification. Then, perform saccharification to obtain the saccharified grain mash.

[0096] Among them, the grains include one or more of sorghum, corn, wheat, and rice; preferably, the grains are sorghum.

[0097] (4) Mix and ferment the saccharified grain mash and the grain mash before saccharification at a weight ratio of 1:2 - 3, and then distill to obtain liquor.

[0098] (2.3) The method for brewing Chinese liquor according to the technical solution (2.1) or (2.2), characterized in that water is used for soaking, wherein the soaking temperature is: 70 - 75 °C, and / or the soaking time is: 20 - 24 h.

[0099] (2.4) The method for brewing Chinese liquor according to any one of the technical solutions (2.1) - (2.3), characterized in that the initial steaming time for steaming the grains is 15 - 20 min, the time for uncovering and simmering water during steaming the grains is 40 - 140 min, and the re-steaming time for steaming the grains is 30 - 80 min.

[0100] (2.5) The method for brewing Chinese liquor according to any one of the technical solutions (2.1) - (2.4), characterized in that the cooling temperature is up to 30 - 35 °C.

[0101] (2.6) The method for brewing Chinese liquor according to any one of the technical solutions (2.1) - (2.5), characterized in that the saccharification temperature is 25 - 30 °C, and / or the saccharification time is 18 - 32 h.

[0102] (2.7) The method for brewing Chinese liquor according to any one of the technical solutions (2.1) - (2.6), characterized in that the fermentation temperature is: 22 - 24 °C, and / or the fermentation time is: 7 - 21 days.

[0103] (2.8) The method for brewing Chinese liquor according to any one of the technical solutions (2.1) - (2.7), characterized in that the volatile substances contained in the Chinese liquor include ester compounds. Preferably, based on each liter of the Chinese liquor, the content of the ester compounds is 1000 - 3500 mg, and preferably, the content of the ester compounds is 1600 - 2500 mg.

[0104] (2.9) The brewing method of Chinese liquor according to any one of technical solutions (2.1)-(2.8), characterized in that the ester compounds include one or more substances selected from the group consisting of ethyl acetate, ethyl lactate and ethyl butyrate, and preferably, the ester compounds are ethyl acetate.

[0105] (2.10) The brewing method of Chinese liquor according to any one of technical solutions (2.1)-(2.9), characterized in that the volatile substances contained in the Chinese liquor further include one or more substances selected from the group consisting of n-propanol, isobutanol, acetal, isoamyl alcohol and phenethyl alcohol.

[0106] (2.11) The brewing method of Chinese liquor according to any one of technical solutions (2.1)-(2.10), characterized in that, based on each liter of the Chinese liquor, the volatile substances contained in the Chinese liquor include 85-400 mg of n-propanol, and / or 100-800 mg of isobutanol, and / or 50-380 of acetal, and / or 150-950 of isoamyl alcohol, and / or 10-50 mg of phenethyl alcohol,

[0107] Preferably, 350-400 mg of n-propanol, and / or 100-250 mg of isobutanol, and / or 300-380 of acetal, and / or 150-200 of isoamyl alcohol, and / or 10-15 mg of phenethyl alcohol.

[0108] In some specific embodiments, the present invention provides a technical solution of a brewing method of Chinese liquor (using the method of brewing Chinese liquor with Daqu in the Qingxiang style), which is specifically as follows:

[0109] (3.1) A technical solution of a brewing method of Chinese liquor, comprising the following steps: mixing the Pichia kudriavzevii active dry yeast, Daqu and grain fermented grains described in any one of technical solutions 1-12 or technical solution 20, saccharifying, fermenting and distilling to obtain Chinese liquor.

[0110] (3.2) The brewing method of Chinese liquor according to technical solution (3.1), characterized in that it comprises the following steps:

[0111] (a) Based on the mass of Daqu, adding 0.1-5 wt% of Pichia kudriavzevii active dry yeast to Daqu and mixing to obtain fermented grains for liquor-making, and preferably, based on the weight of Daqu, the addition amount of Pichia kudriavzevii active dry yeast is 0.1-2 wt%;

[0112] It should be noted that the source of the Daqu used in this application can be any commercially purchased one or can be prepared by conventional methods. For example, the Daqu with light aroma type sold by Angel Yeast Co., Ltd. can be used. For example, it can be prepared by the following method: Mix barley and peas in a weight ratio of 6:3-5, crush them into dough, tread the Daqu, ferment and culture for 26-28 days, and then dry to obtain the Daqu.

[0113] (b) Soak the grains, steam them until cooked, and let them cool. Then, based on the weight of the grains before soaking, inoculate 10wt%-20wt% of the koji obtained in step (1) into the cooled grain mash, mix, ferment, and distill to obtain liquor.

[0114] Among them, the grains include one or more of sorghum, corn, wheat, and rice; preferably, the grains are sorghum.

[0115] (3.3) According to the method for brewing liquor described in technical solution (3.1) or (3.2), it is characterized in that water is used for soaking, wherein the soaking temperature is: 70-75°C, and / or the soaking time is: 20-24h.

[0116] (3.4) According to the method for brewing liquor described in any one of technical solutions (3.1)-(3.3), it is characterized in that the initial steaming time for steaming the grains is 15-20 min, the time for uncovering and simmering water during steaming the grains is 50-55 min, and the re-steaming time for steaming the grains is 30-40 min.

[0117] (3.5) According to the method for brewing liquor described in any one of technical solutions (3.1)-(3.4), it is characterized in that the temperature for cooling is reduced to 30-35°C.

[0118] (3.6) According to the method for brewing liquor described in any one of technical solutions (3.1)-(3.5), it is characterized in that the fermentation temperature is: 15-20°C, and / or the fermentation time is: 25-30 days.

[0119] (3.7) According to the method for brewing liquor described in any one of technical solutions (3.1)-(3.6), it is characterized in that the volatile substances contained in the liquor include ester compounds. Preferably, based on each liter of the liquor, the content of the ester compounds is 1000-3000 mg, and more preferably, the content of the ester compounds is 2500-3500 mg.

[0120] (3.8) According to the method for brewing liquor described in any one of technical solutions (3.1)-(3.7), it is characterized in that the ester compounds include one or more substances selected from the group consisting of ethyl acetate, ethyl lactate, and ethyl butyrate.

[0121] (3.9) The brewing method of baijiu according to any one of technical solutions (3.1)-(3.8), characterized in that, based on each liter of the baijiu, the volatile substances contained in the baijiu include 1000-2500 mg of free ethyl acetate, 700-1300 mg of ethyl lactate, and / or 5-15 mg of ethyl butyrate.

[0122] Preferably, 2000-2500 mg of ethyl acetate, 730-1300 mg of ethyl lactate, and 7-15 mg of ethyl butyrate.

[0123] (3.10) The brewing method of baijiu according to any one of technical solutions (3.1)-(3.9), characterized in that the volatile substances contained in the baijiu further include one or more substances selected from the group consisting of n-propanol, isobutanol, acetal, isoamyl alcohol, and phenethyl alcohol.

[0124] In some specific embodiments, the preparation method of the starch hydrolyzate sugar used in the present invention includes: mixing starch and water at 70-75°C in a weight ratio of starch to water of 1:1.2-1.8, and adjusting the pH value to 5.5-6.5 to obtain a mixture. Then, based on the dry weight of each gram of starch, 8-18 U of amylase is added to the mixture, and then by means of jet liquefaction, jet liquefaction is carried out at a temperature of 90-95°C and then put into a liquefaction tank, and kept warm at 72-75°C for 2-3 h. Then, it is transferred to a saccharification tank and the pH is adjusted to 4.5-5.5. Based on the dry weight of each gram of starch, 130-150 U of glucoamylase is added to the mixture, and saccharification is carried out at 55-65°C for 10-15 h. Then, it is transferred to a hydrolyzate sugar storage tank, and the sugar content is adjusted to 15-20 wt%.

[0125] In some specific embodiments, the malt extract agar medium used in the present invention with a sugar content of 8-15 wt% and a pH value of 4.5-6 includes: 5-10 g of yeast extract powder, 15-20 g of agar, 950-1000 mL of distilled water, and at least 25 g of malt extract powder.

[0126] In some specific embodiments, the preparation method of the malt extract agar medium used in the present invention with a sugar content of 8-15 wt% includes: mixing 5-10 g of yeast extract powder, 15-20 g of agar, 950-1000 mL of distilled water, and 25-30 g of malt extract powder, and adjusting the sugar content to 8-15 wt% using the malt extract powder. After adjusting the pH to 4.5-6, it is boiled and sterilized to obtain a malt extract agar medium with a sugar content of 8-15 wt% and a pH value of 4.5-6.

[0127] In some specific embodiments, the wort liquid medium used in the present invention with a sugar content of 8-15 wt% and a pH value of 4.5-6 comprises: 5-10 g of yeast extract powder, 950-1000 mL of distilled water, and at least 25 g of malt extract powder.

[0128] In some specific embodiments, the preparation method of the wort liquid medium with a sugar content of 8-15 wt% used in the present invention comprises: mixing 5-10 g of yeast extract powder, 950-1000 mL of distilled water, and 25-30 g of malt extract powder, adjusting the sugar content to 8-15 wt% using the malt extract powder, adjusting the pH to 4.5-6, and then boiling and sterilizing to obtain a wort liquid medium with a sugar content of 8-15 wt% and a pH value of 4.5-6.

[0129] In some specific embodiments, when preparing the above-mentioned medium, the raw materials used in the present invention include malt extract powder and / or yeast extract powder. Among them, the malt extract powder is extracted from barley malt and contains rich saccharide substances (such as maltose, glucose, and sucrose), which mainly exist as a carbon source substance in the medium to provide energy for microorganisms during fermentation culture. That is to say, when the present invention uses malt extract powder as a common carbon source to prepare the medium, there is no special limitation on its source, and their sources can be any commercially available ones or prepared by conventional methods. The yeast extract powder mainly exists as an organic nitrogen source substance in the medium to provide the necessary nitrogen element for the growth of microorganisms during fermentation culture. After common organic nitrogen sources such as yeast extract powder are decomposed in the medium, amino acids and small molecular peptides are released to become the nitrogen source required for the growth of microorganisms. That is to say, when the present invention uses yeast extract powder as a common organic nitrogen source to prepare the medium, there is no special limitation on its source, and their sources can be any commercially available ones or prepared by conventional methods. As long as the commercially available or conventionally prepared yeast extract powder has a total nitrogen content of greater than or equal to 10.0 wt% and an amino nitrogen content of greater than or equal to 5.0 wt%, it can be used in the present invention.

[0130] Preferably, in some specific embodiments, based on the weight of the yeast extract powder, the yeast extract powder further comprises: 2-2.5 ppm of vitamin B1, 37-40 ppm of vitamin B2, 113-116 ppm of vitamin B5, 15-20 ppm of vitamin B6, 6-10 ppm of vitamin B7, 25-28 ppm of vitamin B9, 3205-3210 ppm of choline, 1575-1580 ppm of inositol, 325-330 ppm of niacin, and 2-4 μg of vitamin B12 per 100 g of yeast extract powder.

[0131] And / or based on the weight of the yeast extract powder, potassium is 31910 - 31912 mg / kg, sodium is 5735 - 5740 mg / kg, calcium is 354 - 357 mg / kg, magnesium is 2670 - 2675 mg / kg, zinc is 77 - 83 mg / kg, and iron is 77 - 83 mg / kg.

[0132] And / or based on the weight of the yeast extract powder, the yeast extract powder includes: free amino acids 30.3 - 40.85%, and hydrolyzed amino groups 51 - 70.5%.

[0133] Among them, the content of free amino acids includes: based on the weight of the yeast extract powder, free aspartic acid 1 - 2%, free threonine 2 - 3%, free serine 1.5 - 2%, free glutamic acid 6.5 - 7%, free glycine 1 - 1.5%, free alanine 4 - 5%, free cysteine 0.1 - 0.15%, free valine 2 - 3%, free methionine 0.5 - 1%, free isoleucine 2 - 2.5%, leucine 3.3 - 3.7%, free tyrosine 0.5 - 1%, free phenylalanine 1.5 - 2%, free lysine 2 - 2.5%, free histidine 0.1 - 1%, free arginine 1.5 - 2.5%, and free proline 0.5 - 1%.

[0134] Among them, the content of hydrolyzed amino acids includes: based on the weight of the yeast extract powder, hydrolyzed aspartic acid 6 - 6.5%, hydrolyzed threonine 2 - 3%, hydrolyzed serine 2 - 3%, hydrolyzed glutamic acid 10 - 15%, hydrolyzed glycine 2 - 3%, hydrolyzed alanine 5 - 6%, hydrolyzed cysteine 0.5 - 1%, hydrolyzed valine 3 - 4%, hydrolyzed methionine 0.5 - 1%, hydrolyzed isoleucine 3 - 4%, hydrolyzed leucine 4 - 5%, hydrolyzed tyrosine 1 - 2%, hydrolyzed phenylalanine 2 - 3%, hydrolyzed lysine 4 - 5%, hydrolyzed histidine 1 - 2%, hydrolyzed arginine 3 - 4%, and hydrolyzed proline 2 - 3%.

[0135] To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0136] All kinds of reagents / instruments used in the examples and comparative examples of the present invention are conventional commercially available products unless otherwise specified. The information sources of the experimental reagents used in the present invention are shown in Table 1, and the information sources of the experimental instruments used in the present invention are shown in Table 2:

[0137] Table 1. Reagent Information Table

[0138]

[0139]

[0140] Table 2. Instrument Information Table

[0141] Instrument Model Selling Manufacturer Separation by Laminated Separator D424 Hitachi Zosen Corporation Granulator / Changzhou Yimin Drying Equipment Co., Ltd. Fluidized Bed / Jingzhou Wuhuan Container Manufacturing Co., Ltd.

[0142] I. The preparation methods of the wort agar media used in the examples and comparative examples are as follows:

[0143] 1. Wort agar medium with a sugar content of 8 wt% and a pH value of 4.5: Mix 5 g of yeast extract powder, 15 g of agar, 1 L of distilled water, and 30 g of malt extract powder, adjust the sugar content to 8 wt% using malt extract powder, adjust the pH to 4.5, and then boil and sterilize to obtain a wort agar medium with a sugar content of 8 wt% and a pH value of 4.5.

[0144] 2. Wort agar medium with a sugar content of 10 wt% and a pH value of 5: Mix 5 g of yeast extract powder, 15 g of agar, 1 L of distilled water, and 30 g of malt extract powder, adjust the sugar content to 10 wt% using malt extract powder, adjust the pH to 5, and then boil and sterilize to obtain a wort agar medium with a sugar content of 10 wt% and a pH value of 5.

[0145] 3. Wort agar medium with a sugar content of 15 wt% and a pH value of 6: Mix 5 g of yeast extract powder, 15 g of agar, 1 L of distilled water, and 30 g of malt extract powder, adjust the sugar content to 15 wt% using malt extract powder, adjust the pH to 6, and then boil and sterilize to obtain a wort agar medium with a sugar content of 15 wt% and a pH value of 6.

[0146] II. The preparation methods of the wort liquid media used in the examples and comparative examples are as follows:

[0147] 1. Wort liquid medium with a sugar content of 8 wt% and a pH value of 4.5: Mix 5 g of yeast extract powder, 1 L of distilled water, and 30 g of malt extract powder, adjust the sugar content to 8 wt% using malt extract powder, adjust the pH to 4.5, and then boil and sterilize to obtain a wort liquid medium with a sugar content of 8 wt% and a pH value of 4.5.

[0148] 2. Wort liquid medium with a sugar content of 10 wt% and a pH value of 5: Mix 5 g of yeast extract powder, 1 L of distilled water, and 30 g of malt extract powder, adjust the sugar content to 10 wt% using malt extract powder, adjust the pH to 5, and then boil and sterilize to obtain a wort liquid medium with a sugar content of 10 wt% and a pH value of 5.

[0149] 3. Wort liquid medium with a sugar content of 15 wt% and a pH value of 6: Mix 5 g of yeast extract powder, 1 L of distilled water, and 30 g of malt extract powder, adjust the sugar content to 15 wt% using malt extract powder, adjust the pH to 6, and then boil and sterilize to obtain a wort liquid medium with a sugar content of 15 wt% and a pH value of 6.

[0150] III. The preparation of the starch hydrolyzate sugar used in the examples and comparative examples is as follows:

[0151] Mix starch and water at 75°C in a weight ratio of starch to water of 1:1.5, adjust the pH value to 6.0 to obtain a mixture. Then, based on the dry weight of each g of starch, add 15 U of amylase to the mixture, and then use jet liquefaction to carry out jet liquefaction at a temperature of 90°C and place it in a liquefaction tank, keep it at 72°C for 2 h. Then transfer it to a saccharification tank and adjust the pH to 4.5. Based on the dry weight of each g of starch, add 130 U of glucoamylase to the mixture and saccharify it at 60°C for 12 h. Then transfer it to a hydrolyzate sugar storage tank and adjust the sugar content to 15 wt%.

[0152] IV. The preparation of the YPD liquid medium used in the examples is as follows: Mix 10 g of yeast extract powder, 20 g of glucose, 20 g of peptone, and 1000 mL of water, and sterilize at 115°C for 30 min to obtain the YPD liquid medium.

[0153] V. The trace elements contained in the yeast extract powder (model: FM888) used in the examples are as follows: vitamin B1 is 2.3 ppm, vitamin B2 is 38.8 ppm, vitamin B5 is 115.0 ppm, vitamin B6 is 18.0 ppm, vitamin B7 is 7.9 ppm, vitamin B9 is 26.7 ppm, vitamin B12 is 2.3 (μg / 100 g), choline is 3206.0 ppm, inositol is 1577.7 ppm, and niacin is 328.0 ppm.

[0154] The trace elements contained in the yeast extract powder (model: FM888) used in the examples are as follows: potassium is 31911.66 mg / kg, sodium is 5738.91 mg / kg, calcium is 355.25 mg / kg, magnesium is 2673.59 mg / kg, zinc is 80.94 mg / kg, and iron is 80.21 mg / kg.

[0155] The free amino acids contained in the yeast extract powder (model: FM888) used in the examples are 35.1%.

[0156] And the hydrolyzed amino acids are 61.21%.

[0157] Among them, the content of free amino acids is specifically as follows: calculated by the weight of the yeast extract powder, free aspartic acid is 1.6%, free threonine is 2.1%, free serine is 1.7%, free glutamic acid is 6.7%, free glycine is 1.2%, free alanine is 4.2%, free cysteine is 0.1%, free valine is 2.7%, free methionine is 0.8%, free isoleucine is 2.2%, leucine is 3.5%, free tyrosine is 0.9%, free phenylalanine is 1.8%, free lysine is 2.3%, free histidine is 0.5%, free arginine is 2.0%, and free proline is 0.8%.

[0158] Among them, the content of hydrolyzed amino acids is specifically as follows: calculated by the weight of the yeast extract powder, hydrolyzed aspartic acid is 6.23%, hydrolyzed threonine is 2.71%, hydrolyzed serine is 2.73%, hydrolyzed glutamic acid is 12.33%, hydrolyzed glycine is 2.74%, hydrolyzed alanine is 5.17%, hydrolyzed cysteine is 0.61%, hydrolyzed valine is 3.84%, hydrolyzed methionine is 0.84%, hydrolyzed isoleucine is 3.65%, hydrolyzed leucine is 4.72%, hydrolyzed tyrosine is 1.65%, hydrolyzed phenylalanine is 2.68%, hydrolyzed lysine is 4.63%, hydrolyzed histidine is 1.19%, hydrolyzed arginine is 3.30%, and hydrolyzed proline is 2.19%.

[0159] The detection method for the sugar content of the carbon source in the examples and comparative examples is as follows: Take 100 mL of the sugar solution after treatment in a measuring cylinder, and directly measure the sugar degree using a saccharimeter.

[0160] The preparation process of the light-flavor Daqu used in the application examples: Mix barley and peas in a weight ratio of 6:4 and crush them into a state with coarse skin and fine flour, then add water and mix the materials until it can be kneaded into a ball that sticks to the hand but does not drip water; then step on the starter, requiring the starter blocks to be firm, flat, and smooth; then carry out natural fermentation and cultivation, going through stages such as lying the starter, getting moldy, airing the mold, moist fire, strong fire, after fire, and cultivating the starter, strictly controlling the temperature and turning the starter in a timely manner, and cultivating for 26 - 28 days; finally, take the starter out of the room and dry it naturally, and store it ventilated for 3 - 6 months.

[0161] Example 1

[0162] The preparation method of the Pichia kudriavzevii active dry yeast provided in Example 1 is specifically as follows:

[0163] (1) Prepare the carbon source: Add invertase to the storage tank containing cane molasses. Among them, the addition amount of invertase is: based on every g of sucrose contained in cane molasses, add 200 U of invertase, and react at 60 °C for 2 h and adjust the sugar content with water to obtain cane molasses treated with invertase with a pH value of 4 and a sugar content of 15 wt%, that is, the carbon source.

[0164] (2) Cultivate the strain: Inoculate the strain in the glycerol preservation solution into the YPD liquid medium and culture it at 30 °C for 24 h to obtain a bacterial suspension with a viable count of 1×10 8 CFU / mL. Inoculate 1 mL of this suspension onto a malt extract agar slant medium with a sugar content of 8 wt% and a pH value of 4.5, and culture it at 30 °C for 24 h. Then pick a single colony and inoculate it into the YPD liquid medium, and culture it at 30 °C for 24 h to obtain a bacterial suspension of Pichia kudriavzevii C4.12, which is used to complete the activation of the activated strain. Inoculate the activated bacterial suspension of Pichia kudriavzevii C4.12 onto a malt extract agar slant medium (malt extract agar medium) with a sugar content of 8 wt% and a pH value of 4.5, and culture it at 28 °C for 24 h to obtain a slant strain of Pichia kudriavzevii C4.12.

[0165] (3) Prepare the primary liquid seed culture medium: Take 1 loop of the strain of Pichia kudriavzevii C4.12 obtained in step (2) and inoculate it into an F flask containing 100 mL of malt extract liquid medium with a sugar content of 8 wt% and a pH value of 4.5, and culture it at 28 °C for 24 h to obtain 100 mL of the primary liquid seed culture medium of Pichia kudriavzevii C4.12.

[0166] (4) Prepare the secondary liquid seed culture medium: Inoculate the 100 mL of the primary liquid seed culture medium of Pichia kudriavzevii C4.12 obtained in step (3) into a carboys containing 10 L of malt extract liquid medium with a sugar content of 8 wt% and a pH value of 4.5, and culture it at 28 °C for 20 h to obtain 10 L of the secondary liquid seed culture medium of Pichia kudriavzevii C4.12.

[0167] (5) Prepare the fermenter seed culture medium: Inoculate the 10 L of the secondary liquid seed culture medium of Pichia kudriavzevii C4.12 obtained in step (4) into a 1 m3 fermenter containing 6000 kg of the carbon source obtained in step (1), 12.9 kg of ammonium sulfate, 5.6 kg of ammonium phosphate, 9 kg of potassium chloride, 1.4 kg of magnesium sulfate, 20 g of calcium pantothenate, 5 g of nicotinic acid, 10 g of inositol, and 25 g of biotin. Then, under the condition of 24 °C, sterile air is introduced, and the fermentation is carried out for 20 h under the condition that the air flow rate of the introduced sterile air is controlled at 10 m3 air / fermentation broth m3 / hr to obtain 1 m3 of the fermenter seed culture medium of Pichia kudriavzevii C4.12.

[0168] (6) Preparation of fed-batch fermentation culture medium: Inoculate the 1 m3 fermentation tank seed culture medium of Pichia kudriavzevii C4.12 in step (5) into a 15 m3 fermentation tank. Then, under the condition of a feeding rate of 2000 L / hr, continuously feed the carbon source obtained in step (1), and introduce sterile air at 24 °C. Ferment for 20 h under the condition that the air volume of the introduced sterile air is controlled at 30 m3 air / m3 fermentation broth / hr to obtain 10.5 m3 of the fed-batch fermentation culture medium of Pichia kudriavzevii C4.12. Among them, a total of 11200 kg of the carbon source obtained in step (1) is fed until the end of fermentation.

[0169] It should be noted that during the fed-batch fermentation culture process in step (6), the ethanol content in the fed-batch fermentation culture medium of Pichia kudriavzevii C4.12 is detected once every 1 h. When the ethanol content in the fed-batch fermentation culture medium exceeds 0.4%, the feeding rate of the treated molasses is reduced, and at the same time, the air volume of the introduced sterile air is increased to control the ethanol content in the fermentation broth below 0.4%.

[0170] (7) Separation: Use a stack separator to separate the fed-batch fermentation culture medium of Pichia kudriavzevii C4.12 obtained in step (6) at 4000 rpm and retain the precipitate, that is, 21000 kg of yeast milk is obtained.

[0171] (8) Shaping: Add 1 kg of sorbitan monostearate to the yeast milk obtained in step (7), granulate in a granulator at 4 °C, and then extrude it into a sieve with a pore diameter of 0.5 mm to obtain yeast strips in the shape of Chinese long noodles.

[0172] (9) Drying: Dry the yeast strips obtained in step (8) using a fluidized bed dryer to obtain Pichia kudriavzevii active dry yeast; among them, the drying temperature is: the inlet air temperature is 115 °C, the outlet air temperature is 80 °C, and the drying time is 25 min.

[0173] Example 2

[0174] The preparation method of Pichia kudriavzevii active dry yeast provided in Example 2 is specifically as follows:

[0175] (1) Preparation of carbon source: Add invertase to a storage tank containing cane molasses. Among them, the addition amount of invertase is: 200 U of invertase is added per g of sucrose contained in cane molasses, and react at 60 °C for 2 h to obtain cane molasses treated with invertase with a pH value of 4 and a sugar content of 15 wt%, that is, the carbon source.

[0176] (2) Culturing the strain: Inoculate the cell suspension of the activated Pichia kudriavzevii C4.12 onto a malt extract agar slant medium with a sugar content of 10 wt% and a pH value of 5, and culture it at 35 °C for 48 h to obtain the slant strain of Pichia kudriavzevii C4.12. Among them, the activation process of the strain is the same as that in Example 1.

[0177] (3) Preparing the primary liquid seed culture medium: Take 6 loops of the strain of Pichia kudriavzevii C4.12 obtained in step (2) and inoculate it into an F flask containing 250 mL of malt extract liquid medium with a sugar content of 10 wt% and a pH value of 5, and culture it at 35 °C for 48 h to obtain 250 mL of the primary liquid seed culture medium of Pichia kudriavzevii C4.12.

[0178] (4) Preparing the secondary liquid seed culture medium: Inoculate the 250 mL of the primary liquid seed culture medium of Pichia kudriavzevii C4.12 obtained in step (3) into a carboys containing 20 L of malt extract liquid medium with a sugar content of 10 wt% and a pH value of 5, and culture it at 35 °C for 48 h to obtain 20 L of the secondary liquid seed culture medium of Pichia kudriavzevii C4.12.

[0179] (5) Preparing the fermenter seed culture medium: Inoculate the 20 L of the secondary liquid seed culture medium of Pichia kudriavzevii C4.12 obtained in step (4) into a 3 m3 fermenter containing 6000 kg of the carbon source obtained in step (1), 140 kg of ammonia water, 50 kg of diammonium hydrogen phosphate, 20.5 g of potassium chloride, 8 kg of magnesium sulfate, 120 g of calcium pantothenate, 60 g of nicotinic acid, 120 g of inositol and 125 g of biotin. Then, introduce sterile air at 35 °C and ferment for 48 h under the condition that the air flow rate of the introduced sterile air is controlled at 30 m3 air / fermentation broth m3 / hr to obtain 3 m3 of the fermenter seed culture medium of Pichia kudriavzevii C4.12.

[0180] (6) Preparing the fed-batch fermentation culture medium: Inoculate the 3 m3 of the fermenter seed culture medium of Pichia kudriavzevii C4.12 in step (5) into a 180 m3 fermenter. Then, continuously feed the carbon source obtained in step (1) at a feeding rate of 2000 L / hr, introduce sterile air at 35 °C, and ferment for 20 h under the condition that the air flow rate of the introduced sterile air is controlled at 50 m3 air / fermentation broth m3 / hr to obtain 75 m3 of the fed-batch fermentation culture medium of Pichia kudriavzevii C4.12. Among them, a total of 85000 kg of the carbon source obtained in step (1) is fed until the end of fermentation.

[0181] It should be noted that during the fed-batch fermentation culture process in step (6), the ethanol content in the fed-batch fermentation culture broth of Pichia kudriavzevii C4.12 is detected every 1 h. When the ethanol content in the fed-batch fermentation culture broth exceeds 0.4%, the feeding rate of the treated molasses is reduced, and at the same time, the air volume of the sterile air introduced is increased, so that the ethanol content in the fermentation broth is controlled below 0.4%.

[0182] (7) Separation: Using a stack separator, the fed-batch fermentation culture broth of Pichia kudriavzevii C4.12 obtained in step (6) is separated at 4000 rpm, and the precipitate is retained, that is, 30000 kg of yeast cream is obtained.

[0183] (8) Shaping: Add 240 kg of sorbitan laurate to the yeast cream obtained in step (7), granulate in a granulator at 4 °C, and then extrude it into a sieve with a pore size of 0.5 mm to obtain yeast strips in the shape of Chinese dragon beard noodles.

[0184] (9) Drying: The yeast strips obtained in step (8) are dried using a fluidized bed dryer to obtain Pichia kudriavzevii active dry yeast; among them, the drying temperature is: the inlet air temperature is 105 °C, the outlet air temperature is 75 °C, and the drying time is 35 min.

[0185] Example 3

[0186] The preparation method of Pichia kudriavzevii active dry yeast provided in Example 3 is specifically as follows:

[0187] (1) Preparation of carbon source: Add invertase to a storage tank containing beet molasses. Among them, the addition amount of invertase is: based on every g of sucrose contained in the beet molasses, 200 U of invertase is added, and the reaction is carried out at 60 °C for 2 h to obtain beet molasses treated with invertase with a pH value of 4 and a sugar content of 15 wt%, and a mixture obtained by mixing the beet molasses treated with invertase and starch hydrolyzate in equal proportion, that is, the carbon source.

[0188] (2) Culturing of strains: Inoculate the activated cell suspension of Pichia kudriavzevii C4.12 onto a malt extract agar slant medium with a sugar content of 15 wt% and a pH value of 6, and culture at 30 °C for 36 h to obtain the strain of Pichia kudriavzevii C4.12. Among them, the activation process of the strain is the same as that in Example 1.

[0189] (3) Preparation of primary liquid seed culture medium: Take 4 loops of the Pichia kudriavzevii C4.12 strain obtained in step (2) and inoculate them into an F flask containing 500 mL of malt juice liquid medium with a sugar content of 15 wt% and a pH value of 6, and culture at 30 °C for 36 h to obtain 500 mL of the primary liquid seed culture medium of Pichia kudriavzevii C4.12.

[0190] (4) Preparation of secondary liquid seed culture medium: Inoculate the 500 mL of the primary liquid seed culture medium of Pichia kudriavzevii C4.12 obtained in step (3) into a Carlsberg flask containing 40 L of malt juice liquid medium with a sugar content of 15 wt% and a pH value of 6, and culture at 30 °C for 30 h to obtain 40 L of the secondary liquid seed culture medium of Pichia kudriavzevii C4.12.

[0191] (5) Preparation of fermenter seed culture medium: Inoculate the 40 L of the secondary liquid seed culture medium of Pichia kudriavzevii C4.12 obtained in step (4) into a 5 m3 fermenter containing 6000 kg of the carbon source obtained in step (1), 250 kg of ammonia water, 90 kg of ammonium hydrogen phosphate, 10 kg of potassium chloride, 15 kg of magnesium sulfate, 60 g of calcium pantothenate, 20 g of nicotinic acid, 40 g of inositol, and 70 g of biotin. Then, introduce sterile air at 30 °C and ferment for 30 h under the condition that the air flow rate of the introduced sterile air is controlled at 20 m3 of air / m3 of fermentation broth / hr to obtain 3 m3 of the fermenter seed culture medium of Pichia kudriavzevii C4.12.

[0192] (6) Preparation of fed-batch fermentation culture medium: Inoculate the 5 m3 of the fermenter seed culture medium of Pichia kudriavzevii C4.12 in step (5) into a 200 m3 fermenter. Then, continuously feed the carbon source obtained in step (1) at a feeding rate of 2000 L / hr, introduce sterile air at 35 °C, and ferment for 20 h under the condition that the air flow rate of the introduced sterile air is controlled at 80 m3 of air / m3 of fermentation broth / hr to obtain 130 m3 of the fed-batch fermentation culture medium of Pichia kudriavzevii C4.12. Among them, a total of 144000 kg of the carbon source obtained in step (1) is fed until the end of fermentation.

[0193] It should be noted that during the fed-batch fermentation culture process in step (6), the ethanol content in the fed-batch fermentation culture medium of Pichia kudriavzevii C4.12 is detected every 1 h. When the ethanol content in the fed-batch fermentation culture medium exceeds 0.4%, the feeding rate of the treated molasses is reduced, and at the same time, the air flow rate of the introduced sterile air is increased to control the ethanol content in the fermentation broth below 0.4%.

[0194] (7) Separation: Using a laminated separator, the fed-batch fermentation broth of Pichia kudriavzevii C4.12 obtained in step (6) was separated at 4000 rpm, and the precipitate was retained, thus obtaining 58000 kg of yeast cream.

[0195] (8) Shaping: Add 130 kg of sorbitan palmitate to the yeast cream obtained in step (7), granulate it in a granulator at 4 °C, and then extrude it into a sieve with a pore size of 0.5 mm to obtain yeast strips in the shape of longxu noodles;

[0196] (9) Drying: The yeast strips obtained in step (8) were dried using a fluidized bed dryer to obtain Pichia kudriavzevii active dry yeast; among them, the drying temperature was: the inlet air temperature was 70 °C, the outlet air temperature was 40 °C, and the drying time was 65 min.

[0197] Example 4

[0198] The preparation method of Pichia kudriavzevii active dry yeast provided in Example 4 is as follows:

[0199] (1) Preparation of carbon source: Add invertase to a storage tank containing beet molasses. Among them, the addition amount of invertase was: based on every g of sucrose contained in beet molasses, 200 U of invertase was added, and the reaction was carried out at 60 °C for 2 h and the sugar content was adjusted with water to obtain beet molasses treated with invertase with a pH value of 4 and a sugar content of 15 wt%, that is, the carbon source.

[0200] (2) Cultivation of strains: Inoculate the activated cell suspension of Pichia kudriavzevii C4.12 onto a malt extract agar slant medium with a sugar content of 15 wt% and a pH value of 5.5, and cultivate it at 30 °C for 30 h to obtain the strain of Pichia kudriavzevii C4.12. Among them, the activation process of the strain was the same as that in Example 1.

[0201] (3) Preparation of the first-stage liquid seed culture medium: Take 5 loops of the strain of Pichia kudriavzevii C4.12 obtained in step (2) and inoculate it into an F flask containing 400 mL of a malt extract liquid medium with a sugar content of 15 wt% and a pH value of 5.5, and cultivate it at 30 °C for 30 h to obtain 400 mL of the first-stage liquid seed culture medium of Pichia kudriavzevii C4.12.

[0202] (4) Preparation of secondary liquid seed culture medium: Inoculate the 400 mL of the primary liquid seed culture medium of Pichia kudriavzevii C4.12 obtained in step (3) into a carboys filled with 25 L of malt extract liquid medium with a sugar content of 15 wt% and a pH value of 5.5, and culture at 30 °C for 20 h to obtain 25 L of the secondary liquid seed culture medium of Pichia kudriavzevii C4.12.

[0203] (5) Preparation of fermenter seed culture medium: Inoculate the 25 L of the secondary liquid seed culture medium of Pichia kudriavzevii C4.12 obtained in step (4) into a 4 m3 fermenter, which contains 6000 kg of the carbon source obtained in step (1), 320 kg of ammonium sulfate, 110 kg of phosphoric acid, 25 kg of potassium chloride, 12 kg of magnesium sulfate, 90 g of calcium pantothenate, 15 g of niacin, 30 g of inositol, and 120 g of biotin. Then, sterile air is introduced at 30 °C, and the fermentation is carried out for 20 h under the condition that the air flow rate of the sterile air is controlled at 25 m3 air / m3 of fermentation broth / hr to obtain 4 m3 of the fermenter seed culture medium of Pichia kudriavzevii C4.12.

[0204] (6) Preparation of fed-batch fermentation culture medium: Inoculate the 4 m3 of the fermenter seed culture medium of Pichia kudriavzevii C4.12 obtained in step (5) into a 150 m3 fermenter. Then, under the condition of a feeding rate of 200 L / hr, continuously feed the carbon source obtained in step (1), and introduce sterile air at 30 °C. Stir for 28 h under the condition that the air flow rate of the introduced sterile air is controlled at 60 m3 air / m3 of fermentation broth / hr to obtain 105 m3 of the fed-batch fermentation culture medium of Pichia kudriavzevii C4.12. Among them, a total of 115900 kg of the carbon source obtained in step (1) is fed until the end of fermentation.

[0205] It should be noted that during the fed-batch fermentation culture process in step (6), the ethanol content in the fed-batch fermentation culture medium of Pichia kudriavzevii C4.12 is detected every 1 h. When the ethanol content in the fed-batch fermentation culture medium exceeds 0.4%, the feeding rate of the treated molasses is reduced, and at the same time, the air flow rate of the introduced sterile air is increased to control the ethanol content in the fermentation broth below 0.4%.

[0206] (7) Separation: Use a stack separator to separate the fed-batch fermentation culture medium of Pichia kudriavzevii C4.12 obtained in step (6) at 4000 rpm and retain the precipitate to obtain 52000 kg of yeast milk.

[0207] (8) Shaping: Add 190 kg of sorbitan stearate to the yeast milk obtained in step (7), granulate it in a granulator at 4 °C, and then extrude it through a sieve with a pore size of 0.5 mm to obtain yeast strips in the shape of long, thin noodles.

[0208] (9) Drying: Dry the yeast strips obtained in step (8) using a fluidized bed dryer to obtain Pichia kudriavzevii active dry yeast; among them, the drying temperature is: the inlet air temperature is 80 °C, the outlet air temperature is 45 °C, and the drying time is 50 min.

[0209] Example 5

[0210] The preparation method of Pichia kudriavzevii active dry yeast provided in Example 5 is different from that in Example 4 in that: the carbon source used is different. The carbon source used in Example 6 is: beet molasses treated with sucrase with a pH value of 4 and a sugar content of 35 wt%, that is, the carbon source.

[0211] In step (6), a total of 114,700 kg of the carbon source obtained in step (1) was added dropwise until the end of fermentation. In step (7), 48,000 kg of yeast milk was obtained.

[0212] Example 6

[0213] The preparation method of Pichia kudriavzevii active dry yeast provided in Example 6 is different from that in Example 4 in that: the carbon source used is different. The carbon source used in Example 6 is: beet molasses treated with sucrase with a pH value of 4 and a sugar content of 10 wt%, that is, the carbon source. In step (6), a total of 117,600 kg of the carbon source obtained in step (1) was added dropwise until the end of fermentation. In step (7), 46,800 kg of yeast milk was obtained.

[0214] Comparative Example 1

[0215] The preparation method of Pichia kudriavzevii active dry yeast provided in Comparative Example 1 is different from that in Example 1 in that: the carbon source used is different. The carbon source used in Comparative Example 1 is: starch hydrolysate with a pH value of 4 and a sugar content of 15 wt%. In step (7), 19,300 kg of yeast milk was obtained.

[0216] Comparative Example 2

[0217] The preparation method of Pichia kudriavzevii active dry yeast provided in Comparative Example 2 is different from that in Example 2 in that: the carbon source used is different. The carbon source used in Comparative Example 2 is: cane molasses without sucrase treatment with a pH value of 4 and a sugar content of 15 wt%. In step (7), 21,000 kg of yeast milk was obtained.

[0218] Comparative Example 3

[0219] The preparation method of Pichia kudriavzevii active dry yeast provided in Comparative Example 3 is different from that in Example 3 in that: the carbon sources used are different. The carbon source used in Comparative Example 3 is a mixture obtained by mixing sugar beet raw molasses with a pH of 4 and a sugar content of 15 wt% that has not been treated with invertase and starch hydrolyzate in equal proportions. In step (7), 52000 kg of yeast cream is obtained.

[0220] Comparative Example 4

[0221] The preparation method of Pichia kudriavzevii active dry yeast provided in Comparative Example 4 is different from that in Example 4 in that: the carbon sources used are different. The carbon source used in Comparative Example 4 is sugar beet molasses with a pH of 4 and a sugar content of 15 wt% that has not been treated with invertase. In step (7), 38600 kg of yeast cream is obtained.

[0222] Comparative Example 5

[0223] The preparation method of Pichia kudriavzevii active dry yeast provided in Comparative Example 5 is different from that in Example 5 in that: the carbon sources used are different. The carbon source used in Comparative Example 5 is sugar beet molasses with a pH of 4 and a sugar content of 35 wt% that has been treated with invertase. In step (7), 37200 kg of yeast cream is obtained.

[0224] Comparative Example 6

[0225] The preparation method of Pichia kudriavzevii active dry yeast provided in Comparative Example 6 is different from that in Example 5 in that: the carbon sources used are different. The carbon source used in Comparative Example 6 is sugar beet molasses with a pH of 4 and a sugar content of 10 wt% that has been treated with invertase. In step (7), 36500 kg of yeast cream is obtained.

[0226] Technical effect evaluation:

[0227] The total cell count, viable cell rate, and moisture content of the Pichia kudriavzevii active dry yeast obtained in Examples 1-6 and Comparative Examples 1-5 were detected, and the detection results are shown in Table 3.

[0228] Among them, the method for measuring the total cell count: After activating the dry yeast with sterile physiological saline, the total number of cells measured using a microscope magnified 40 times and a hemocytometer is the sum of the dead cell count and the viable cell count.

[0229] The method for measuring the viable cell rate: After adding the dry yeast to sterile physiological saline for activation, it is stained with methylene blue solution, and then the percentage of the ratio of the viable yeast cell count to the total cell count measured using a microscope magnified 40 times and a hemocytometer. The cells stained blue are dead cells, and the cells not stained blue are viable cells.

[0230] Method for measuring moisture content: The measurement is carried out with reference to the method in GB / T 20886.1-2021.

[0231] Table 3. Detection results of total cell count, viable cell rate and moisture content of Pichia kudriavzevii active dry yeast

[0232] Total Cell Count (100 million / g) Viable Cell Ratio (%) Moisture (%) Example 1 260 76 5.0 Example 2 280 77 5.0 Example 3 300 82 4.7 Example 4 350 90 4.5 Example 5 290 83 4.6 Example 6 280 74 5.0 Comparative Example 1 180 68 4.9 Comparative Example 2 190 63 5.0 Comparative Example 3 220 71 4.8 Comparative Example 4 198 72 4.5 Comparative Example 5 190 65 4.6 Comparative Example 6 170 61 5.0

[0233] As shown in Table 3, the total cell count of Pichia kudriavzevii active dry yeast prepared in Examples 1-6 is between 26 and 35 billion / g, while the total cell count of dry yeast in Comparative Examples 1-6 is between 17 and 22 billion / g. The results show that the Pichia kudriavzevii active dry yeast obtained by the preparation method provided in the examples of the present invention has good quality, and the parameters of its total cell count, viable cell rate and moisture are all stable within a certain range, which fully indicates that the quality of Pichia kudriavzevii active dry yeast obtained by the preparation method of the present invention is stable.

[0234] In addition, as shown in Table 3, since the cell count and viable cell rate of Pichia kudriavzevii active dry yeast obtained in Example 4 are higher than those in Examples 1-6. Therefore, the Pichia kudriavzevii active dry yeast prepared in Example 4 is selected and used to prepare light-flavor Rhizopus koji, light-flavor bran koji and light-flavor fortified Daqu and applied in the solid-state light-flavor Xiaoqu brewing process, bran koji light-flavor brewing process and Daqu light-flavor brewing process; and the wines prepared by the above brewing processes are subjected to detection of volatile substances to evaluate the quality of the wines. The specific steps of the brewing process and the detection results of volatile substances in the wines are shown in Application Examples 1-6 below.

[0235] Application Example 1 (Method for brewing light-flavor Xiaoqu with solid state, raw material is sorghum)

[0236] (1) Preparation of light-flavor Rhizopus koji: Based on the weight of bran, inoculate 10 wt% of the activated Rhizopus Q303 strain and culture at 30 °C for 72 h to obtain a fermented product. Based on the weight of the fermented product, add 1 wt% of the Pichia kudriavzevii active dry yeast obtained in Example 1 to the fermented product and mix to obtain koji, that is, light-flavor Rhizopus koji.

[0237] (2) Soaking grains: Soak high-quality sorghum without mildew and insect damage in water at 70 °C for 22 h and filter the water to obtain soaked sorghum.

[0238] (3) Steaming grains: Steam the soaked sorghum for 20 min at a temperature of 100 °C and a pressure of 1.5 kPa for the first time, cover and simmer for 50 min, and then steam again for 40 min to obtain steamed sorghum.

[0239] (4) Cooling: The cooked sorghum is cooled to 30 °C to obtain the cooled sorghum.

[0240] (5) Saccharification:

[0241] Experimental group: Based on the weight of the sorghum before soaking the grains, 0.5 wt% of the Rhizopus koji for light-flavor liquor is added and mixed evenly with the grain mash to obtain fresh grain mash. Then, the grain mash is spread evenly on the stacking saccharification bed for saccharification to obtain the saccharified grain mash. Among them, the initial temperature of saccharification is 26 °C, the saccharification time is 24 h, and ventilation is maintained. During the saccharification process, the temperature is maintained at 26 - 33 °C.

[0242] Control group: Based on the weight of the sorghum before soaking the grains, 0.5 wt% of the commercially available Rhizopus koji shown in Table 1 is added and mixed to obtain fresh grain mash. Then, the grain mash is spread evenly on the stacking saccharification bed for saccharification to obtain the saccharified grain mash. Among them, the initial temperature of saccharification is 26 °C, the saccharification time is 24 h, and ventilation is maintained.

[0243] (6) Fermentation:

[0244] Experimental group: The saccharified grain mash and fresh grain mash are mixed evenly at a ratio of 1:3 by weight, then cooled by ventilation to 23 °C, and then sealed and fermented in a fermentation box for 15 d and then distilled to obtain liquor.

[0245] Control group: The same as the experimental group.

[0246] (7) Detection of volatile substances: The liquor obtained in step (6) is detected by the detection method of volatile substances. Among them, the detection parameters are: chromatographic column: Baijiu column 21134 - 5 (25 m × 320 μm × 1 μm); carrier gas (high-purity nitrogen) flow rate: 0.6 mL / min; split ratio: 40:1; tail gas blow 25 mL / min; H2 flow rate 30 mL / min; air flow rate 400 mL / min; injection volume: 1 μL; detector temperature: 250 °C; injection port temperature 250 °C; column oven temperature programming conditions are: 50 °C for 2 min of equilibration, programmed temperature rise at 5 °C / min to 110 °C, then at 3 °C / min to 130 °C, and at 15 °C / min to 230 °C and hold for 2 min. The detection results are shown in Table 4.

[0247] Table 4. Detection results of volatile substances

[0248]

[0249] As shown in Table 4, the results show that the liquor prepared with the Rhizopus koji for light-flavor liquor in the experimental group has nearly 10 times higher ethyl acetate content than that in the control group and meets the national standard for high-quality light-flavor liquor, indicating that the liquor body prepared with the Rhizopus koji for light-flavor liquor using Pichia kudriavzevii as the raw material with sorghum has better quality.

[0250] Application Example 2 (Method of Brewing with Solid-State Aroma-Type Small Qu) with Corn as Raw Material

[0251] (1) Preparation of Aroma-Type Rhizopus Qu: Based on the weight of bran, inoculate 10 wt% of the activated Rhizopus Q303 strain and culture it at 30 °C for 72 h to obtain a fermented product. Based on the weight of the fermented product, add 1 wt% of the Pichia kudriavzevii active dry yeast obtained in Example 2 to the fermented product and mix to obtain the qu, i.e., the aroma-type Rhizopus qu.

[0252] (2) Soaking the Grains: Soak high-quality corn without mildew and insect damage in water at 70 °C for 22 h and then filter the water to obtain the soaked corn.

[0253] (3) Steaming the Grains: Steam the soaked corn at a temperature of 100 °C and a pressure of 1.5 kPa for 20 min for the first steaming. After opening the lid and simmering in water for 120 min, then steam again for 60 min to obtain the steamed corn.

[0254] (4) Cooling: Cool the steamed corn to 30 °C to obtain the cooled corn.

[0255] (5) Saccharification:

[0256] Experimental Group: Based on the weight of the corn before soaking, add 0.5 wt% of the aroma-type Rhizopus qu and mix it evenly with the grain mash to obtain fresh grain mash. Then, spread the grain mash evenly on the stacking saccharification bed for saccharification to obtain the saccharified grain mash. Among them, the initial saccharification temperature is 26 °C, the saccharification time is 24 h, and ventilation is maintained.

[0257] Control Group: Based on the weight of the corn before soaking, add 0.5 wt% of the commercially available Rhizopus qu shown in Table 1 and mix to obtain fresh grain mash. Then, spread the grain mash evenly on the stacking saccharification bed for saccharification to obtain the saccharified grain mash. Among them, the initial saccharification temperature is 26 °C, the saccharification time is 24 h, and ventilation is maintained.

[0258] (6) Fermentation:

[0259] Experimental Group: Mix the saccharified fermented grains and fresh fermented grains evenly at a ratio of 1:3 by weight, then ventilate and cool to 23 °C, put them into the fermentation tank, seal and ferment for 15 d, and then distill to obtain wine.

[0260] Control Group: The same as the experimental group.

[0261] (7) Detection of Volatile Substances: Detect the wine obtained in step (6) by the detection method of volatile substances. Among them, the detection method of volatile components is the same as that in Application Example 1, and the detection results are shown in Table 5.

[0262] Table 5 Detection Results of Volatile Substances

[0263]

[0264] As shown in Table 5, the results indicate that compared with the control group, the content of ethyl acetate in the liquor prepared by the experimental group has increased by nearly 10 times, reaching the national standard for high-quality light-aroma liquor, indicating that the liquor quality prepared with the light-aroma rhizopus koji using corn as the raw material and Pichia kudriavzevii is better.

[0265] Application Example 3 (Method for brewing light-aroma liquor with small starter cakes in solid state, with wheat as the raw material)

[0266] (1) Preparation of light-aroma rhizopus koji: Based on the weight of bran, inoculate 10 wt% of the activated Rhizopus Q303 strain and culture it at 30 °C for 72 h to obtain a fermented product. Based on the weight of the fermented product, add 1 wt% of the Pichia kudriavzevii active dry yeast obtained in Example 3 to the fermented product and mix to obtain the koji, namely the light-aroma rhizopus koji.

[0267] (2) Soaking grains: Soak high-quality wheat without mildew and insect damage in water at 70 °C for 22 h and filter the water to obtain the soaked wheat.

[0268] (3) Steaming grains: Steam the soaked wheat at a temperature of 100 °C and a pressure of 1.5 kPa for 20 min for the first steaming. After uncovering the lid and simmering in water for 50 min, then perform a second steaming for 60 min to obtain the steamed wheat.

[0269] (4) Cooling: Cool the steamed wheat to 30 °C to obtain the cooled wheat.

[0270] (5) Saccharification:

[0271] Experimental group: Based on the weight of the wheat before soaking grains, inoculate 0.5 wt% of the light-aroma rhizopus koji and mix it evenly with the grain mash to obtain fresh grain mash. Then, spread the grain mash evenly on the stacking saccharification bed for saccharification to obtain the saccharified grain mash. Among them, the initial temperature of saccharification is 26 °C, the saccharification time is 24 h, and ventilation is maintained.

[0272] Control group: Based on the weight of the wheat before soaking grains, inoculate 0.5 wt% of the commercially available rhizopus koji shown in Table 1 and mix to obtain fresh grain mash. Then, spread the grain mash evenly on the stacking saccharification bed for saccharification to obtain the saccharified grain mash. Among them, the initial temperature of saccharification is 26 °C, the saccharification time is 24 h, and ventilation is maintained.

[0273] (6) Fermentation:

[0274] Experimental group: Mix the saccharified fermented grains and fresh fermented grains evenly at a ratio of 1:3 by weight, then cool the temperature by ventilation to 23 °C, put them into the fermentation tank, seal and ferment for 15 d, and then distill to obtain the liquor.

[0275] Control group: The same as the experimental group.

[0276] (7) Detection of volatile substances: The wine obtained in step (6) is detected by the detection method of volatile substances. Among them, the detection method of volatile components is the same as that in Application Example 1, and the detection results are shown in Table 6.

[0277] Table 6 Detection results of volatile substances

[0278]

[0279]

[0280] As shown in Table 6, the results show that compared with the control group, the content of ethyl acetate in the wine body prepared by the experimental group has increased significantly, reaching the national standard of high-quality light-aroma liquor, indicating that the wine body prepared with wheat as the raw material and using the light-aroma type Rhizopus koji with Pichia kudriavzevii has better quality.

[0281] Application Example 4 (Method for brewing light-aroma Xiaoqu liquor with rice as raw material)

[0282] (1) Preparation of light-aroma type Rhizopus strain: Based on the weight of bran, inoculate 10 wt% of the activated Rhizopus Q303 koji and culture at 30 °C for 72 h to obtain a fermented product. Based on the weight of the fermented product, add 1 wt% of the Pichia kudriavzevii active dry yeast obtained in Example 4 to the fermented product and mix to obtain koji, that is, light-aroma type Rhizopus koji.

[0283] (2) Soaking the grains: Soak high-quality rice without mildew and insect damage in water at 70 °C for 8 h and filter the water to obtain soaked rice.

[0284] (3) Steaming the grains: Steam the soaked rice at 100 °C for 30 min to obtain steamed rice.

[0285] (4) Cooling: Cool the steamed rice to 30 °C to obtain cooled rice.

[0286] (5) Saccharification:

[0287] Experimental group: Based on the weight of the rice before soaking the grains, inoculate 0.5 wt% of the light-aroma type Rhizopus koji and mix evenly with the grain mash to obtain fresh grain mash, and then spread the grain mash evenly on the stacking saccharification bed for saccharification to obtain saccharified grain mash. Among them, the initial temperature of saccharification is 26 °C, the saccharification time is 24 h, and ventilation is maintained.

[0288] Control group: Based on the weight of the rice before soaking the grains, inoculate 0.5 wt% of the commercially available Rhizopus koji shown in Table 1 and mix to obtain grain mash, and then spread the grain mash evenly on the stacking saccharification bed for saccharification to obtain saccharified grain mash. Among them, the initial temperature of saccharification is 26 °C, the saccharification time is 24 h, and ventilation is maintained.

[0289] (6) Fermentation:

[0290] Experimental group: After uniformly mixing the saccharified grain mash and water in a weight ratio of 1:2.5, the temperature was lowered to 23 °C by ventilation, and then put into a fermentation box and sealed for fermentation for 15 days, and then distilled to obtain wine.

[0291] Control group: The same as the experimental group.

[0292] (7) Detection of volatile substances: The wine obtained in step (6) was detected by the detection method of volatile substances. Among them, the detection method of volatile components was the same as that in Application Example 1, and the detection results are shown in Table 7.

[0293] Table 7 Detection results of volatile substances

[0294]

[0295] As shown in Table 7, the results show that compared with the control group, the content of ethyl acetate in the wine body prepared in the experimental group increased by nearly 10 times, reaching the national standard for high-quality light-flavor liquor, indicating that the wine body prepared with rice as the raw material and using the light-flavor rhizopus koji of Pichia kudriavzevii has better quality.

[0296] Application Example 5 (Method for brewing light flavor with bran koji, raw material is sorghum)

[0297] (1) Preparation of light-flavor bran koji: Based on the weight of bran, inoculate 10 wt% of the activated Aspergillus niger UV-11 strain and culture at 35 °C for 5 days to obtain a fermentation product. Based on the weight of the fermentation product, add 1 wt% of the Pichia kudriavzevii active dry yeast obtained in Example 5 to the fermentation product and mix to obtain koji, that is, light-flavor bran koji.

[0298] (2) Soaking grains: Soak high-quality sorghum without mildew and insect damage in water at 70 °C for 22 h and filter the water to obtain soaked sorghum.

[0299] (3) Steaming grains: Steam the soaked sorghum at a temperature of 100 °C and a pressure of 1.5 kPa for 20 min for the first steaming. After opening the lid and simmering in water for 50 min, then perform a second steaming for 40 min to obtain steamed sorghum.

[0300] (4) Cooling: Cool the steamed sorghum to 30 °C to obtain cooled sorghum.

[0301] (5) Saccharification:

[0302] Experimental group: Based on the weight of the sorghum before soaking the grains, 10 wt% of the Qingxiang-type bran koji was added to the cooled sorghum obtained in step (4) and mixed evenly to obtain fresh fermented grains. Then, the fresh fermented grains were spread evenly on a stacking saccharification bed for saccharification to obtain saccharified fermented grains. Among them, the initial temperature of saccharification was 26 °C, the saccharification time was 24 h, and ventilation was maintained.

[0303] Control group: Based on the weight of the sorghum before soaking the grains, 10 wt% of the commercially available bran koji shown in Table 1 was added and mixed to obtain fresh fermented grains. Then, the fresh fermented grains were spread evenly on a stacking saccharification bed for saccharification to obtain saccharified fermented grains. Among them, the initial temperature of saccharification was 26 °C, the saccharification time was 24 h, and ventilation was maintained.

[0304] (6) Fermentation:

[0305] Experimental group: After mixing evenly at a ratio of 1:3 by weight of the saccharified fermented grains to the fresh fermented grains and cooling the temperature to 23 °C by ventilation, it was put into a fermentation tank and sealed for fermentation for 15 d, and then distilled to obtain wine.

[0306] Control group: The same as the experimental group.

[0307] (7) Detection of volatile substances: The wine obtained in step (6) was detected by the detection method of volatile substances. Among them, the detection method of volatile components was the same as that in Application Example 1, and the detection results are shown in Table 8.

[0308] Table 8 Detection results of volatile substances

[0309]

[0310] As shown in Table 8, the results show that compared with the control group, the content of ethyl acetate in the wine prepared by the experimental group increased significantly, with a growth rate of 167%, reaching the national standard for high-quality Qingxiang-type wine. This indicates that using the Qingxiang-type bran koji of Pichia kudriavzevii with sorghum as the raw material can produce better-quality wine.

[0311] Application Example 6 (Method for brewing Qingxiang-type liquor with daqu, raw material is sorghum)

[0312] (1) Preparation of fortified Qingxiang-type daqu: Based on the weight of the Qingxiang-type daqu (this Qingxiang-type daqu is sold by Angel Yeast Co., Ltd.), 0.5 wt% of the Pichia kudriavzevii active dry yeast obtained in Example 6 was added to the Qingxiang-type daqu and mixed to obtain fortified Qingxiang-type daqu.

[0313] (2) Soaking the grains: High-quality sorghum without mildew and insect damage was soaked in water at 70 °C for 22 h to obtain soaked sorghum;

[0314] (3) Steaming the grains: The soaked sorghum is initially steamed for 20 min under the conditions of a temperature of 100 °C and a pressure of 1.5 kPa. After uncovering the lid and simmering for 50 min, it is re-steamed for 40 min to obtain the steamed sorghum.

[0315] (4) Cooling: The steamed sorghum is cooled to 30 °C to obtain the cooled sorghum.

[0316] (5) Fermentation:

[0317] Experimental group: Based on the weight of the sorghum before soaking, 10 wt% of fortified light-flavor Daqu is added to the cooled sorghum obtained in step (4). Then, the temperature is lowered by ventilation to 17 °C and it is sealed and fermented in a fermentation box for 28 d, and then distilled to obtain liquor.

[0318] Control group: The difference from the experimental group is that: based on the weight of the sorghum before soaking, 10 wt% of the said light-flavor Daqu (this light-flavor Daqu is sold by Angel Yeast Co., Ltd.) is added to the cooled sorghum obtained in step (4). Then, the temperature is lowered by ventilation to 17 °C and it is sealed and fermented in a fermentation box for 28 d, and then distilled to obtain liquor.

[0319] (6) Detection of volatile substances: The liquor obtained in step (5) is detected by the detection method of volatile substances. Among them, the detection method of volatile components is the same as that in Application Example 1, and the detection results are shown in Table 9.

[0320] Table 9. Detection results of volatile substances

[0321]

[0322] As shown in Table 9, the results show that compared with the control group, the total ester and ethyl acetate contents in the liquor body prepared in the experimental group are significantly increased, increasing by 24% and 38% respectively. This shows that the fortified light-flavor Daqu prepared by using Pichia kudriavzevii active dry yeast can effectively improve the main flavor substances and liquor quality of light-flavor Daqu liquor.

[0323] The above embodiments are only for further elaboration and understanding of the technical solution of the present invention, and are not a limitation to the present invention. Any improvements made by those skilled in the art on this basis that do not involve outstanding substantive features and non-significant progress shall fall within the protection scope of the present invention.

Claims

1. A Pichia kudriavzevii active dry yeast, characterized in that, The total number of cells of Pichia kudriavzevii in each gram of the active dry yeast is greater than or equal to 23 billion and less than or equal to 40 billion, the viable cell rate of Pichia kudriavzevii in the active dry yeast is greater than or equal to 74% and less than or equal to 95%, and the moisture content is 4.5 - 5 wt%.

2. The active dry yeast according to claim 1, characterized in that, The total number of cells of Pichia kudriavzevii in each gram of the active dry yeast is greater than or equal to 26 billion and less than or equal to 35 billion, and / or the viable cell rate of Pichia kudriavzevii in the active dry yeast is greater than or equal to 74% and less than or equal to 90%.

3. The active dry yeast according to claim 1 or 2, characterized in that, The Pichia kudriavzevii is Pichia kudriavzeii C4.12 4. The active dry yeast according to any one of claims 1 to 3, characterized in that, It is prepared by a method comprising the following steps: culturing a seed culture solution of Pichia kudriavzevii in a mixture of a carbon source and a nutrient source to obtain a culture solution, then feeding the carbon source into the culture solution for culturing to obtain a fermentation culture solution, emulsifying the fermentation culture solution to obtain yeast cream, and granulating and drying the yeast cream to obtain Pichia kudriavzevii active dry yeast. Wherein, the carbon source is one or a combination of two of molasses and starch hydrolysate sugar treated with sucrase. Wherein, the nutrient source includes one or more substances selected from the group consisting of ammonium sulfate, ammonium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, nicotinic acid, inositol, biotin, ammonia water, diammonium hydrogen phosphate, and ammonium hydrogen phosphate.

5. The active dry yeast according to any one of claims 1-4, characterized in that, The sugar content of the carbon source is 10 - 35 wt%, preferably, the sugar content of the carbon source is 10 - 20 wt%.

6. The active dry yeast according to any one of claims 1-5, characterized in that, The molasses is one or a combination of two of cane molasses and beet molasses.

7. The active dry yeast according to claim 6, wherein The molasses treated with sucrase is prepared by a method comprising the following steps: mixing molasses with sucrase and reacting at 40 - 80 °C for 2 - 5 h to obtain molasses treated with sucrase. Preferably, based on each gram of sucrose in the molasses, the addition amount of sucrase is 100 - 1000 U.

8. The active dry yeast according to any one of claims 1-7, characterized in that, The nutrient source is one or more substances selected from the group consisting of ammonium sulfate, ammonium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, nicotinic acid, inositol, biotin, ammonia water, diammonium hydrogen phosphate, and ammonium hydrogen phosphate.

9. The active dry yeast according to claim 8, characterized in that, By weight, relative to 5500 - 6500 parts of the carbon source, the addition amount as the nutrient source includes 12 - 325 parts of ammonium sulfate, and / or 5 - 115 parts of ammonium phosphate, and / or 5 - 30 parts of potassium chloride, and / or 1 - 20 parts of magnesium sulfate, and / or 0.015 - 0.125 parts of calcium pantothenate, and / or 0.001 - 0.065 parts of nicotinic acid, and / or 0.005 - 0.125 parts of inositol, and / or 0.02 - 0.13 parts of biotin, and / or 135 - 260 parts of ammonia water, and / or 45 - 55 parts of diammonium hydrogen phosphate, and / or 58 - 59 parts of ammonium hydrogen phosphate.

10. The active dry yeast according to any one of claims 1-9, characterized in that, The emulsification is to mix the yeast milk and the emulsifier according to a weight ratio of 20,000 - 60,000:0.1 - 245 to emulsify the yeast milk. Among them, the emulsifier includes one or more substances selected from the group consisting of vegetable oil, sorbitan monostearate, sorbitan laurate, sorbitan palmitate, sorbitan stearate, sorbitan stearyl glycerol, sorbitan oleate, and sorbitan trioleate.

11. The active dry yeast according to claim 10, wherein, The emulsifier is one of sorbitan monostearate, sorbitan laurate, sorbitan palmitate, or sorbitan stearate.

12. The active dry yeast according to claim 10 or 11, characterized in that, Calculated by weight, the emulsifier includes: 0.1 - 2 parts of sorbitan monostearate, and / or 235 - 245 parts of sorbitan laurate, and / or 125 - 135 parts of sorbitan palmitate, and / or 185 - 195 parts of sorbitan stearate.

13. A method for preparing the active dry yeast of Pichia kudriavzevii according to any one of claims 1-12, characterized in that, It includes the following steps: Culturing the seed culture solution of Pichia kudriavzevii in a mixture of a carbon source and a nutrient source to obtain a culture solution, then feeding the carbon source into the culture solution for culturing to obtain a fermentation culture solution, emulsifying the fermentation culture solution to obtain yeast milk, granulating and drying the yeast milk to obtain Pichia kudriavzevii active dry yeast. Among them, the carbon source is one or a combination of two of molasses and starch hydrolyzate treated with sucrase. Among them, the nutrient source includes one or more substances selected from the group consisting of ammonium sulfate, ammonium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, nicotinic acid, inositol, biotin, ammonia water, diammonium hydrogen phosphate, and ammonium hydrogen phosphate.

14. The preparation method according to claim 13, characterized in that, It includes the following steps: (1) Inoculating the activated Pichia kudriavzevii yeast strain into a liquid medium for culturing to obtain a seed culture solution. Preferably, the liquid medium is a wort liquid medium; (2) Inoculating the seed culture solution obtained in step (1) into the mixture of the carbon source and the nutrient source for primary culture, then feeding the carbon source into the culture solution and introducing sterile air for secondary culture to obtain a fermentation culture solution; (3) Separating the fermentation culture solution obtained in step (2) to obtain yeast milk, then mixing the yeast milk and the emulsifier, granulating and drying to obtain Pichia kudriavzevii active dry yeast. Preferably, the drying temperature is: the inlet air temperature is 80 - 115 °C, the outlet air temperature is 40 - 80 °C, and / or the drying time is 25 - 65 min.

15. The preparation method according to claim 13 or 14, characterized in that, In step (1), the sugar content of the liquid medium is 5 - 25 wt%, preferably 8 - 15 wt%.

16. The preparation method according to any one of claims 13-15, characterized in that, The culture temperature in step (1) is: 25 - 35 °C, and / or the primary culture temperature in step (2) is: 20 - 35 °C, and / or the secondary culture temperature in step (2) is: 20 - 35 °C.

17. The preparation method according to any one of claims 13-15, characterized in that, The culture time in step (1) is: 20 - 50 h, and / or the primary culture time in step (2) is: 20 - 50 h, and / or the secondary culture time in step (2) is: 15 - 30 h.

18. The preparation method according to any one of claims 13-15, characterized in that, In step (2), the flow rate of the carbon source is 200 - 2500 L / hr, and / or the air volume of the sterile air introduced is 28 - 32 m 3 air / fermentation broth m 3 / hr, and / or during the secondary culture process, the ethanol content in the fermentation culture broth is 0.4% or less.

19. The preparation method according to any one of claims 13-15, characterized in that, In step (3), the separation is to separate the fermentation culture solution under the condition of 3500 - 4500 rpm, retain the precipitate, and obtain the yeast milk.

20. Komagataella phaffii active dry yeast prepared by the preparation method according to any one of claims 13 - 19.

21. Use of the Komagataella phaffii active dry yeast according to any one of claims 1 - 12 or claim 20 in the preparation of koji.

22. A distiller's yeast, characterized in that, Containing the Komagataella phaffii active dry yeast according to any one of claims 1 - 12 or claim 20.

23. The koji according to claim 22, characterized in that, The koji is prepared by a method comprising the following steps: after mixing the Komagataella phaffii active dry yeast and the bacterial strain, koji is obtained. Preferably, the bacterial strain is a Rhizopus bacterial strain or an Aspergillus niger bacterial strain.

24. The koji according to claim 22 or 23, characterized in that, When the bacterial strain is a Rhizopus bacterial strain, the koji is prepared by a method comprising the following steps: (1) Based on the weight of bran, inoculate 5 - 15 wt% of the Rhizopus bacterial strain and ferment and culture to obtain a fermented product. Preferably, the temperature for fermentation and culture is 28 - 33 °C and / or the time for fermentation and culture is 70 - 73 h; (2) Based on the weight of the fermented product, add 0.1 - 5 wt% of the Komagataella phaffii active dry yeast to the fermented product and mix to obtain koji.

25. The distiller's yeast according to any one of claims 22-24, characterized in that, When the bacterial strain is an Aspergillus niger bacterial strain, the koji is prepared by a method comprising the following steps: (1) Based on the weight of bran, inoculate 5 - 15 wt% of the Aspergillus niger bacterial strain and ferment and culture to obtain a fermented product. Preferably, the temperature for fermentation and culture is 28 - 33 °C and / or the time for fermentation and culture is 4 - 5 d; (2) Based on the weight of the fermented product, add 1 - 2 wt% of the Komagataella phaffii active dry yeast to the fermented product and mix to obtain koji.

26. The koji according to claim 22, characterized in that, The koji is prepared by a method comprising the following steps: after mixing the Komagataella phaffii active dry yeast and the daqu, koji is obtained, wherein the daqu is prepared by a method comprising the following steps: after mixing barley and peas in a weight ratio of 6:3 - 5, crushing, forming into groups, fermenting and culturing, and drying, daqu is obtained. Preferably, based on the quality of the daqu, the addition amount of the Komagataella phaffii active dry yeast is 0.1 - 5 wt%. More preferably, based on the weight of the daqu, the addition amount of the Komagataella phaffii active dry yeast is 0.1 - 2 wt%.

27. Use of the koji according to any one of claims 22 - 26 in the preparation of liquor. Preferably, the liquor is Chinese liquor.

28. A Chinese liquor, characterized in that, Containing the koji according to any one of claims 22 - 26.

29. The Chinese liquor according to claim 28, wherein By including the following steps: after mixing the koji according to any one of claims 22 - 25 with the grain mash, saccharifying, fermenting, and distilling, Chinese liquor is obtained.

30. The Chinese liquor according to claim 28 or 29, characterized in that, The volatile substances contained in the Chinese liquor include ester compounds. Preferably, based on each liter of the Chinese liquor, the content of the ester compounds is 1000 - 3500 mg. Preferably, the ester compounds include one or more substances selected from the group consisting of ethyl acetate, ethyl lactate, and ethyl butyrate.

31. The baijiu according to any one of claims 28-30, characterized in that, The volatile substances contained in the liquor further include one or more substances selected from the group consisting of n-propanol, isobutanol, acetal, isoamyl alcohol, and phenethyl alcohol. Preferably, per liter of the liquor, the volatile substances contained in the liquor include ethyl acetate 1000 - 2500 mg, and / or ethyl lactate 700 - 1300 mg, and / or ethyl butyrate 5 - 15 mg, and / or n-propanol 85 - 400 mg, and / or isobutanol 100 - 800 mg, and / or acetal 50 - 380 mg, and / or isoamyl alcohol 150 - 950 mg, and / or phenethyl alcohol 10 - 50 mg. More preferably, the ethyl acetate is 1000 - 2350 mg, and / or the n-propanol is 90 - 390 mg, and / or the isobutanol is 150 - 800 mg, and / or the acetal is 50 - 380 mg, and / or the isoamyl alcohol is 180 - 950 mg, and / or the phenethyl alcohol is 10 - 45 mg.

32. The brewing method of the Chinese liquor according to any one of claims 28-31, characterized in that, It includes the following steps: mixing the Pichia kudriavzevii active dry yeast, the strain, and the grain mash, saccharifying, fermenting, and distilling to obtain the liquor.

Images