Complex microbial inoculant reinforced distiller's yeast as well as preparation method and application thereof

Abstract

The invention relates to the technical field of white spirit brewing, and particularly provides a complex microbial inoculant enhanced distiller's yeast and a preparation method and application thereof, and microbial inoculants adopted by the enhanced distiller's yeast disclosed by the invention comprise rhizopus oryzae, saccharomyces cerevisiae, Wickerhamomyces anomalus and Kluyveromyces marxianus. The preparation method of the strengthened distiller's yeast comprises the following steps: respectively and independently culturing the microbial agents to prepare solid microbial agents, then mixing the solid microbial agents with production distiller's yeast according to a certain proportion to obtain the strengthened distiller's yeast, and applying the strengthened distiller's yeast to mechanical production of fen-flavor liquor. The content of ethyl acetate in the brewed raw wine is obviously increased by 33.67% (p is less than 0.01) compared with that of a production control group; the rate of a superior product reaches 37.05% and is obviously improved by 14.38% compared with that of a control group. And the produced wine body has pure faint scent, prominent ester fragrance, vinasse fragrance, soft, sweet and harmonious taste and prominent style.

Description

Technical Field

[0001] The invention relates to the technical field of liquor brewing, and in particular to a compound bacterial agent-enhanced distiller's yeast, and a preparation method and application thereof. Background Art

[0002] Chinese liquor carries China's long history and unique culture. It is one of the pillar industries for the development of the national economy, and its development is of vital importance. As one of the four basic flavors of Chinese liquor, light-fragrance liquor is made from sorghum as raw material, using koji as saccharification and fermentation agent, and carefully brewed through special processes under the action of microorganisms. It is widely loved by consumers for its pure fragrance, mellow sweetness, natural harmony, and clean and refreshing aftertaste. Therefore, the development of light-fragrance liquor production technology is of great significance.

[0003] In liquor, the flavor substances that determine its style account for about 2% of the total liquor. Ethyl acetate is one of the main flavor substances of light-fragrance liquor. The fruity aroma it presents greatly improves the quality and style of light-fragrance liquor. The types and contents of flavor substances in liquor are affected by the types of microorganisms and fermentation conditions during the fermentation process, and have certain fluctuations. It is well known that liquor brewing belongs to a natural fermentation process, and the main source of liquor fermentation microorganisms is koji. However, the open fermentation environment and complex brewing technology used in liquor production lead to a very complex composition of the brewing microbial community, resulting in unstable liquor production quality and low production efficiency. Literature reports that using single or multiple microorganisms to regulate and strengthen the liquor fermentation process is an important way to maintain the stability of liquor production. It is worth noting that strengthening koji by inoculating microorganisms with specific functions has been developed and applied to improve fermentation performance and liquor quality.

[0004] At present, there are few patents that use functional microorganisms to strengthen koji to improve the quality of liquor. For example, patent CN202010448141.5 discloses a microbial composite agent, which is added together with Daqu to the fermentation process of Maotai-flavor liquor. The content of tetramethylpyrazine is as high as 882.6μg / L, the content of 4-methylguaiacol is as high as 853.1μg / L, and the content of 4-ethylguaiacol is as high as 200.4μg / L; Patent CN20221053 2280.5 discloses a high-temperature koji fortified with a composite microbial agent and its application in sauce-flavor liquor. The flavor precursors of the fortified mash and the aroma substances in the finished liquor are improved, but there is no significant change in ethyl acetate and ethyl lactate in the original liquor; Patent CN202311131577.1 inoculates Zymomonas mobilis in the traditional koji-making process to produce a fortified koji, and the fortified koji is used to brew sauce-flavor liquor, which can effectively improve the liquor yield and improve the quality of the liquor. There are even fewer patents for the application of fortified koji in light-flavor liquor. Only patent CN202210501009.5 discloses that a strain of saccharomyces cerevisiae is fortified into traditional koji and applied to the brewing of light-flavor original liquor of traditional process, which significantly increases the ethyl acetate content in the original liquor.

[0005] The unique characteristics and style of liquor flavor are not only affected by the natural environment and geographical region, but also by the production process, such as raw materials, type of koji, fermentation container, etc. The differences in these abiotic factors lead to the unique microbial community and differences in the metabolism of synergistic microorganisms in the liquor fermentation process. Therefore, on the basis of isolating and obtaining core functional microorganisms, it is an effective method to enhance the quality of raw liquor by utilizing the synergistic effect between microorganisms to strengthen them into koji and finally apply them to brewing production. Ethyl acetate is the main aroma component in light-fragrance liquor, and its content directly affects the taste of the raw liquor. At present, the raw liquor brewed by mechanized light-fragrance liquor has the problem of low raw liquor superiority rate caused by low ethyl acetate content. How to solve this technical problem has become one of the technical problems to be solved in this field. Summary of the invention

[0006] In view of this, the present invention proposes a composite microbial agent fortified distiller's yeast and its preparation method and application, and solves the problem of low ethyl acetate content and low premium rate of raw liquor brewed by mechanized light-fragrance liquor by strengthening different functional microorganisms in distiller's yeast. At the same time, it also solves the problem of unstable quality of traditional distiller's yeast fermentation, improves the quality of distiller's yeast, and ensures the production stability, high efficiency and convenience of different batches of distiller's yeast.

[0007] The technical solution of the present invention is achieved in this way. The present invention provides a composite bacterial agent, which includes: Rhizopus oryzae G1 solid bacterial agent, Saccharomyces cerevisiae Y1 solid bacterial agent, Kluyveromyces marxianus Y245 solid bacterial agent and Wickerhamomyces anomalus Y348 solid bacterial agent.

[0008] In some embodiments, in the composite bacterial agent, the mass ratio of Rhizopus oryzae G1 solid bacterial agent, Saccharomyces cerevisiae Y1 solid bacterial agent, Kluyveromyces marxianus Y245 solid bacterial agent and Wickerhamomyces anomalus Y348 solid bacterial agent is (15-20): (1-5): (0.5-2): (0.5-2).

[0009] In some embodiments, the deposit number of Rhizopus oryzae G1 is CCTCC NO: M2019425, the deposit number of Saccharomyces cerevisiae Y1 is CCTCC NO: M 20241943, the deposit number of Kluyveromyces marxianus Y245 is CCTCC NO: M 2021225, and the deposit number of Wickerhamomyces anomalus Y348 is CCTCC NO: M20241944.

[0010] In some embodiments, the preparation method of Rhizopus oryzae G1 solid bacterial agent is: inoculating the Rhizopus oryzae G1 bacteria into a rice koji juice culture medium for culturing to obtain a first-class Rhizopus oryzae species; inoculating the first-class species into a bran solid culture medium for culturing to obtain a second-class Rhizopus oryzae species; inoculating the second-class Rhizopus oryzae species into a rice flour solid culture medium for culturing, and drying to prepare the Rhizopus oryzae G1 microbial solid bacterial agent.

[0011] In some embodiments, the preparation method of the Saccharomyces cerevisiae Y1 microbial solid inoculant is: inoculate the Saccharomyces cerevisiae Y1 cells into a malt juice medium for culturing to prepare a primary yeast species; transfer the primary yeast species culture fluid to a molasses medium for culturing to prepare a secondary yeast species; inoculate the prepared secondary seed fluid into a rice flour solid culture medium for culturing, and dry to prepare a yeast Y1 microbial solid inoculant.

[0012] In some embodiments, the preparation method of Kluyveromyces marxianus Y245 solid inoculant is as follows: inoculating Kluyveromyces marxianus Y245 cells into a malt juice medium for cultivation to prepare a primary yeast species; transferring the primary yeast species culture fluid to a molasses medium for cultivation to prepare a secondary yeast species; inoculating the prepared secondary seed fluid into a rice flour solid culture medium for cultivation, and drying to prepare a yeast Y245 microbial solid inoculant.

[0013] In some embodiments, the preparation method of the abnormal Wickham yeast (Wickerhamomyces anomalus) Y348 solid bacterial agent is: inoculating the abnormal Wickham yeast Y348 bacteria into a malt juice medium for cultivation to prepare a yeast primary species; transferring the yeast primary species culture liquid to a molasses medium for cultivation to prepare a yeast secondary species; inoculating the prepared secondary seed liquid into a rice flour solid culture medium for cultivation, and drying to prepare a yeast Y348 microbial solid bacterial agent.

[0014] In a second aspect of the present invention, there is also provided a compound bacterial agent-fortified koji, which comprises a traditional koji and the aforementioned compound bacterial agent, wherein the mass ratio of the traditional koji to the compound bacterial agent is (0.5-1):(4-6).

[0015] In a third aspect, the present invention also provides a method for preparing the above-mentioned composite bacterial agent-fortified koji, comprising: mixing traditional koji and composite bacterial agent in a mass ratio to obtain composite bacterial agent-fortified koji.

[0016] In a fourth aspect, the present invention also provides an application of the above-mentioned composite bacterial agent, including using the composite bacterial agent in food fermentation, especially in the field of winemaking, including white wine, yellow wine, etc.

[0017] In some embodiments, the composite bacterial agent is used for the fermentation of mechanized light-fragrance liquor, and the method of use is to directly add it to the fermentation system of the mechanized light-fragrance liquor.

[0018] In some embodiments, by using the above-mentioned composite bacterial agent in the fermentation of mechanized light-flavor liquor, the ethyl acetate content in the base liquor of the mechanized light-flavor liquor and the superior grade rate of the base liquor can be increased.

[0019] In some embodiments, the brewing method of the above-mentioned mechanized light-fragrance liquor is as follows:

[0020] After soaking glutinous sorghum in water at 65-90℃ for 16±6h, it is steamed in a ball pot with pressure. The cooked grain is cooled to 22-26℃ by a cooling machine. The fortified wine yeast containing microbial agents is added by a yeast adding machine at an inoculation rate of 0.9-1.4% of the dry grain weight. The yeast and grain are mixed evenly by an auger or a rake, and transported to a sealed saccharification room by a plate chain for bacterial culture and saccharification. The in-box temperature is controlled at 23-32℃, and the temperature difference at each point in the box bed is controlled within 5℃. After bacterial culture and saccharification for 22-29h, the grain-slag ratio is 1: (1.6-1.8) and the slag is added. The grain-slag mixing equipment, conveying and cold air equipment are turned on to mix the grain-slag evenly, and then cooled to 12-19℃ before loading into a 2m 3 The stainless steel tank truck is transported to the constant temperature fermentation room by forklift, and the temperature of the fermentation room is controlled at 19-25℃. The fermentation cycle is 15 days, and the robot is used to steam the wine, and the steaming time is controlled at 25-50min. The single-hanging distillation time is controlled at 35-70min to obtain the light-fragrant original wine.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a microbial agent, a fortified distiller's yeast and the application thereof in mechanized light-fragrance liquor. The ethyl acetate content in the light-fragrance liquor obtained by the method of the present invention reaches 1.31 g / L, which is significantly increased by 33.67% (p < 0.05) compared with the production control group; the superior product rate reaches 37.05%, which is significantly increased by 14.38 percentage points (p < 0.05) compared with the control group. The produced liquor has a positive fragrance, prominent ester fragrance, lees fragrance, sweet and harmonious taste, and prominent style, which has important application value for improving the quality of light-fragrance liquor.

[0023] Biomaterial Information:

[0024] Biomaterial Deposit

[0025] Kluyveromyces marxianus Y245 was deposited in the China Center for Type Culture Collection on March 15, 2021, with the deposit number CCTCC NO: M 2021225. The deposit address is China Center for Type Culture Collection, Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0026] Rhizopus oryzae G1 was deposited in the China Center for Type Culture Collection on June 4, 2019, with the deposit number CCTCC NO: M 2019425. The deposit address is China Center for Type Culture Collection, Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0027] Saccharomyces cerevisiae Y1 was deposited in the China Center for Type Culture Collection on September 9, 2024, with the deposit number CCTCC NO: M 20241943. The deposit address is China Center for Type Culture Collection, Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0028] Abnormal Wickham yeast (Wickerhamomyces anomalus) Y348 was deposited in the China Center for Type Culture Collection on September 9, 2024, with the deposit number CCTCC NO: M 20241944, and the deposit address is China Center for Type Culture Collection, Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0031] Unless otherwise specified, the methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art, and can be obtained by those skilled in the art through commercial channels.

[0032] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range. In the present specification and claims, range definitions can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.

[0033] The culture medium designed in the following examples is as follows:

[0034] The composition of YPD liquid medium is: 2% glucose, 1% yeast extract powder and 2% peptone, pH is natural.

[0035] Malt extract medium: Mix malt extract powder and water in a weight ratio of 1:8, control the sugar content at 11-12BX, and keep the pH natural.

[0036] Rice koji juice culture medium: grind the seed koji into powder and pass it through a 20-mesh rice sieve, add 5 times the volume of water, place it in a 65℃ oven for saccharification for 24-36 hours, centrifuge and take the supernatant. Use water to adjust the sugar content of the supernatant to 5-6BX, and use 10% sodium hydroxide solution to adjust the pH value to 5-6.

[0037] Molasses culture medium: Weigh molasses, add 2 times of hot water at about 60°C, stir until the molasses is dissolved, then add 0.1% concentrated sulfuric acid, stir well and let stand for about 12 hours, then add the supernatant to the seed tank or fermentation tank, and add water until the sugar content of the molasses culture medium is 14-20BX.

[0038] Bran solid culture medium: weigh the materials according to the weight ratio of bran to rice flour of 10:1, add 30-40% water, and the pH is natural.

[0039] Yeast rice flour solid culture medium: Weigh the materials according to the weight ratio of corn flour to bran of 10:1, add water adjusted with acid to pH 5-6 and stir to control the water content of the materials at 55-60%.

[0040] Mold rice flour solid culture medium: weigh materials according to the weight ratio of rice flour to bran of 10:1, add water adjusted with acid to pH 5-6 and stir to control the water content of the materials at 30-40%.

[0041] All the above culture media need to be sterilized before use.

[0042] Traditional koji preparation:

[0043] The early indica rice is soaked for 8-10 hours, and the slurry is ground. The early indica rice flour and rice bran that have passed through a 20-mesh sieve are added to the rice slurry, wherein the weight ratio of the early indica rice: the early indica rice flour: the rice bran is 63:35:2. After being evenly stirred, Angel Rhizopus is inoculated, and the inoculation amount is 3.0-3.5% of the total weight of the early indica rice, the rice flour and the rice bran; and the koji blanks are kneaded into cake-shaped koji blanks with a diameter of 6-7 cm by hand, and the koji blanks are arranged on a wooden box in a culture room, with a spacing of about 2-3 cm between the koji blanks, and covered with bamboo mats; the temperature of the culture room is controlled at 30-32° C., and when the koji blank product temperature rises to 35° C., the bamboo mat is removed; the koji blanks are continuously cultured until the product temperature of the koji blanks reaches 25-28° C., and then dried at 35° C., and the koji blanks are seed koji. The Guanyin soil is crushed and passed through a 14-mesh sieve, wherein the Guanyin soil is produced in Linli County, Hunan Province. The rice bran is passed through a 16-mesh sieve. The prepared seed koji is crushed and passed through a 20-mesh sieve, and then mixed with the rice bran and stirred evenly. Then bentonite is added and stirred evenly, wherein the weight ratio of the bentonite: rice bran: seed koji is 75:25:1.4-1.8, to obtain a mixture. Water is added to the mixture so that the water content in the mixture is 50%. After stirring evenly, the mixture is made into spherical koji blanks with a diameter of 7-8 cm, and the prepared koji blanks are neatly placed in a culture box in a culture room, bamboo mats are spread on the koji blanks, and the temperature of the culture room is controlled to be between 28-30° C. After 16 hours of incubation, check the temperature of the koji in the incubator. When the temperature of the koji rises to 30-35℃, uncover the bamboo mat and prop up the incubator. When the temperature of the koji drops to room temperature, pick it out. The koji should not be squeezed with each other and continue to incubate for 5 days. After the incubation, place the koji in a drying room and dry it at 35-40℃ until the moisture content is ≤5%, which is the traditional wine koji. The finished koji should meet the following requirements:

[0044] a. Sensory perception: Green skin on the surface, a small amount of black core and a large amount of red core in the center, with small and numerous cracks.

[0045] b. Physical and chemical indicators: moisture ≤ 5, saccharification power ≥ 19g / 100g, fermentation power ≥ 30.0%.

[0046] Example 1

[0047] In this example, a small-scale solid-state fermentation was carried out on a strengthened single strain.

[0048] The preserved Rhizopus oryzae G1, Saccharomyces cerevisiae Y1, Kluyveromyces marxianus Y245 and Wickham's yeast Y348 were inoculated into YPD liquid culture medium respectively, and cultured at 160r / min and 30℃ for 24h to obtain the seed liquid of each strain. The seed liquid was inoculated into the bran culture medium in a certain proportion, and cultured at a constant temperature of 30℃ in an incubator for 3-5 days to prepare bran koji containing a single strain. After mixing the single strain bran koji with the traditional craft wine koji, it was used for laboratory small-scale fragrance solid-state fermentation effect verification. The specific steps are as follows:

[0049] (1) Soaking grains: Weigh 500 g of glutinous sorghum for each sample, add warm water at 60°C to 70°C (the water level should be one finger higher than the grain surface), and soak the grains in a 65°C oven for 20 to 24 hours.

[0050] (2) Steaming the grain: First, drain the soaked grain and place it in a sterilizing steamer for initial steaming at 115°C for 30 minutes. After the initial steaming, add 80°C water to simmer the grain for 10 minutes. After draining the water, place it in a sterilizing steamer for re-steaming at 110°C for 10 minutes.

[0051] (3) Mixing grain and koji: The re-steamed grain is cooled to 30°C and divided into No. 12 self-sealing bags, with 1050g of cooked grain in each self-sealing bag, and fully mixed with the koji. The amount of koji used is about 1% of the weight of the dry grain. The koji used are Rhizopus oryzae G1, Saccharomyces cerevisiae Y1, Kluyveromyces marxianus Y245, Abnormal Wickham yeast Y348 and traditional koji.

[0052] (4) Saccharification: Place the mixed koji in an incubator at 30°C for saccharification for about 24 hours. Keep the ziplock bag open and seal the opening with a wet towel to ensure the supply of oxygen and water required for the saccharification process.

[0053] (5) Sterilization of mash: Weigh the mash used according to 500 g of a single sample and put it into a sterilizing steamer for sterilization at 121°C for 30 min.

[0054] (6) Fermentation in bags: Mix the saccharified grains with the sterilized mash in a weight ratio of 1:1, put them into a gas sampling bag, seal it, evacuate it, and put it into an incubator at 30°C for fermentation for 7 days.

[0055] (7) Distillation: The fermented mash was placed in a special conical flask for distillation. The first 100 ml of the distilled wine was sampled for alcohol content and gas chromatography determination. Three parallel samples were made for each koji, and three rounds of small-scale solid-state fermentation were repeated. The gas chromatography results of solid-state fermentation after strengthening each strain are shown in Table 1.

[0056] Table 1 Data of solid-state fermentation of wine produced by strengthening single strain in small-scale trials

[0057]

[0058] From the data in Table 1, we can know that adding the above strains to the original wine yeast can increase the alcohol content, especially adding Saccharomyces cerevisiae Y1 and Rhizopus oryzae G1 has the largest increase, but the content of n-propanol and fusel oil also increases accordingly. Adding strains of abnormal Wickham yeast Y348 and Kluyveromyces marxianus Y245 can increase the ethyl acetate content and reduce the fusel oil content in the original wine. In addition, strain Y245 can also reduce the n-propanol content, but the wine rate of adding these two strains is reduced.

[0059] Example 2

[0060] This example uses a composite bacterial agent to carry out small-scale solid-state fermentation

[0061] Since the fermentation process of liquor involves very complex microbial interactions and metabolism, the conventional single-bacteria culture method cannot present the actual microbial metabolism in the actual liquor production. Therefore, the combined fermentation method has become a powerful tool for studying the function of liquor brewing microorganisms. The above strains were combined, and a total of 10 combinations were designed, and the original distiller's yeast was used as a control. After the laboratory small-scale solid-state fermentation in the step of Example 1, the best combination was found according to the wine data, and the specific results are shown in Table 2.

[0062] Table 2 Comparison of solid-state fermentation wine production data in combined fermentation laboratory test

[0063]

[0064] As can be seen from the data in Table 2, the microbial composite agent obtained by combining Rhizopus oryzae G1, Saccharomyces cerevisiae Y1, Kluyveromyces marxii Y245 and Wickham's yeast Y348 can significantly improve the wine yield and the aroma components in the original wine, especially the ethyl acetate content is significantly higher than that of other experimental groups, which may be due to the synergistic effect of the strains. Therefore, it is preliminarily judged that the composite agent has a better effect in improving the wine yield and improving the quality of the wine, and the next step is to verify this combination in large-scale production.

[0065] Example 3

[0066] In order to further verify the effect of the optimal composite microbial agent obtained in Example 2 on the alcohol content and quality of the original wine, this example needs to verify the combination in large-scale production, so it is necessary to mass-produce each strain of microbial agent to meet production needs. Therefore, this example conducts step-by-step expansion culture in the koji making workshop to obtain the microbial agent, and the specific steps are as follows:

[0067] (1) Preparation of solid bacterial agent of Rhizopus oryzae

[0068] 1.1 Cultivation of primary species of Rhizopus oryzae

[0069] Under sterile conditions, a small amount of Rhizopus oryzae mycelium is picked up with an inoculation needle and inoculated into a triangular bottle filled with rice koji juice medium, placed in a constant temperature 30°C shaker, cultured at 140r / min for 2 days, taken out, placed in a constant temperature 30°C incubator for static culture for 2-3 days, and the first-class Rhizopus oryzae is obtained when the white mycelium covers the entire liquid surface and there is no contamination by other bacteria. The first-class Rhizopus oryzae is stored in a 4°C refrigerator for standby use, and the storage time shall not exceed 1 week.

[0070] 1.2 Cultivation of secondary species of Rhizopus oryzae

[0071] In the clean bench, pour the first-level species of Rhizopus oryzae and sterile water into the homogenizer, break the mycelium, mix the mycelium and sterile water thoroughly, and then pour them into the inoculation tank. Connect the inoculation tank to the inoculation port of the koji seeding machine, enter the inoculation operation interface, use compressed air to blow all the bacterial liquid in the inoculation tank into the koji seeding machine, close the valve, and enter the cultivation and drying stage. It is divided into five stages, the first stage is the static stage, the second and third stages are the cultivation stage, and the fourth and fifth stages are the drying stage. After the koji seeding machine is cultivated for 48 hours, the material is discharged and the saccharification power is tested to be ≥28g / 100g. After passing the test, the second-level species of Rhizopus oryzae is obtained.

[0072] 1.3 Preparation of solid inoculant of Rhizopus oryzae

[0073] The sterilized rice flour solid culture medium is cooled by an air cooler. After the material temperature drops to 35°C, the secondary root mold species are added. The inoculation amount is controlled at 1%. Stir evenly and blown into the disc koji making machine through a Roots blower. After the material enters the disc and is flattened, turn on the fan, control the material temperature at 27-32°C according to the ambient temperature, and turn off the fan, damper, heating wire, etc. after the material temperature is consistent to start static cultivation. When the material temperature rises to 32°C, turn on the automatic control, the disc starts to automatically control the temperature, and the average temperature of the material is controlled at 32±0.5°C by controlling the wind speed, wind temperature, and damper. During the entire cultivation process, the disc is rotated once 6 hours after the feeding is completed and a sample is taken to observe the appearance. After that, the disc is rotated once every 2 hours, with a rotation amplitude of 90 degrees, and samples are taken to observe the appearance until the cultivation is completed. The cultured koji material is blown into the dryer through a Roots blower, and the drying is started after the air inlet temperature and time are set. During the drying process, the average temperature of the material is controlled not to exceed 45°C, and the material can be discharged when the moisture content reaches ≤13%. After the crushing, samples are taken for testing, and the solid Rhizopus oryzae inoculum is obtained when the saccharification power is ≥28g / 100g.

[0074] (2) Preparation of solid yeast inoculant

[0075] 2.1 Cultivation of primary yeast

[0076] Under sterile conditions, three cultured test tube slant strains of Saccharomyces cerevisiae Y1, Kluyveromyces marxianus Y245 and Wickham's anomalous yeast Y348 were taken respectively, and the yeasts were picked up with three inoculation loops and inoculated into the malt extract triangular flask culture medium, and then placed in a constant temperature shaking incubator at 30°C, 140r / min, and cultured for 30h to obtain the primary yeast strain.

[0077] 2.2 Cultivation of secondary yeast species

[0078] Place the inoculation ring at the inoculation port and light it. Wipe your hands with 75% alcohol, open the inoculation port, and pour in the first-level yeast. After completion, tighten the inoculation port and open the manual valve of the air inlet flow meter. Set the pressure of the 1000L fermentation tank to 0.02MPa, open the seed tank, and let the seed liquid enter the fermentation tank. Set the culture temperature to 30°C, the speed to 100-500r / min, the dissolved oxygen value to 55%, the measured value to 25%-100%, the pH to 4.50-6.0, and click "Fermentation" on the control panel to start the culture. When the dissolved oxygen data line tends to be stable, the value is ≥80%, the speed is 100-150r / min, the yeast growth tends to stop, the quality is best, and the number of brewer's yeast detected is 1×10 9 cfu / ml, the number of Saccharomyces cerevisiae, Kluyveromyces marxianus and Wickham's yeast was 2×10 9 If the concentration is above cfu / ml, the secondary yeast species is obtained.

[0079] 2.3 Preparation of solid yeast inoculant

[0080] The sterilized rice flour solid culture medium is cooled by an air cooler. After the material temperature drops to 35°C, the secondary yeast is added, and the moisture content of the material is controlled at 50%. Stir evenly and blow it into the disc koji making machine through a Roots blower. Set the wind temperature to 27.0-31.0°C and the average temperature to 32°C before feeding. Start feeding and level the material in the disc after feeding is completed. When the material temperature rises to 32°C, turn on the automatic control disc to start automatic temperature control. By controlling the wind speed, wind temperature, and air door, the average temperature of the material is controlled at 30-35°C. During the cultivation process, turn over the koji once every 5 hours or so, and turn the disc once every 2.5 hours or so. Blow the cultivated koji material into the dryer through a Roots blower, set the air inlet temperature and time, and start drying. During the drying process, the average temperature is controlled not to exceed 45°C. When the moisture content of the material is ≤14%, it can be discharged. After crushing, samples are taken for testing, and the number of brewer's yeast reaches 2×10 9 cfu / g, the number of Saccharomyces cerevisiae, Kluyveromyces marxianus and Wickham's yeast all reached 2×10 9 If the content is above 1.5 cfu / g, the solid yeast agent is obtained.

[0081] (3) Preparation of microbial agents

[0082] The Rhizopus oryzae solid inoculant, Saccharomyces cerevisiae solid inoculant, Kluyveromyces marxianus solid inoculant and Wickhamia anomala solid inoculant prepared in steps 1 and 2 are mixed at a weight ratio of 17:3:1:1 to prepare a microbial inoculant.

[0083] The prepared microbial agent is mixed with traditional distiller's yeast in a weight ratio of 5:1 to obtain a fortified distiller's yeast containing the microbial agent.

[0084] The sensory properties of the obtained fortified distiller's yeast are light yellow, without any other color, odor or foreign matter.

[0085] The physical and chemical indicators of the obtained fortified distiller's yeast are as follows: fineness ≥85%, water content ≤10.5, saccharification power between 26-32g / 100g, and fermentation power ≥33.0%.

[0086] Example 4

[0087] This embodiment provides an application of fortified wine yeast in mechanized light-fragrance liquor

[0088] In this embodiment, the koji used are the fortified koji prepared in Example 3 and the traditional koji.

[0089] The specific steps are as follows:

[0090] (1) Soaking grains

[0091] Select the soaking barrel for grain feeding, set the liquid level of soaking water, start the water pump and water inlet valve, add 70-90℃ soaking water into the soaking barrel, and after adding water, start the grain feeding system to evenly distribute the glutinous sorghum into each soaking barrel. After feeding, manually salvage the grain husk, rake the grain in the soaking barrel flat, and let the grain soak for 18±6h.

[0092] (2) Steamed grain

[0093] After draining the soaking water, steam the grains for the first time. The holding time for the first time is 5-40 minutes. The pressure and time for the first time are adjusted according to the glutinous nature of the grains. The stewing time is determined according to the effect of the first steaming of the grains. The water temperature for the stewing of the grains is controlled at 50-60℃. After the stewing of the grains is completed, the grains should not touch the hand. After the stewing of the grains is completed, open the bottom cover of the steamer to drain the soaking water and then steam again. The holding time for the second time is 5-60 minutes. The pressure and time for the second time are adjusted according to the glutinous nature of the grains.

[0094] Sensory standards for cooked grains: soft and cooked to the core, with a skin breakage rate ≥ 80%, and small grain clumps.

[0095] Testing of physical and chemical indicators of cooked grains: The standard for moisture content of cooked grains is 45% to 60%.

[0096] (3) Saccharification

[0097] The proportion of koji added is 0.9-1.4% of the dry grain weight, the temperature entering the box is controlled at 20-32℃, the temperature difference at each point in the box bed is controlled within 5℃, and the saccharification and incubation time is controlled at 22-29h. When unpacking, the sorghum should be loose and not clump, shiny, have a koji aroma, taste sweet, and the highest temperature should not exceed 45℃.

[0098] Testing of physical and chemical indicators of saccharified mash: reducing sugar content is between 0.5-5.0g / 100g.

[0099] (4) Grain residue mixing and tank truck fermentation

[0100] Turn on the lees fan to cool the lees. Turn on the lees mixing equipment, conveying and cooling equipment to mix the lees evenly, and then cool it to 13-20℃ before loading it into the tank truck. After loading into the tank truck, cover the lees with a clean film and seal it with sandbags on all sides. The temperature of the mash entering the tank truck is controlled at 13-20℃, and the temperature in the fermentation room is controlled at 20-25℃. The temperature rise curve of the fermentation process in the truck follows the principle of "slow at the beginning, firm in the middle, and falling back at the end". The temperature of the tank truck reaches its peak on the 4th to 7th day, and then the temperature of the tank truck drops appropriately. Distillation is carried out after the fermentation period of 14-21 days. The entire fermentation temperature rise range is 14-36℃.

[0101] Sensory experience of fermentation: loose, shiny, moderate moisture, no peculiar smell.

[0102] Physical and chemical indicators of mash: moisture content of mash is controlled at 66-73%, acidity is 0.5-1.4mmol.

[0103] (5) Distillation

[0104] After the fermentation is completed, the tank truck will open the film at the designated place and shovel out the moldy mash. Open the steam valve and adjust the steam valve of each wine steamer to control the steam pressure according to the air pressure. The steaming is done by a robot, the time is controlled at 25-50 minutes, and the single-hanging distillation time is controlled at 35-70 minutes. After the steaming is completed, adjust the size of the steam valve to control the distillation pressure, take 3-5kg of head liquor for each hang, the high-alcohol liquor is ≤12kg, the main liquor is controlled between 60%vol-68%vol, and the alcohol content of the main liquor of a single hang is not less than 62.5%vol.

[0105] (6) Raw liquor testing and data comparison and analysis

[0106] The obtained light-fragrance raw liquor is subjected to quantitative analysis and detection by GC-FID gas chromatograph to obtain liquor chromatographic data. Among them, the liquor rate is calculated based on the mass of 55° alcohol per kilogram of dry food. The specific calculation formula is: first convert the weight and degree of the main liquor and the tail liquor into the 55° alcohol weight M alcohol, and combine the feed amount to calculate the liquor rate η = M alcohol weight / M dry food weight * 100%. The calculation method of the superior grade rate is: superior grade rate = the amount of superior liquor (55% vol, kg) / the amount of raw materials (kg) * 100%. The specific results are shown in Table 3:

[0107] Table 3 The output of light-flavored base liquor using fortified distiller's yeast

[0108]

[0109] From the data in the table above, it can be seen that the fortified koji containing microbial agents can significantly increase the superiority rate and ethyl acetate content of the light-flavored base liquor, among which the superiority rate increased significantly by 14.38 percentage points (p < 0.05) and the ethyl acetate content increased by 33.67% (p < 0.01). After evaluation by national judges, the liquor produced by fortified koji has a positive fragrance, prominent ester aroma, lees aroma, sweet taste, coordination, and outstanding style.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A composite bacterial agent, characterized in that: The composite bacterial agent comprises: Rhizopus oryzae G1 solid bacterial agent, Saccharomyces cerevisiae Y1 solid bacterial agent, Kluyveromyces marxianus Y245 solid bacterial agent and Wickerhamomyces anomalus Y348 solid bacterial agent.

2. The composite bacterial agent according to claim 1, characterized in that In the composite bacterial agent, the mass ratio of Rhizopus oryzae G1 solid bacterial agent, Saccharomyces cerevisiae Y1 solid bacterial agent, Kluyveromyces marxianus Y245 solid bacterial agent and Wickerhamomyces anomalus Y348 solid bacterial agent is (15-20): (1-5): (0.5-2): (0.5-2).

3. The composite bacterial agent according to claim 1, characterized in that The deposit number of the Rhizopus oryzae G1 is CCTCC NO: M 2019425, the deposit number of the Saccharomyces cerevisiae Y1 is CCTCC NO: M 20241943, the deposit number of the Kluyveromyces marxianus Y245 is CCTCC NO: M 2021225, and the deposit number of the Wickerhamomyces anomalus Y348 is CCTCC NO: M 20241944.

4. The composite bacterial agent according to claim 1, characterized in that The preparation method of Rhizopus oryzae G1 solid bacterial agent comprises the following steps: inoculating Rhizopus oryzae G1 bacterial bodies into a rice koji juice culture medium for culturing to obtain a first-class Rhizopus oryzae species; inoculating the first-class species into a bran solid culture medium for culturing to obtain a second-class Rhizopus oryzae species; inoculating the second-class Rhizopus oryzae species into a rice flour solid culture medium for culturing, and drying to prepare the Rhizopus oryzae G1 microbial solid bacterial agent.

5. The composite bacterial agent according to claim 1, characterized in that The preparation method of Saccharomyces cerevisiae Y1 microbial solid inoculant is as follows: inoculating Saccharomyces cerevisiae Y1 cells into a malt juice medium for cultivation to prepare a primary yeast species; transferring the primary yeast species culture solution into a molasses medium for cultivation to prepare a secondary yeast species; inoculating the prepared secondary seed solution into a rice flour solid medium for cultivation, and drying to prepare a yeast Y1 microbial solid inoculant.

6. The composite bacterial agent according to claim 1, characterized in that The preparation method of Kluyveromyces marxianus Y245 solid bacterial agent is as follows: inoculating Kluyveromyces marxianus Y245 bacterial bodies into a malt juice medium for cultivation to prepare a primary yeast species; transferring the primary yeast species culture solution to a molasses medium for cultivation to prepare a secondary yeast species; inoculating the prepared secondary seed solution into a rice flour solid culture medium for cultivation, and drying to prepare a yeast Y245 microbial solid bacterial agent.

7. The composite bacterial agent according to claim 1, characterized in that The preparation method of the abnormal Wickham yeast (Wickerhamomyces anomalus) Y348 solid bacterial agent is as follows: inoculating the abnormal Wickham yeast Y348 bacteria into a malt juice medium for cultivation to prepare a primary yeast species; transferring the primary yeast species culture liquid to a molasses medium for cultivation to prepare a secondary yeast species; inoculating the prepared secondary seed liquid into a rice flour solid culture medium for cultivation, and drying to prepare a yeast Y348 microbial solid bacterial agent.

8. A compound bacterial agent fortified distiller's yeast, characterized in that: The compound bacterial agent-fortified koji comprises traditional koji and the compound bacterial agent described in any one of claims 1 to 7, and the mass ratio of the traditional koji to the compound bacterial agent is (0.5-1):(4-6).

9. The method for preparing the composite bacterial agent-fortified distiller's yeast according to claim 8, characterized in that: The method comprises mixing traditional distiller's yeast with compound bacterial agent according to a mass ratio to obtain distiller's yeast fortified with the compound bacterial agent.

10. The use of the composite bacterial agent according to any one of claims 1 to 7, characterized in that: The composite bacterial agent is used for food fermentation.

Images