CN106591160A - Compound Xiaoqu and Xiaoqu Baijiu production method - Google Patents

Abstract

The present invention provides a compound Xiaoqu, which comprises mother koji and yeast, wherein the microorganisms in the mother koji comprise Aspergillus niger, Rhizopus nigricans, Rhizopus oryzae and Aspergillus oryzae, the yeast comprises ester-producing yeast and Saccharomyces cerevisiae, a weight ratio of the Aspergillus niger to the Rhizopus nigricans to the Rhizopus oryzae to the Aspergillus oryzae is 14:2:3:1, a weight volume ratio of the mother koji to the ester-producing yeast is 100 g:20 ml, a weight volume ratio of the mother koji to the Saccharomyces cerevisiae is 100 g:10 ml, and the yeast numbers in the ester-producing yeast and the Saccharomyces cerevisiae are not less than 1*10<8>/mL. According to the present invention, the wind producing capacity of the compound Xiaoqu is improved; the ester production ability is storing; and through the gas chromatography analysis, the number and the type of the produced flavor substances are increased compared to the ordinary Xiaoqu.

Description

A kind of production method of compound Xiaoqu and Xiaoqu Baijiu

technical field

The invention provides a production method of compound Xiaoqu and Xiaoqu liquor, which belongs to the technical field of brewing.

Background technique

At present, Xiaoqu liquor has the problems of low liquor yield and insufficient aroma. The essence is that the wine-making microorganisms cannot play their roles well. The traditional Xiaoqu wine production process only starts from several excellent strains, adding molds with high saccharifying power and yeast with high fermenting power. This kind of ditty song made by simple compounding must have insufficient alcohol yield and fragrance.

The traditional glutinous rice wine production process mainly uses Aspergillus niger as the saccharification agent, and the liquor yield is relatively high. Rhizopus niger has the strongest saccharification ability, Aspergillus niger has strong saccharification ability, good persistence, high alcohol yield and acid resistance, while Aspergillus oryzae has fast saccharification, is not acid-resistant, and has poor saccharification persistence. When using wild raw materials, because Aspergillus niger has more tannin-decomposing enzymes, which can decompose tannins in raw materials to reduce the impact on fermentation, it is more suitable to use Aspergillus niger as a saccharification agent. Considering the quality of liquor, the enzyme system contained in Aspergillus niger is relatively complex, and the taste of finished wine is relatively poor; enough, the wine quality is higher. Therefore, in order to take into account the advantages of both and solve the contradiction between alcohol yield and aroma, they should be cultivated separately to make koji and mixed in a certain proportion.

In recent years, great progress has been made in the screening of wine-making microorganisms, the selection of strains and the compounding of koji. Colony composition also varied greatly.

Rhizopus nigricans mycelium without septum, colony black gray, dioecious, mycelium is white, without septum multinucleated, mostly flocculent. Rhizopus oryzae is one of the important molds in Chinese koji. Rhizopus plays an extremely important role in wine production. In addition to producing a large amount of amylase and protease during the growth process, it can also produce organic acids such as citric acid, lactic acid, and succinic acid. Therefore, flavor substances such as ethyl acetate and ethyl lactate can be produced under certain conditions.

Aspergillus is a large group of microorganisms in liquor brewing, and it accounts for the most types of molds in liquor production. It has relatively strong heat resistance, and can produce a small amount of hydrolytic enzymes with high enzymatic activity, such as glucoamylase, amylase, protease, etc. Due to the acidic environment during the fermentation process, Aspergillus mainly secretes acid hydrolytic enzymes, which enable Aspergillus to grow, reproduce and metabolize normally in an acidic environment, catalyze the hydrolysis of starch and protein in the raw materials in a timely manner, and provide the materials required for subsequent fermentation. Among them, Aspergillus oryzae and Aspergillus niger are important sources of acid protease and α-amylase. These two enzymes can also function under acidic or strongly acidic conditions in the fermentation process, and can produce glucoamylase with high enzyme activity.

At present, the research on the production of Xiaoqu liquor is mainly concentrated in China. Due to the characteristics of less koji used, short fermentation cycle, high yield of starch, wide selection of raw materials and simple process equipment, Xiaoqu liquor is distributed in the southern and southwestern regions of my country and small workshops in urban and rural areas. In the early 1930s, Fang Xinfang used mold to make bran yeast for wine making, but it failed to be popularized. After the founding of New China, the former local Ministry of Industry organized and summarized the "Operation of Liquor Brewing in Yantai", with "bran koji wine mother, reasonable ingredients, low temperature storage, steaming at a fixed temperature" as the main points of operation. The most important thing in the brewing of bran koji liquor is the joint action of saccharification and fermentation of Aspergillus and yeast. The process conditions of the entire liquor fermentation depend on the physiological characteristics of Aspergillus and yeast. The selection of good molds has a great influence on the production of liquor. Shandong Linyi Distillery, as the first national new craft liquor pilot, increased the yield of liquor. At present, different distilleries use different strains to make moldy bran koji. Some wineries isolate and screen koji medicine themselves, and some directly buy it from other koji makers. Among them, Aspergillus oryzae, Aspergillus niger and the like can be directly used as strains for preparing bran koji. Screening of excellent mold strains is generally based on high glucoamylase activity and good growth performance as indicators to select the best strains.

Ester-producing yeast, also known as aromatic yeast, is a general term for a class of yeasts that can produce a large amount of ester substances in the metabolic process, mainly including Candida and Hansenula heterosexual. The ester-producing yeast is cultured statically in a liquid medium When , a layer of white bacterial film will be formed on the surface of the liquid, so ester-producing yeast is also called film-producing yeast. Esterogenic yeast has the ability to ferment alcohol and acetic acid, and also has the ability to ferment ethyl acetate. Studies have shown that the total ester content produced by ester-producing yeast is higher when the fermentation pH is lower. The suitable fermentation temperature is between 25°C and 30°C. When cultured at a constant temperature of 28°C, the amount of total ester produced is the highest; when the temperature is higher than 37°C, the metabolic ability of ester-producing yeast is weak, and the ester-producing ability will be greatly reduced. decline. Adding ester-producing yeast in liquor production can increase the content of ethyl acetate in liquor. Currently, liquor companies often add Saccharomyces cerevisiae in the production process to improve the quality of liquor.

In recent years, my country has made great achievements in the screening of ester-producing yeasts. Breeding excellent ester-producing yeasts and optimizing their fermentation conditions have gradually become research hotspots. Li Ruili et al. changed the culture environment of ester-producing yeasts to make The yield of ethyl acetate reached 2.15g/L; the ester-producing yeast screened by Wanshilv et al. from Quyao was fermented with sorghum fermentation medium, so that the total ester content reached 3.27g/L; Liu Yuancai et al. optimized the ester-producing yeast Under the conditions of ester production of bacteria, under the culture conditions of 0.6% ethanol, 0.4% acetic acid, 25% rice husk, temperature 28°C, time 72h and crushing degree 25%, the ethyl acetate production reached 4.81g/L; Xu Ling et al. In liquor production, ester-producing yeast was added and screened, so that the content of ethyl acetate reached 7.36g/L, which improved the quality of original wine; after Li Guohong and others optimized the culture conditions of ester-producing yeast, the amount of ethyl acetate produced by fermentation with sorghum compound medium up to 8.00g/L; it is necessary to screen and separate ester-producing yeast from brewer's koji and apply it in the production of liquor to improve the quality of liquor. Therefore, it is of great practical significance to develop and screen yeast strains with good ester-producing ability. However, using ester-producing yeast alone, it was found that although the quality of liquor was greatly improved, the wine production rate would decrease.

In recent years, my country has made great progress in strain compounding. Wu Qun from Jiangnan University successfully improved the efficiency and quality of sesame-flavored liquor production by comparing several mold strains from different sources and then interacting with yeast. Hu Yinchuan et al. mixed strains to brew small koji rice wine, which increased the yield by 6.26% compared with the traditional method Rate. Ke Wenfu et al showed that the production of Xiaoqu Baijiu by the multi-microqu semi-solid method can improve the yield of liquor by 10% compared with the production of whole rhizopus koji, the mouthfeel is obviously improved, and the fragrance is pleasant. Hu Jianhua and others screened 13 strains of wine-making microorganisms from Qingxiang Daqu, compounded them, distilled and picked the wine, and the yield and flavor substances of the obtained liquor were improved. Under the suggestion of Wang Yuantai, director of Yuanshan Food Industry Fermentation Research Institute, Baotou Distillery started the cultivation and application of multi-micro-bran koji, and found that multi-micro-bran koji can increase the aroma. Liu Mingming and others compounded bran koji and Daqu not only improved the quality of liquor, but also increased the yield of liquor and the content of flavor substances. Zhang Donglin compounded Rhizopus and AS 3.4309 strains, which not only improved the quality of liquor, but also increased the yield of liquor and the content of flavor substances. Xu Mingming and others compounded 4 kinds of molds and yeasts to increase the content of flavor substances in rice wine. Therefore, koji making with mixed bacteria is worth studying.

Contents of the invention

The purpose of the invention is to overcome the deficiencies of the prior art and provide a production method for compounding Xiaoqu and Xiaoqu liquor.

The first object of the present invention is to provide an ester-producing yeast, and the preservation number of the ester-producing yeast is CCTCC M 2016400.

The second invention object of the present invention is to provide a kind of composite ditty, described composite ditty is made up of mother song and yeast,

The microorganism in described parent song is made up of Aspergillus niger, Rhizopus niger, Rhizopus oryzae and Aspergillus oryzae;

The yeast is composed of ester-producing yeast and Saccharomyces cerevisiae;

The weight ratio of described Aspergillus niger, Rhizopus niger, Rhizopus oryzae and Aspergillus oryzae is 14:2:3:1;

The weight-to-volume ratio of the mother song and ester-producing yeast is 100g: 20ml;

The weight-to-volume ratio of the mother song and Saccharomyces cerevisiae is 100g: 10ml;

The number of yeasts in the ester-producing yeast and Saccharomyces cerevisiae is not less than 1× ¹⁰⁸ /mL;

The Aspergillus niger preservation number is CCTCC M 2016410;

The Aspergillus oryzae preservation number is CCTCC M 2016411;

The Rhizopus niger preservation number is CCTCC M 2016412;

The Rhizopus oryzae preservation number is CCTCC M 2016413;

The deposit number of the ester-producing yeast is CCTCC M 2016400.

The third object of the invention of the present invention is to provide the preparation method of the compound ditty, comprising the steps of:

A, the preparation of mother koji: inoculate the mold spores of Rhizopus niger, Rhizopus oryzae, Aspergillus niger, and Aspergillus oryzae respectively into culture bottles with bran, cultivate for 24-48h, when the surface of bran is covered with mycelia , cultured for 3-6 days in the bottle; take it out and dry it at 40°C for 4 hours, mix Aspergillus niger, Rhizopus niger, Rhizopus oryzae and Aspergillus oryzae in a weight ratio of 14:2:3:1, and then store it at 4°C for For brewing;

B. Yeast preparation: the ester-producing yeast and Saccharomyces cerevisiae were expanded and cultivated separately to make the number of yeasts reach 1×10 ⁸ /mL, and then set aside.

The preparation method of described parent song is as follows:

Take wheat to spray moist grain, crush it, mix it with fresh wheat bran, add water, and mix the ingredients evenly; divide the evenly mixed ingredients into triangular bottles, the thickness of the filling should not exceed 2cm, wrap the seal with 8 layers of gauze, and then use Wrap and sterilize with kraft paper, put it into a sterile room, and when it is cooled to 30°C, use an inoculation loop to pick out the fungal spores of Rhizopus niger, Rhizopus oryzae, Aspergillus niger, and Aspergillus oryzae into the triangular flask respectively, and mix well;

Put Rhizopus nigricans and Rhizopus oryzae in an incubator at 28°C for 24 hours, and when the surface of the bran is covered with hyphae, culture them in bottles for 3 days;

Aspergillus niger and Aspergillus oryzae were cultured in a 30°C incubator for 48 hours, and when the surface of the bran was covered with hyphae, they were bottled and cultured for 6 days;

After culturing, take them out and dry them at 40°C for 4 hours, mix Aspergillus niger, Rhizopus niger, Rhizopus oryzae and Aspergillus oryzae in a weight ratio of 14:2:3:1, and then store them at 4°C for winemaking .

The fourth object of the present invention is to provide a method for producing Xiaoqu baijiu by using the compound Xiaoqu, comprising the steps of:

1) Steamed rice husk: weigh the rice husk, add water to moisten it, and heat and sterilize it at 115°C for 20 minutes;

2) Steaming material: Mix the steamed rice husk and the weighed rice in a white porcelain plate, add water, let it stand for 10 minutes, put it into a triangular bottle, seal it with 6 layers of gauze, and sterilize the steaming material for 20 minutes;

3) Saccharification: Pour the material into a white porcelain plate, cool to about 30°C, add 2% of the mother koji, that is, add 2g of the mother koji per 100g of raw materials, mix well, then put it into a triangular bottle, and wrap it with 6 layers of gauze , saccharified at 32°C for 24h;

4) Fermentation: Put the saccharified grains into a white porcelain plate, add 10% yeast liquid, mix well, put them into a triangular flask, connect the cork and catheter, and ferment for 7 days at 30°C; observe every day Fermentation and weighing;

5) Distillation: add water to the pressure cooker, heat it with an electric furnace, and sprinkle the fermented raw materials in the pressure cooker layer by layer to ensure that the material is loose and even. The water is sealed, and at this time, the wine is steamed on a low heat to keep the pot boiling, and at the same time, the circulating water is turned on to start to condense. When the wine is out, the first 10mL wine head is received separately, which contains a large amount of low-boiling methanol and other harmful substances to the human body. Stop receiving when the alcohol concentration is lower than 30°C, and then pick up the tail;

The raw materials are rice husk, rice and water; the 10% yeast liquid is cultured by ester-producing yeast and Saccharomyces cerevisiae until the number of yeasts reaches 1× ¹⁰⁸ /mL, and then added 100g of mother koji , add 20ml of ester-producing yeast liquid and 10ml of Saccharomyces cerevisiae liquid, then add sterile water to dilute to 10% of the raw material, which is 10% yeast liquid; that is, for every 100g of raw material, add 2g of mother koji, add 0.2 g of ester-producing yeast and Saccharomyces cerevisiae ml and 0.1ml, and then diluted to 10ml, which is 10% yeast solution.

The preservation information of several microorganisms involved in the present invention is as follows:

1) Aspergillus niger

Storage date: 2016-7-28

Deposit unit: China Center for Type Culture Collection (CCTCC);

Deposit number: CCTCC M 2016410;

Taxonomic designation: Aspergillas niger FLW-1.

Strain characteristics: The surface of the colony is granular, with radioactive grooves and concentric rings. The reverse side is slightly yellowish brown in the central part, with radioactive cracks corresponding to the surface grooves. Cultured at 28°C for 7 days, the colony diameter was 1.9-2.5cm. Diaphragm can be seen sometimes in hyphae, podocytes are not obviously enlarged, apical capsule is obviously enlarged, mostly spherical, radial stalks grow on the surface, conidia are clustered at the top of the stalks, and conidia are spherical and brown.

2) Aspergillus oryzae

Storage date: 2016-7-28

Deposit unit: China Center for Type Culture Collection (CCTCC);

Deposit number: CCTCC M 2016411;

Classification name: Aspergillus oryzae (Aspergillus oryzae) FLW-2.

Strain characteristics: dense colony, initially white and then dark brown; creeping hyphae crawling, colorless, with well-developed brown finger-like rhizoids; cystic peduncles are erect, rarely solitary, mostly in bundles of 2-4 plants, The wall is smooth, brown, and F1 has cystic enlargement. The sporangia are spherical, with slight spines on the wall, black, 60-250um in diameter; the axis of the cyst is light brown, subspherical, oval or spherical, 50-90um in diameter; Striped and angular, yellow-gray in color, 5-8um in diameter; chlamydospores are spherical.

3) Rhizopus niger

Storage date: 2016-7-28

Deposit unit: China Center for Type Culture Collection (CCTCC);

Deposit number: CCTCC M 2016412;

Taxonomic designation: Rhizopus nigricans FLC-3.

Strain characteristics: the colony is relatively dense, initially white and then taupe; rhizoids are very developed; creeping hyphae crawling, colorless. Sporangia 2-7 in bundles, slightly curved, dark brown, unbranched, smooth surface, length 1500-3000um, diameter 20-25um; 100-200um; the axis of the cyst is smooth and taupe, with a diameter of 70-120um; the cystic receptacle is large and obvious, wedge-shaped; the surface of cyst spores has obvious stripes, corners, irregular shape, mostly ovate, gray; thick Mycospores ovoid or spherical.

4) Rhizopus oryzae

Storage date: 2016-7-28

Deposit unit: China Center for Type Culture Collection (CCTCC);

Deposit number: CCTCC M 2016413;

Classification name: Rhizopus oryzae (Rhizopus oryzae) FLC-4.

Strain characteristics: dense colony, initially white and then dark brown; creeping hyphae crawling, colorless, with well-developed brown finger-like rhizoids; cystic peduncles are erect, rarely solitary, mostly in bundles of 2-4 plants, The wall is smooth, brown, and F1 has cystic enlargement. The sporangia are spherical, with slight spines on the wall, black, 60-250um in diameter; the axis of the cyst is light brown, subspherical, oval or spherical, 50-90um in diameter; Striped and angular, yellow-gray in color, 5-8um in diameter; chlamydospores are spherical.

5) Ester-producing yeast

Storage date: 2016-7-19

Deposit unit: China Center for Type Culture Collection (CCTCC);

Deposit number: CCTCC M 2016400;

Classification name: Saccharomyces cerevisiae (Saccharomyces cerevisiae) QXC-2.

Strain characteristics: The colony of the yeast strain is raised, larger, with slightly irregular edges, the surface is moist and smooth, and is off-white; the cell shape is oval, and single-terminal budding.

Since ancient times, there has been a saying in wine making that "if you want to make wine, you must first make koji", and people vividly call koji "the bone of wine". Rhizopus is widely used in liquor production mainly because of its strong amylase-producing ability, which can saccharify starchy raw materials into dextrin and glucose. Rhizopus also has the ability to produce alcohol, which can be fermented while saccharifying, and can produce organic acids such as citric acid during the fermentation process. In addition, under certain conditions, it can also produce flavor substances such as ethyl acetate and ethyl lactate; Aspergillus is a large group of microorganisms in liquor brewing, and it accounts for the most types of molds in liquor production. Aspergillus oryzae and Aspergillus niger are important sources of acid protease and α-amylase, which can decompose protein and starch in raw materials, not only can produce wine, but also increase flavor substances in liquor.

The compound Xiaoqu produced by multiple strains is not just a simple mixture of several microorganisms. Due to the interaction of microbial growth and mutual influence, there are great differences in the number, type and enzyme system of microorganisms in the various processes of liquor production. The final products are also very different. The microorganisms used in this study were selected from dozens of Xiaoqu and combined after mutagenesis. For example, the ester-producing ability of the screened ester-producing yeast alone can reach 9.51g/L, The ester-producing ability of the ester-producing yeast reported in China is higher. Secondly, the growth and metabolic conditions of various microorganisms are studied. After selecting the most suitable culture conditions, they are cultured separately, and then the mother song is determined by orthogonal saccharification experiments. The ratio of yeast, and then use the saccharification conditions and time of the mother song to determine the type and quantity of yeast to be added. This is a ratio method that no one else has done. The results of fermentation experiments using these methods show that: 1. The wine-producing ability of the compound Xiaoqu has been improved; 2. The ester-producing ability is strong; 3. Through gas chromatography analysis, it can be seen that the number and types of flavor substances produced are higher than those of ordinary Xiaoqu. more.

Description of drawings

Figure 1-1 is the dilution coating result of the brewer's koji medicine QXC;

Figure 1-2 is the diluted coating result of brewer's koji medicine HX;

Fig. 1-3 is the dilute coating result of distiller's koji medicine CN;

Figure 2-1 is the yeast respiration intensity measured by the TTC method of the red yeast with high alcohol production capacity;

Figure 2-2 is the yeast respiration intensity measured by the TTC method of the pink yeast with low alcohol production capacity;

Figure 3-1 is the colony characteristics of QXC-2 yeast strain on YPD medium;

Figure 3-2 is the colony characteristics of HX-1 yeast strain on YPD medium;

Fig. 3-3 is the bacterium colony characteristic of CN-5 yeast strain on YPD medium;

Figure 4-1 is the cell morphology of the QXC-2 yeast strain;

Figure 4-2 is the cell morphology of 2HX-1 yeast strain;

Figure 4-3 is the cell morphology of CN-5 yeast strain;

Fig. 5 is the electrophoresis diagram of yeast genome DNA;

Fig. 6 is the electrophoresis diagram of the 5.8S rDNA-ITS sequence PCR amplification product in the yeast genome;

Figure 7 shows the sequencing results of QXC-2;

Fig. 8 is the gas chromatogram of Wangxian Xiaoqu Liquor; 1. methanol; 2. ethanol; 3. ethyl acetate; 4. unknown; 5. isobutanol; 6 acetic acid.; 7. n-butanol; 8. unknown; 9 .furfural; 10. unknown;

Figure 9 is a gas chromatogram of No. 12 compound Xiaoqu Baijiu. Among them, 1. Acetaldehyde; 2. Methanol; 3. Propanol; 4. Ethyl acetate; 5. Acetal; 6. Unknown; Ethyl butyrate; 11. Isoamyl alcohol; 12. Unknown; 13. Ethyl lactate.

detailed description

Sources of Materials Used in the Invention.

Drugs are listed in Table 1.

Table 1 Drugs

The instruments are shown in Table 2.

Table 2 Instruments

Angel Saccharomyces cerevisiae, commercially available, Hubei Yichang Angel Biological Group Co., Ltd.

The varieties of Xiaoqu are shown in Table 3.

Table 3 Types of ditties and their sources

1. Screening of Aspergillus niger, Rhizopus niger, Rhizopus oryzae and Aspergillus oryzae, and preparation of mother koji

1.1 Separation method

Use the dilution coating method to dilute and coat the Xiaoqu suspension, weigh 1g of the ground Xiaoqu and add it to 100mL sterile water and mix evenly. After gradient dilution, take 0.1mL of the diluted bacterial solution on Tiger Red Agar, PDA and Chapei’s Medium Spread and incubate at 28°C for 48h. Take out the flat plate without miscellaneous bacteria, pick the mold colony and inoculate it on the tiger red agar medium, and repeat the operation 3 to 4 times to obtain a relatively pure strain. Cultivate the strain obtained in the previous step for 4-5 days to obtain mature spores and apply gradient dilution according to the above method, and repeat the application 2 to 3 times to obtain a more pure strain. According to the colony morphology and microscopic examination results, the pure strains were inoculated into inclined planes and stored in the refrigerator for later use. The impure strains were further isolated and stored, and were initially classified.

1.2 Strain identification

1) Insert culture

Prepare the PDA medium in advance, and insert about 2-6 pieces of sterilized glass slides at an angle of 45° by aseptic operation. Pick a small amount of spores from the slope with an inoculation needle, and place them as close to the root of the slide as possible. Place the petri dish upside down in an incubator, culture at 28°C for a certain period of time (5-6 days for Rhizopus and Mucor, 10-14 days for Aspergillus penicillium), observe and record the characteristics of the colonies.

2) Colony morphology and characteristics

(1) Growth or development speed: after culturing for a certain number of days, the diameter of the colony is measured, which is usually described as extremely slow, slow, medium, fast, etc.

(2) Color of the colony: the color and change of the hyphae on the surface and the bottom of the colony and the back of the colony.

(3) The surface of the colony: record the shape of the whole colony, the center part, part and the edge part respectively.

(4) Colony texture: Common types of colony appearance are wool-like, felt-like, leather-like, fluffy, gelatin-like, cotton-wool-like, powder-like or bundle-like.

(5) The edge of the colony: ciliated, jagged, entire, dendritic, etc.

(6) The height of the colony: the central part of the colony is raised or sunken or mounded, the colony is flat, sunken, etc.

(7) Color change of the culture medium: whether the range of color change is only the part covered by the mycelium, or is it extended to other parts.

(8) Exudate: Some fungi often secrete colored droplets on the surface of the colony. Pay attention to whether there is such a phenomenon, quantity and color when observing.

(9) Odor: aromatic, earthy or musty, etc., of course there are also odorless ones.

3) Microscopic morphology and characteristics

Put an appropriate amount of lactic acid carbolic acid cotton blue staining solution in the center of the clean glass slide, take out the cultured colony plate, take out the cover glass from the plate under the sterile range of the alcohol lamp and cover it lightly on the glass slide, and place it after staining for a certain period of time. Observe and record the following characteristics under a microscope.

(1) Mycelium: Whether there are transverse septa, color, width, shape, etc., and whether there are special structures such as roots, podocytes, nodules or bulges.

(2) Morphology of fruiting bodies: observe the color, shape, size, and structural characteristics of ascus, ascus shells, sporangia, and basidiocarps.

(3) Spores: the shape, color, and surface features of asexual and sexual spores (such as ornamentation or protrusions, etc.), whether the spores are separated (unicellular or multicellular), and the type of spore germination (unipolar germination, bipolar stage budding or multi-stage budding, etc.).

1.3 Determination of the performance of glucoamylase and liquefaction enzyme

(1) Preparation of single-strain bran koji

Mix bran and water evenly in a ratio of 1:1, and pack them into 500ml triangular flasks with a thickness of about 1cm. Be careful not to get bran on the mouth of the triangular flask. After sealing, sterilize at 115°C for 1 hour. Cool to below 45°C, shake well to make it loose, inoculate each bottle with 3 rings of slant strains, place in an incubator, culture at 28°C for 5 days, shake once every 12 hours during the period, and stop shaking after the bran hardens. .

Take the bran koji out of the triangular bottle, put it into 4 layers of gauze, wrap the number, and dry it at 35°C for 40 hours to obtain an absolutely dry bran koji.

(2) Determination of saccharification power

① Preparation of 5% solid koji extract: Accurately weigh 5.00g of dry bran koji, put it into a 250ml beaker, separate the koji lumps, add 10mL of acetic acid-sodium acetate buffer solution with pH=4.6 and 90ml of distilled water, shake well Place it in a constant temperature water bath at 35°C for 1 hour, take it out every 15 minutes, shake it and mix it once, collect the filtrate after filtration and wait for determination.

②Saccharification: Accurately measure 25.0mL of 20g/L starch solution in a 50ml volumetric flask, keep it in a constant temperature water bath at 35°C for 10min, make the temperature reach the measured temperature, draw 5.00ml sample extract into the volumetric flask, shake well and put it back into the water bath Accurately saccharify in medium for 1 hour, quickly add 1ml of NaOH solution, take out the volumetric flask, shake well, and cool to room temperature.

③Pre-experiment: In order to correctly grasp the volume of the standard solution to be added and try to complete the titration within 1 min, a pre-experiment is required. In a 150ml Erlenmeyer flask, add 5ml of film reagent A solution, B solution, and test solution in turn, add a certain amount of glucose standard solution in advance, heat it to boiling on an electric furnace with a power of 1000w, and add 1.0% A solution dropwise in the boiling state. Take 1-2 drops of base blue indicator, titrate with 0.25% standard glucose solution to blue light yellow, which is the titration end point, and record the total amount of glucose V used for titration.

④ Formal measurement: Add 5ml of film reagent A solution, B solution, and test solution to a 150ml conical flask in turn, and 10ml of distilled water. Add glucose standard solution less than about 1ml of V in the pre-experiment in advance, and place the conical flask in a power Heat it to boiling on a 1000w electric furnace, add 1-2 drops of 1.0% methylene blue indicator dropwise in the boiling state, and titrate with 0.25% standard glucose solution to blue light yellow, which is the titration end point. Quantity V ₁ .

⑤Blank test: replace the liquid to be tested in the formal titration with distilled water, repeat the above operation, and record the consumed volume V ₀ .

⑥ calculation

Saccharification power of koji medicine: At 35°C, pH=4.6, 1 g of dry koji is enzymatically hydrolyzed with soluble starch for 1 hour to produce 1 mg of glucose as one unit of saccharification power.

Formula 1

In the formula:

X──saccharification power (mg/g h);

V0──The amount of glucose standard solution consumed by blank titration, mL;

V1──The amount of glucose standard solution consumed when the sample is formally measured, mL;

C ──concentration of glucose standard solution, g/mL;

t——Enzymolysis time, h.

(3) Determination of liquefaction force

① Sample pretreatment: Accurately weigh 5.00g of dry koji, put it into a beaker with a capacity of 250ml, separate the agglomeration of koji, add 10mL of acetic acid-sodium acetate buffer solution with pH=4.6 and 90ml of distilled water, shake well and place at constant temperature Incubate in a water bath at 35°C for 1 hour, take it out every 15 minutes, shake and mix well, collect the filtrate after filtration and wait for determination.

② Liquefaction: Take 25ml of starch solution with a concentration of 20.0g/L and acetic acid-sodium acetate buffer solution with pH=4.6 in a 100ml test tube, keep it in a constant temperature water bath at 35°C for 10min, make the temperature reach the measurement temperature, and absorb 5.00ml Put the leaching solution of the sample in the test tube, shake it well, put it back into the water bath, and record the liquefaction start time. Dip the reaction solution with a glass rod at intervals on the white porcelain titration plate pre-added with iodine solution until the color does not change, which is the end time of liquefaction.

③ calculation

The definition of liquefaction power of solid koji: the mass of soluble starch liquefied by 1 g of dry koji for 1 hour under the reaction conditions of 35°C and pH=4.6.

Formula 2

In the formula:

X──Liquefaction force (g/g.h);

25.0──The amount of soluble starch solution, ml;

0.020──Concentration of soluble starch solution, g/ml;

0.25──The sample amount of absolute dry koji corresponding to 5.0ml leachate, g;

60──Conversion coefficient of liquefaction soluble starch time into 1h;

t——liquefaction time, min.

Four molds with good morphology and the best saccharification performance and wine-producing flavor were screened out and preserved respectively: Aspergillus niger preservation number is CCTCC M 2016410; Aspergillus oryzae preservation number is CCTCC M 2016411; Rhizopus niger preservation number is CCTCC M 2016411; It is CCTCC M 2016412; the deposit number of Rhizopus oryzae is CCTCC M 2016413.

1.5 Expanded culture of mold

Expanded cultivation: Inoculate the four kinds of Rhizopus selected into the slant medium of the test tube, cultivate at 28°C for one week, observe the growth of the mycelium, and use it for future use.

1.6 Preparation of mother song

Weigh 1 kg of fresh wheat bran in a white porcelain plate, add 800 mL of tap water, and mix the ingredients evenly. The uniformly mixed material was divided into 40 250mL conical flasks, the thickness of which was not more than 2cm, wrapped and sealed with 8 layers of gauze, and then wrapped with kraft paper for sterilization. Sterilize at 0.1Mpa, 121°C for 30min. At this time, turn on the ultraviolet light in the sterile room for 30 minutes. After the sterilization is completed, take it out and put it into the sterile room. When it is cooled to 30°C, use the inoculation loop to pick up the mold spores and put them into the conical flask, and mix them well. Rhizopus niger, Rhizopus oryzae, Aspergillus niger, and Aspergillus oryzae each received 10 triangular flasks. Put Rhizopus niger and Rhizopus oryzae in an incubator at 28°C for 24 hours, and when the surface of the bran is covered with hyphae, culture them in bottles for 3 days; When the surface is covered with hyphae, culture the bottle for 6 days; after culture, take it out and dry it at 40°C for 4 hours, mix Aspergillus niger, Rhizopus niger, Rhizopus oryzae and Aspergillus oryzae, and store it at 4°C for wine making use.

2. Screening and identification of ester-producing yeast

2.1 Isolation and purification of ester-producing yeast

Add 1 g of the selected brewer’s koji medicine (Drosophila distiller’s yeast, Yichang City, Hubei Province) into 100 mL of enrichment medium, and incubate in a constant temperature shaker incubator at 28°C for 48 hours. Dilute to 10-6 times, draw 0.1mL respectively and apply in YPD medium, and incubate at 28°C for 48h. Select yeasts with different colonies and cell shapes, number them according to the source and the order of selection, and repeat the separation and purification until pure colonies are obtained.

The isolated and purified yeast strains were inoculated on PDA slant medium in a sterile environment, cultured at 28°C for 48 hours, stored in a refrigerator at 4°C, and transferred regularly every month.

2.2 Screening of ester-producing yeast

1) Determination of yeast respiratory enzyme activity by TTC method

Inoculate the purified yeast on the TTC lower layer medium, and culture it upside down in a constant temperature incubator at 28°C for 48 hours, then pour about 12mL of TTC upper layer medium into the plate under a sterile environment, shake it left and right, so that the medium covers After wrapping all the colonies on the plate with a black cloth bag, continue to incubate in a constant temperature incubator at 28°C in the dark for 2 to 3 hours, judge the alcohol production capacity of the yeast according to the color of the colony, and select the red colony to determine its ester production capacity.

2) Determination of ester-producing ability of ester-producing yeast

The selected ester-producing yeasts were inoculated into the ester-producing medium, and fermented at 28° C. for 6 days. After the fermentation, the content of total esters in the fermentation broth was measured, and yeast strains with high ester-producing ability were selected for molecular identification.

The determination method of total ester content is as follows:

The quality of the flavor of liquor brewed with distiller's yeast is mainly based on the ester-producing ability of the yeast. For brewer's koji with high total ester content, the yeast in the corresponding koji has a high ester-producing ability.

Take 50 mL of fermented liquid in a 50 mL centrifuge tube, centrifuge at 4000 r/min for 10 min, absorb the supernatant, and measure the content of total esters in the fermented liquid according to the determination method of total esters in liquor (calculated as ethyl acetate, g/L).

Formula 3

In the formula, 0.0935——the actual concentration of NaOH standard titration solution, mol/L;

15.00——the volume of NaOH standard titration solution added during saponification, mL;

0.1047—— _The actual concentration of _H2SO4 standard titration solution, mol/L;

V1——the volume of H ₂ SO ₄ standard titration solution consumed during back titration, mL;

0.08812——The mass of ethyl acetate equivalent to 1.00mL NaOH standard titration solution [c(NaOH)=1.000mol/L], g;

5——Sampling volume, mL.

2.3 Identification of ester-producing yeast

1) Morphological identification

The screened yeasts were cultured in YPD solid medium, and a small number of colonies were picked in the logarithmic growth phase and stained with methylene blue to make a microscopic observation under a 100×10 oil lens. Combined with the colony characteristics and cell morphology of the yeast in the "Handbook of Fungal Identification", the ester-producing yeast was identified.

2) Molecular identification

Inoculate 1 loop of a strain with high ester-producing ability into 10 mL of YPD liquid medium, and culture overnight at 28°C.

A, Genomic DNA extraction

Use a pipette gun to draw 1.5mL of yeast liquid into a 1.5mL sterile PE centrifuge tube, centrifuge at 10000r/min for 1min at 4°C, and discard the supernatant. Use the Ezup Column Yeast Genomic DNA Extraction Kit to extract yeast DNA according to the instruction manual, and store the extracted yeast genomic DNA in a -20°C refrigerator for later use.

B, Genomic DNA purity test

The DNA solution was diluted 5 times, and 5 mL of the DNA dilution was taken for agarose gel electrophoresis at a gel mass concentration of 0.8% and a voltage of 80 V for 80 min. After electrophoresis, observe the gel in the gel imaging analysis system to judge the purity of the extracted DNA.

C, PCR amplification

Yeast PCR amplification object is the 5.8S rDNA gene of the yeast strain, and the extracted yeast genomic DNA is used as a template, and the fungus is amplified by primers ITS (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') Ribosomal ITS region gene.

D, PCR reaction system

The PCR reaction system was: 12.5 μL 2×Taq PCR MasterMix, 0.5 μL primer ITS1 (10 μmol/L), 0.5 μL primer ITS4 (10 μmol/L), 2 μL DNA template, and finally replenished to 25 μL with double distilled water.

E, PCR reaction program

The PCR reaction program was: pre-denaturation at 94°C for 4 min, followed by 34 cycles including denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 1 min, and the last cycle of extension at 72°C for 10 min.

F, PCR product agarose gel electrophoresis

Take 5 μL of PCR products and electrophoresis with 1.4% agarose gel at 80V voltage for 1 h, detect in gel imaging analysis system, compare the position of DNA band and DNA Marker, and preliminarily judge the size of PCR product fragments. Finally, the amplified product containing the target band was sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for sequencing.

G, sequencing and result analysis

The sequence obtained by sequencing was entered into www.ncbi.nlm.nih.gov, and the GenBank nucleic acid sequence database was searched by BLAST to determine its species.

2.4 Results and analysis

2.4.1 Screening results of high-yielding koji drugs

The screening results of the high-yield koji drugs are shown in Table 4.

Table 4 Determination of the ester-producing ability of distiller's koji medicine

As can be seen from Table 4, among the 13 kinds of koji medicines (listed in the above table), there are 9 kinds of koji medicines whose ester production ability is below 2.00g/L; 1 kind of koji medicine is 2.06g/L, and the ester production ability belongs to the downstream level; The ester production capacity of the three koji medicines reached above 5.00g/L, and the ester production capacity was higher than other varieties. Therefore, the three high-yielding koji drugs were separated and purified, and the ester-producing yeasts were screened out.

2.4.2 Primary screening results of ester-producing yeast

The results of primary screening of ester-producing yeast are shown in Figure 1-1, Figure 1-2, and Figure 1-3.

2.4.3 Re-screening results of ester-producing yeast

A, TTC method to measure yeast respiratory enzyme activity

The results of the determination of yeast respiratory enzyme activity by TTC method are shown in Figure 2-1 and Figure 2-2. It can be seen from the figure that darker dark red yeasts appear on the plate, indicating that the selected yeast strains have higher respiratory enzyme activity and better ethanol production ability; lighter pink yeasts have lower ethanol production ability.

B. Determination of Ester Production

The test results of ester production are shown in Table 5.

Table 5 acid-base titration method to determine the ester-producing ability of yeast strain

Nine strains of yeast were screened out from the three kinds of koji, and six of them had low ester-producing ability and provided alcohol for the production of liquor, and the other three not only provided alcohol for the production of liquor, but also produced a large amount of ester substances in the production of liquor .

2.4.4 Identification results of ester-producing yeast

A. Morphological identification

The identification results of colony characteristics of the yeast strains are shown in Figures 3-1, 3-2, and 3-3. It can be seen from the figure that there are obvious differences in the colonies of the three yeast strains: the colonies of the QXC-2 yeast strain are raised, larger, with slightly irregular edges, and the surface is moist and smooth, and it is off-white, which is initially determined to be Saccharomyces cerevisiae; HX The colony of yeast strain -1 is raised, with regular edges, moist and smooth surface, white, and it is initially identified as Saccharomyces cerevisiae; the colony of yeast strain CN-5 is relatively flat, with a large area, and the whole is round with "burrs" on the surface. Smooth, off-white, initially identified as heterosexual Hansenula.

The cell morphology identification results of the yeast strains are shown in Figures 4-1, 4-2, and 4-3. It can be seen from the figure that the cell shape of QXC-2 yeast is oval, with single-terminal buds, combined with the characteristics of the colony, it is identified as Saccharomyces cerevisiae; the cell shape of HX-1 yeast is round, with single-terminal buds According to the characteristics of the colony, it was identified as Saccharomyces cerevisiae; the cell shape of CN-5 yeast was short rod-shaped, unilateral budding, and according to the characteristics of the colony, it was identified as Hansenula heterosexual.

B. Molecular identification

The results of yeast DNA electrophoresis detection are shown in Figure 5. It can be seen from the figure that in the automatic gel imaging analysis system, the DNA bands of the three yeast strains are clear and there are no miscellaneous bands, indicating that the extracted yeast genome DNA is of high purity.

The electrophoresis of the amplified product is shown in Figure 6. It can be seen from the figure that in the automatic gel imaging analysis system, the amplified DNA fragment band is single, the amplified fragment size of CN-5 is about 550bp, and the amplified fragments of QXC-2 and HX-1 The size is about 600bp. And no band appeared in the blank group (not shown), indicating that there was no contamination and no non-specific amplification.

GenBank identification results are shown in Figure 7 and Table 6.

Table 6 yeast identification results

From the determined gene sequence, the nucleotide sequence of QXC-2 is shown in SEQ ID No.1. The sequences of QXC-2 and HX-1 are completely consistent. Combined with morphological identification, they are finally identified as the same Saccharomyces cerevisiae, CN The molecular identification of -5 was also H. heterosexual, but no significant similarity was found in the GenBank database. The QXC-2 strain with high ester production was selected for preservation, and the preservation number was CCTCC M 2016400. It was combined with Saccharomyces cerevisiae Angelica, and then compounded with the mother koji.

2.5 Expanded culture of yeast

Wort slant strains were cultured at 28°C for 2 days, and 3 single colonies were picked, inoculated into a 250mL Erlenmeyer flask at 30°C, counted after 2 days of culture, so that the number of bacteria reached 1× ¹⁰⁸ /mL, and set aside.

Three, ditty recombination

For the compounding of four kinds of fungi and two kinds of yeasts, compare it with Xiaoqu, the largest Xiaoqu distillery in the region (Wangxian Liquor Industry), and do an orthogonal experiment design, as shown in Table 7.

Table 7 Orthogonal ratio table of bran

4. Fermentation of Xiaoqu Baijiu

1. The fermentation of Xiaoqu baijiu comprises the following steps:

Steamed rice husk: Weigh 630g of rice husk, add water to moisten it, and heat and sterilize it at 115°C for 20 minutes.

Steaming material: Mix the steamed rice husk and 4200g of rice in a white porcelain plate, add 6300mL of water, let it stand for 10min, put it into 14 1000mL triangular flasks, seal with 6 layers of gauze, and extinguish at 115℃ Bacteria steamed for 20 minutes.

Saccharification: Pour the material into a white porcelain plate, cool to about 30°C, add 2% of the mother koji, that is, add 2g of the mother koji per 100g of raw materials, mix well, then put it into a triangular bottle, wrap it with 6 layers of gauze, and Saccharification at 32°C for 24h.

Fermentation: Put the saccharified grains into a white porcelain plate, add 10% yeast liquid, mix well, put them into Erlenmeyer flasks, connect with corks and catheters, and ferment for 7 days at 30°C. Observe the fermentation situation and weigh every day.

Distillation: add water to the pressure cooker, heat it with an electric furnace, and sprinkle the fermented raw materials in the pressure cooker layer by layer to ensure that the material is loose and even. , At this time, steam the wine on a low heat, keep the pot boiling, and at the same time turn on the circulating water to start to condense. When the wine is out, the first 10mL wine head is received separately, which contains a large amount of low-boiling methanol and other harmful substances to the human body. Stop receiving when the alcohol concentration is lower than 30°C, and then pick up the tail of the wine.

The raw materials are rice husk, rice and water; the 10% yeast liquid is cultured by ester-producing yeast and Saccharomyces cerevisiae until the number of yeasts reaches 1× ¹⁰⁸ /mL, and 100 g of mother koji is added , add 20ml of ester-producing yeast liquid and 10ml of Saccharomyces cerevisiae liquid, then add sterile water to dilute to 10% of the raw material, which is 10% yeast liquid; that is, for every 100g of raw material, add 2g of mother koji, add 0.2 g of ester-producing yeast and Saccharomyces cerevisiae ml and 0.1ml, and then diluted to 10ml, which is 10% yeast solution.

Measure its alcohol content, then convert it into the alcohol content at 20°C, and calculate the alcohol rate.

Calculation of alcohol yield: alcohol yield includes theoretical alcohol yield, starch utilization rate, starch alcohol yield, and raw material alcohol yield. The raw material alcohol yield is often used in production. The calculation method is as follows: View 20°C Alcohol volume fraction, mass fraction, density comparison table, volume fraction is converted into mass fraction, calculate the quality that produces 65% volume fraction, alcohol yield=(65% mass fraction mass/raw material total mass)*100%

2. Chromatographic analysis of Xiaoqu Baijiu

Determination of flavor components in Xiaoqu liquor (GC): Chromatographic conditions: chromatographic column is DB-WAX capillary column; inlet temperature is 230°C; column oven temperature program: keep at 60°C for 3min, rise to 200°C at 5°C/min, then Then rise to 230°C at 10°C/min and keep for 1min; carrier gas: 99.999% high-purity helium, carrier gas flow rate: 1mL/min.

Mass spectrometry conditions: EI ion source, ion source temperature of 230°C, ionization voltage of 70eV, quadrupole temperature of 150°C, ionization mode: EI, mass scanning range of m/z: 20-500; spectral library NIST.

Qualitative and quantitative analysis of aroma substances: comparative analysis with the NIST spectral library, and comprehensive reference materials and related background information to conduct qualitative analysis of the substances. Measure the content of trace components such as ethyl acetate, methanol, and isobutanol.

3. Results

3.1 Liquor yield of Xiaoqu: as shown in Table 8

Table 8 Alcohol yield and yield after fermentation

From the above table it can be concluded that:

① No. 12 has the highest alcohol content and the highest alcohol yield, and the alcohol yield has increased by 11.64%.

②The quality of raw materials consumed by fermentation with different Xiaoqu ratios is different. Generally speaking, the more raw materials consumed by respiration, the more vigorous the microbial metabolism, and the more alcohol produced. No. 12 and No. 18 consumed 0.14 kg, the most; No. 10 to No. 20 consumed almost 0.12 kg of mold respiration; and No. 1 to 10 and No. 20 to 25 only consumed 0.10 kg of rice raw materials, of which No. 4 and No. 21 only consumed 0.08 kg.

③ No. 12, No. 13, No. 14, No. 17, No. 18, and No. 20 have high alcohol yield and complex compounding. Screen them and No. 26 to measure the main flavor substances, compare their contents, and determine their optimal ratio .

3.2 Contents of main flavor substances in liquor: as shown in Table 9

Table 9 Contents of main flavor substances in 100mL liquor

Comparing Figure 8 and Figure 9, it can be seen that not only the content of ethyl acetate in No. 12 is much higher than that of No. 26 (Wangxian Xiaoqu Liquor), but also more flavor substances than No. 26.

It can be seen from the above table that No. 12 and No. 14 do not contain methanol, and No. 17 and No. 18 have the highest content of 0.040g/100mL. Higher alcohol is one of the most important aroma and taste substances in wine. An appropriate amount of higher alcohol can make the aroma Harmonious but out of range is the main substance of miscellaneous flavors. Methanol is a substance that is poisonous to the human body and is very harmful to the vision, so its content should be strictly controlled.

The results showed that No. 12 had the highest liquor yield and the most flavor substances content, so it was the best compound group.

4 Conclusion

The research results show that in the production of liquor, the saccharification power of koji determines the yield of liquor, but it is not proportional. When the mold is used in a compound form for winemaking, it not only promotes saccharification, but also improves yeast fermentation.

On the 12th, when Aspergillus niger: Rhizopus niger: Rhizopus oryzae: Aspergillus oryzae = 14:2:3:1, add 20ml and 10ml of ester-producing yeast and Saccharomyces cerevisiae per 100g of koji, the wine yield is the highest, which is higher than the local single strain It has increased by 11.64%, and has the most types of flavor substances, among which the ester content has increased by 0.148g/100mL, which is the best compound group. Selecting excellent strains to compound koji and re-fermentation can increase the yield of liquor. At the same time, the content of flavor substances is greatly increased, and the aroma of liquor is the strongest. Usually, the higher the alcohol yield, the lower the ester-producing ability, but in this study, both the alcohol yield and the ester-producing ability of No. 12 were the strongest.

Organization Applicant

----------------------

<110> OrganizationName : Changjiang Normal University

Application Project

-------------------

<120> Title : A Production Method of Compound Xiaoqu and Xiaoqu Baijiu

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<141> CurrentFilingDate : ____-__-__

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Claims (5)

1. An ester-producing yeast, characterized in that, the preservation number of the ester-producing yeast is CCTCC M 2016400. 2. a composite ditty, is characterized in that, described composite ditty is made up of mother song and yeast, The microorganism in described parent song is made up of Aspergillus niger, Rhizopus niger, Rhizopus oryzae and Aspergillus oryzae; The yeast is composed of ester-producing yeast and Saccharomyces cerevisiae; The weight ratio of described Aspergillus niger, Rhizopus niger, Rhizopus oryzae and Aspergillus oryzae is 14:2:3:1; The weight-to-volume ratio of the mother song and ester-producing yeast is 100g: 20ml; The weight-to-volume ratio of the mother song and Saccharomyces cerevisiae is 100g: 10ml; The number of yeasts in the ester-producing yeast and Saccharomyces cerevisiae is not less than 1× ¹⁰⁸ /mL; The Aspergillus niger preservation number is CCTCC M 2016410; The Aspergillus oryzae preservation number is CCTCC M 2016411; The Rhizopus niger preservation number is CCTCC M 2016412; The Rhizopus oryzae preservation number is CCTCC M 2016413; The deposit number of the ester-producing yeast is CCTCC M 2016400. 3. the preparation method of composite ditty as claimed in claim 2, is characterized in that, comprises the steps: A, the preparation of mother koji: inoculate the mold spores of Rhizopus niger, Rhizopus oryzae, Aspergillus niger, and Aspergillus oryzae respectively into culture bottles with bran, cultivate for 24-48h, when the surface of bran is covered with mycelia , cultured for 3-6 days in the bottle; take it out and dry it at 40°C for 4 hours, mix Aspergillus niger, Rhizopus niger, Rhizopus oryzae and Aspergillus oryzae in a weight ratio of 14:2:3:1, and then store it at 4°C for For brewing; B. Yeast preparation: the ester-producing yeast and Saccharomyces cerevisiae were expanded and cultivated separately to make the number of yeasts reach 1×10 ⁸ /mL, and then set aside. 4. the preparation method of compound ditty according to claim 3 is characterized in that, the preparation method of described parent song is as follows: Take wheat to spray moist grain, crush it, mix it with fresh wheat bran, add water, and mix the ingredients evenly; divide the evenly mixed ingredients into triangular bottles, the thickness of the filling should not exceed 2cm, wrap the seal with 8 layers of gauze, and then use Wrap and sterilize with kraft paper, put it into a sterile room, and when it is cooled to 30°C, use an inoculation loop to pick out the fungal spores of Rhizopus niger, Rhizopus oryzae, Aspergillus niger, and Aspergillus oryzae into the triangular flask respectively, and mix well; Put Rhizopus nigricans and Rhizopus oryzae in an incubator at 28°C for 24 hours, and when the surface of the bran is covered with hyphae, culture them in bottles for 3 days; Aspergillus niger and Aspergillus oryzae were cultured in a 30°C incubator for 48 hours, and when the surface of the bran was covered with hyphae, they were bottled and cultured for 6 days; After culturing, take them out and dry them at 40°C for 4 hours, mix Aspergillus niger, Rhizopus niger, Rhizopus oryzae and Aspergillus oryzae in a weight ratio of 14:2:3:1, and then store them at 4°C for winemaking . 5. utilize the method for compounding Xiaoqu described in claim 2 to produce Xiaoqu liquor, it is characterized in that, comprises the steps: 1) Steamed rice husk: weigh the rice husk, add water to moisten it, and heat and sterilize it at 115°C for 20 minutes; 2) Steaming material: Mix the steamed rice husk and the weighed rice in a white porcelain plate, add water, let it stand for 10 minutes, put it into a triangular bottle, seal it with 6 layers of gauze, and sterilize the steaming material for 20 minutes; 3) Saccharification: Pour the material into a white porcelain plate, cool to about 30°C, add 2% of the mother koji, that is, add 2g of the mother koji per 100g of raw materials, mix well, then put it into a triangular bottle, and wrap it with 6 layers of gauze , saccharified at 32°C for 24h; 4) Fermentation: Put the saccharified grains into a white porcelain plate, add 10% yeast liquid, mix well, put them into a triangular flask, connect the cork and catheter, and ferment for 7 days at 30°C; observe every day Fermentation and weighing; 5) Distillation: add water to the pressure cooker, heat it with an electric furnace, and sprinkle the fermented raw materials in the pressure cooker layer by layer to ensure that the material is loose and even. The water is sealed, and at this time, the wine is steamed on a low heat to keep the pot boiling, and at the same time, the circulating water is turned on to start to condense. When the wine is out, the first 10mL wine head is received separately, which contains a large amount of low-boiling methanol and other harmful substances to the human body. Stop receiving when the alcohol concentration is lower than 30°C, and then pick up the tail; The raw materials are rice husk, rice and water; the 10% yeast liquid is cultured by ester-producing yeast and Saccharomyces cerevisiae until the number of yeasts reaches 1× ¹⁰⁸ /mL, and then added 100g of mother koji , add 20ml of ester-producing yeast liquid and 10ml of Saccharomyces cerevisiae liquid, then add sterile water to dilute to 10% of the raw material, which is 10% yeast liquid; that is, for every 100g of raw material, add 2g of mother koji, add 0.2 g of ester-producing yeast and Saccharomyces cerevisiae ml and 0.1ml, and then diluted to 10ml, which is 10% yeast solution.