CN105400652A - Method for rapidly preparing man-made pit mud through functional microbial group of high-yield butyric acid and caproic acid - Google Patents

Abstract

The invention relates to a method for rapidly preparing artificial pit mud by using Clostridium genus butyric acid and caproic acid functional microbial flora. The invention belongs to the technical field of brewing, and the technical problem to be solved by the present invention is to provide a method for rapidly preparing artificial pit mud by using functional microbial flora, and the cellar pool using the artificial pit mud can produce high-quality Luzhou-flavor liquor. The specific implementation of the present invention comprises the following steps: a, the cultivation of the fermented liquid of the functional microbial flora; b, the accumulation and fermentation of the initial cultivation pit mud for 20 to 25 days, and the temperature control is at 25 to 35°C; c, the accumulation and fermentation of the artificial pit mud for 30 to 35 days Days, you can get the finished product. The method of the invention uses the fermented liquid of functional microbial flora as one of the main inoculum, protects high-quality pit mud resources, can quickly obtain high-quality artificial pit mud, and improves the quality of liquor in new pits. The invention provides a new scheme for the preparation of new cellar mud, and has broad application prospects.

Description

A method for rapid preparation of artificial pit mud by using functional microbial flora with high production of butyric acid and caproic acid

technical field

The invention belongs to the technical field of brewing, and in particular relates to a method for rapidly preparing artificial pit mud by utilizing functional microbial flora with high production of butyric acid and caproic acid.

Background technique

In the production process of Luzhou-flavor liquor, the cellar is not only a fermentation vessel, but also the cellar mud is one of the main sources of wine-making functional microorganisms. Most of the microorganisms in the pit mud are anaerobic bacteria, and the butyric acid, caproic acid and fatty acid esters produced by their metabolism are the main flavor substances of Luzhou-flavor liquor, so the acid-producing ability of the pit mud directly determines The quality of the cellar pool ultimately affects the quality and body style of liquor. The acid-producing microorganisms in the pit mud are domesticated and gradually enriched during the production process. Therefore, there is a saying in the industry that "a hundred-year-old cellar produces good wine".

With the expansion of the scale of my country's liquor industry, various wine companies have built a considerable number of cellars, but the functional microorganisms in the cellars are not formed overnight, so the preparation of artificial cellar mud has become the main factor restricting the increase in the production of high-quality liquor Limiting factor. At present, the microorganisms in the preparation process of artificial pit mud mainly come from inoculated old high-quality pit mud. Due to the scarcity of high-quality pit mud, the inoculation amount of pit mud is limited, and finally the preparation time and quality of artificial pit mud are greatly prolonged. .

Contents of the invention

The technical problem to be solved by the present invention is to provide a functional microbial flora culture with high-efficiency production of butyric acid and caproic acid, which can be applied to the rapid preparation of artificial pit mud.

The invention provides a method for preparing artificial pit mud by using functional microbial flora. The inventive method comprises the following steps:

a, with semi-synthetic yellow water culture medium, anaerobic fermentation culture Clostridium genus produces butyric acid and hexanoic acid microorganisms respectively, then the culture solution is mixed to get pit mud to produce butyric acid, hexanoic acid functional microbial flora fermented liquid;

b. Mix the 1:1 mixture of yellow water and water with a pH value of 5.0, the fermentation broth of functional microbial flora, and high-quality pit mud according to the mass ratio of 4-5:1:1 to make a suspension;

c. Mix the yellow mud, pit sealing mud and Daqu powder according to the mass ratio of 9-12:6:1, add the suspension prepared in step b, and control the moisture content of the mixture between 45-50%;

d. Pile up the pit mud that has been mixed, pat it tightly, and seal it for anaerobic fermentation for 20-25 days to form the primary pit mud;

e. Mix the 1:1 mixture of yellow water and water with a pH value of 5.0, and the fermented liquid of functional microbial flora according to the mass ratio of 4 to 5:1 and mix evenly;

f. According to the mass ratio of 30-40:20:1, mix the yellow mud, primary pit mud and Daqu powder, add the suspension prepared in step e, mix well to make a pit mud mixture, and control the moisture content of the pit mud mixture at Between 45 and 50%;

g. Pat the pit mud mixture obtained in step f to the other side, seal and ferment for 30 to 35 days to become artificial pit mud.

Wherein, the high-yielding butyric acid and caproic acid functional microbial bacteria described in step a of the above method is at least one of Clostridium butyricum, Clostridium tyrobutyricum or Clostridium krusii.

Wherein, the formula of the semi-synthetic yellow water medium described in step a of the above method is: sodium acetate 4-6 g/L, ammonium sulfate 0.3-0.7 g/L, dipotassium hydrogen phosphate 0.2-0.6 g/L, magnesium sulfate 0.1～0.3g/L, yeast extract 0.5～1.5g/L, cysteine hydrochloride 0.3～0.7g/L, dissolve in water and yellow water, which account for half of the volume ratio, and the pH value is 5.5 to 6.5, get it after sterilization.

Wherein, the inoculum amount of the Clostridium genus butyric acid and caproic acid-producing microorganisms described in step a of the above method is 5-10%, the fermentation temperature is 33-37° C., and the time for anaerobic fermentation culture is 7 days.

Wherein, during the sealed anaerobic fermentation of the initial cultivation pit mud described in step d of the above method, the fermentation temperature of the pit mud core is controlled between 25°C and 35°C.

Wherein, during the sealed fermentation of the pit mud mixture described in step g of the above method, the core fermentation temperature is controlled between 25°C and 35°C.

Wherein, the fermented liquid obtained after the fermentation of each bacterial strain in step a of the above method is mixed in equal proportions to obtain a mixed fermented liquid.

It can be understood that the high-quality pit mud mentioned in step b of the above method is derived from the existing old pit pit mud that has been proved capable of producing high-quality liquor through production practice. Preferably, the high-quality pit mud is pit mud derived from an old pit over 100 years old.

The present invention also provides the artificial pit mud prepared by the above method.

The beneficial effect of the present invention is that: the method of the present invention utilizes the fermented liquid of functional microbial flora as one of the main inoculum, increases the initial content of functional microorganisms in the artificial pit mud, and simultaneously uses 1:1 yellow water with a pH value of 5 as the The main source of nutrients for microorganisms in artificial pit mud. In addition, a unique pit mud preparation method has been established, which greatly shortens the time for preparing artificial pit mud. It only takes 50 to 60 days to obtain high-quality artificial pit mud. At the same time, the method of the present invention reduces the inoculation amount of high-quality pit mud, and 60 to 70 parts of artificial pit mud can be obtained from 1 part of high-quality pit mud, which protects high-quality pit mud resources, and can quickly obtain high-quality artificial pit mud, which improves the quality of new pits. quality of liquor. The invention provides a new scheme for the preparation of new cellar mud, and has broad application prospects.

Instructions attached

Figure 1. Three samples of bottom mud were taken from each of the four pits. After the total DNA was extracted, high-throughput sequencing was used to analyze the bacterial community structure in the pit mud, and the results of principal component analysis (PCA) were performed on 12 samples.

detailed description

The specific implementation of the present invention will be further described below in conjunction with the examples, and the present invention is not limited to the scope of the examples.

The present invention uses functional microbial flora fermentation liquid as one of the main inoculum, which can increase the initial content of functional microorganisms in artificial pit mud, and greatly shorten the time for preparing artificial pit mud, and only need 50-60 days to obtain high-quality artificial pit mud. It is preferred that the microorganism used is at least one of Clostridium butyricum, Clostridium tyrobutyricum or Clostridium krusei.

In addition, the present invention also uses 1:1 yellow water with a pH value of 5 (1:1 yellow water in the present invention refers to an equal-volume mixture of yellow water and water, that is, yellow water after equal proportion dilution) as The main source of nutrition for microorganisms in artificial pit mud mainly utilizes carbon sources rich in lower alcohols and short-chain fatty acids. Studies have found that these carbon sources can be quickly utilized by pit mud microorganisms to accelerate the growth and reproduction of microorganisms. And the present invention creatively adjusts the pH value of the yellow water culture medium from about 3.5 to weakly acidic 5.0, so as to maintain an acidic environment and inhibit the growth of miscellaneous bacteria, and prevent overacid from inhibiting the activity of functional microorganisms. At the same time, according to the preliminary research of the present invention, the concentration of ethanol can be reduced to about 2.5% by dilution, which removes the inhibition of high-concentration ethanol on the target microorganisms of the present invention and other beneficial fermentation microorganisms. The above various measures are combined to reduce the inoculation amount of high-quality pit mud, protect high-quality pit mud resources, shorten the preparation time and improve the yield of artificial pit mud.

Concrete inventive method can be carried out in the following steps:

a. Cultivation of functional microbial flora

Functional microorganisms producing butyric acid and hexanoic acid were respectively grown in semi-synthetic yellow water medium (the formula of semi-synthetic yellow water medium was sodium acetate 4-6g/L, ammonium sulfate 0.3-0.7g/L, dipotassium hydrogen phosphate 0.2-0.6 g/L, magnesium sulfate 0.1~0.3g/L, yeast extract 0.5~1.5g/L, cysteine hydrochloride 0.3~0.7g/L, water and yellow water account for half by volume, and the pH value is 5.5 to 6.5, ready to use after sterilization), the inoculum size is 5 to 10%, the temperature is 33 to 37°C, and anaerobic culture is carried out for 7 days. Then, the culture liquid is mixed in equal proportions to obtain the fermentation liquid of the butyric acid and hexanoic acid functional microbial flora in the pit mud.

b. Mix 1:1 yellow water with a pH value of 5.0, functional microbial flora fermentation broth, and high-quality pit mud at a mass ratio of 4 to 5:1:1 to make a suspension.

c. Crush the blocky yellow mud and pit sealing mud into particles with a particle size of 1 to 3 cm, and mix the yellow mud, pit sealing mud and Daqu powder evenly according to the mass ratio of 9 to 12:6:1.

d. Add the suspension prepared in step b to the material pile prepared in step c, and control the moisture content of the material pile between 45% and 50%.

e. Pile the mixed pit mud into a conical shape, pat it tightly, and seal and ferment for 20 to 25 days to form the primary pit pit mud. During this period, the fermentation temperature of the pit mud core is controlled between 25 and 35°C.

f. Take 1:1 yellow water adjusted to pH 5.0, and functional microbial flora fermentation broth according to the mass ratio of 4-5:1 and mix evenly.

g. Crush the lumpy yellow mud and primary pit mud into particles with a particle size of 1 to 3 cm, and mix the yellow mud, primary pit mud and Daqu powder according to the mass ratio of 30 to 40:20:1.

h. Add the suspension prepared in step f to the material heap obtained in step g, and control the moisture content of the material heap between 45% and 50%.

i. Pile the processed pit mud into a conical shape, pat it tightly, and seal and ferment for 30 to 35 days to become artificial pit mud. During this period, the fermentation temperature of the pit mud core is controlled between 25 and 35°C.

Those skilled in the art can adopt the method of hierarchical expansion culture when preparing a large amount of functional microbial fermentation broth in step a, and finally carry out intermittent shaking culture in fermentors or airtight containers. These are all easily realized by those skilled in the art according to the prior art. In this field, the inoculum size refers to the ratio of the volume of the seed solution to the volume of the inoculated system.

In step a and step f, the temperature of the yellow water should be controlled between 30°C and 40°C in order to allow the microorganisms in the pit mud to grow as soon as possible.

In step b, high-quality cellar mud should come from cellars with high wine quality, preferably cellars with a cellar age of more than 100 years. The yellow mud selected in step c and step g should be selected from the farmland soil layer with a depth of 10-30 cm, in which phosphorus, potassium and humus are rich, which is conducive to the growth of pit mud microorganisms. In step c and step g, the Daqu powder can be commercially available conventional Daqu. In order to further improve the quality of pit mud, medium temperature Daqu is preferred.

The artificial pit mud prepared by the method of the present invention is an excellent artificial pit mud product, which can be used in the fermentation process of high-quality liquor.

The method of the present invention is described more specifically below with examples.

Embodiment one, adopt the method of the present invention to prepare artificial pit mud

1. Select the bacterial strain Clostridium butyricum (Clostridium butyricum, preservation number: ATCC19398) and Clostridium tyrobutyricum (Clostridium tyrobutyricum, preservation number: ATCC25755) with the ability to produce butyric acid and the bacterial strain Clostridium kluyveri (Clostridium kluyveri) with the ability to produce caproic acid , preservation number: ATCC8527) were inoculated and fermented in semi-synthetic yellow water medium in a 50L fermenter, and the formula of the used semi-synthetic yellow water medium was sodium acetate 5gg/L, ammonium sulfate 0.5gg/L, hydrogen phosphate Potassium 0.4gg/L, magnesium sulfate 0.2gg/L, yeast extract 1gg/L, cysteine hydrochloride 0.5gg/L, add 1:1 sterile yellow water (yellow water diluted with equal volume of water) to 1000mL , adjust the pH to 6.0, and sterilize at 115°C for 20 minutes. The liquid volume of the fermentation tank is 20L, the inoculation amount is 10%, the temperature is 35°C, anaerobic fermentation is carried out for 7 days, and the culture liquid of each strain is mixed in equal proportions to obtain the functional microbial flora fermentation liquid.

2. Take 60kg of 100-year-old cellar mud, 60kg of fermented liquid of functional microbial flora, and 300kg of 1:1 yellow water with a pH value of 5.0 (yellow water diluted with an equal volume of water), and stir it into a suspension.

3. Take 300kg of crushed pit sealing mud, 450kg of Datian yellow mud, and 50kg of medium-temperature Daqu, and mix the three evenly, with a water content of about 25%.

4. Add the suspension in step 1 to the solid material pile, and mix thoroughly while adding, so that the soil particles, fungi, and koji powder are evenly mixed, and the water content is about 50%.

5. After the materials are piled tightly, seal and ferment for 25 days, and control the temperature between 25 and 35 to obtain 1100 kg of primary pit mud with a water content of about 46%.

6. Add 2,200kg of yellow mud (with a water content of about 20%), 55kg of koji powder, 200kg of functional microbial flora fermentation liquid, and 800kg of 1:1 yellow water with a pH value of 5.0 to the primary pit mud.

7. After the materials are piled tightly, seal and ferment for 35 days, and control the temperature between 25 and 35 to obtain about 4000 kg of artificial pit mud with a water content of about 45%.

8. Spread artificial pit mud in the newly built cellar pool, and put in grain grains to ferment to produce wine.

The cellar pool (artificial old cellar), the new cellar (yellow mud, cellar bottom mud, and sealing cellar mud mixed) of the artificial cellar mud prepared by the inventive method, the 30-year cellar pond, and the 50-year cellar pond, in the same raw material Under the conditions of production and process, Luzhou-flavor liquor was produced, and the content of main aroma substances in liquor was compared. The results are shown in Table 1. Three samples of pit bottom mud were taken from each of the four cellar ponds. After extracting the total DNA, high-throughput sequencing was used to analyze the bacterial community structure in the pit mud, and principal component analysis (PCA) was performed on 12 samples. The results are shown in Figure 1 .

Table 1 Contents of main aroma substances in wines from each cellar (unit: g/L)

ethyl hexanoate ethyl butyrate ethyl acetate caproic acid butyric acid Acetic acid Xinjiao 1.90 0.24 1.22 0.41 0.10 0.27 Artificial old cellar 2.27 0.35 1.39 0.63 0.13 0.30 30 years cellar 2.35 0.38 1.53 0.65 0.18 0.33 50 years cellar 3.55 0.42 2.08 0.78 0.17 0.48

As can be seen from Table 1, the quality of the Luzhou-flavor liquor produced by the artificial old pits using the artificial pit mud prepared by the present invention is obviously higher than that of the new pits, and the content of main aroma substances is close to that of the 30-year-old pits, and still has a certain level with the 50-year-old pits. gap. It can also be seen from the cluster analysis results in Figure 1 that the samples from the 4 cellars clustered into 3 categories in the PCA graph. Cellars vary greatly. In addition, this method can obtain 60-70 parts of artificial pit mud for every 1 part of high-quality pit mud. Compared with the prior art, for example, 1 part of high-quality pit mud disclosed in patent document CN102181342B produces 7-8 parts of artificial pit mud, and the inoculum amount of high-quality pit mud is reduced by nearly 90%, which protects the resources of high-quality pit mud.

Claims (7)

1. Utilize the method for fast preparation of artificial pit mud by the functional microbial flora of high yield butyric acid and hexanoic acid, it is characterized in that comprising the following steps: a, with semi-synthetic yellow water culture medium, anaerobic fermentation culture Clostridium genus produces butyric acid and hexanoic acid microorganisms respectively, then the culture solution is mixed to get pit mud to produce butyric acid, hexanoic acid functional microbial flora fermented liquid; b. Mix the 1:1 mixture of yellow water and water with a pH value of 5.0, the fermentation broth of functional microbial flora, and high-quality pit mud according to the mass ratio of 4-5:1:1 to make a suspension; c. Mix the yellow mud, pit sealing mud and Daqu powder according to the mass ratio of 9-12:6:1, add the suspension prepared in step b, and control the moisture content of the mixture between 45-50%; d. Pile up the pit mud that has been mixed, pat it tightly, and seal it for anaerobic fermentation for 20-25 days to form the primary pit mud; e. Mix the 1:1 mixture of yellow water and water with a pH value of 5.0, and the fermentation broth of functional microbial flora according to the mass ratio of 4 to 5:1 to make a suspension; f. According to the mass ratio of 30-40:20:1, mix the yellow mud, primary pit mud and Daqu powder, add the suspension prepared in step e, mix well to make a pit mud mixture, and control the moisture content of the pit mud mixture at Between 45 and 50%; g. Pat the pit mud mixture obtained in step f to the other side, seal and ferment for 30 to 35 days to become artificial pit mud. 2. The method according to claim 1, characterized in that: the high-yield butyric acid and hexanoic acid functional microbial bacteria in step a are at least one of Clostridium butyricum, Clostridium tyrobutyricum or Clostridium kruseri. 3. The method according to claim 1, wherein the formula of the semi-synthetic yellow water medium described in step a is: sodium acetate 4～6g/L, ammonium sulfate 0.3～0.7g/L, dipotassium hydrogen phosphate 0.2～0.6g/L, magnesium sulfate 0.1～0.3g/L, yeast extract 0.5～1.5g/L, cysteine hydrochloride 0.3～0.7g/L, soluble in water and yellow water according to volume ratio Half of the mixed diluent has a pH value of 5.5 to 6.5 and can be obtained after sterilization. 4. The method according to claim 1, characterized in that: the Clostridium genus described in step a produces butyric acid, the inoculum size of caproic acid microorganisms is 5-10%, fermentation temperature is 33-37 DEG C, anaerobic The time of fermentation and cultivation is 7 days. 5. The method according to claim 1, characterized in that: during the sealed anaerobic fermentation of the initial cultivation pit mud in step d, the fermentation temperature of the pit mud core is controlled between 25°C and 35°C. 6. The method according to claim 1, characterized in that: during the sealed fermentation of the pit mud mixture in step g, the core fermentation temperature is controlled at 25-35°C. 7. The artificial pit mud prepared by the method according to any one of claims 1 to 6.