CN115305164A

CN115305164A - High-microbial-activity pit mud culture solution and preparation method thereof

Info

Publication number: CN115305164A
Application number: CN202210849721.4A
Authority: CN
Inventors: 张梦梦; 吴玉轩; 李小杰; 赵巧珍; 崔新莹; 商海林; 石国亮
Original assignee: Jinan Baotu Spring Wine Co ltd
Current assignee: Jinan Baotu Spring Wine Co ltd
Priority date: 2022-07-19
Filing date: 2022-07-19
Publication date: 2022-11-08

Abstract

The invention relates to a high-microbial-activity pit mud culture solution and a preparation method thereof, belonging to the technical field of pit mud culture solutions. The technical problem that in the prior art, a single caproic acid bacterium is cultured and added into a cellar, and the expected effect on the quality improvement of the strong aromatic white spirit is not achieved is solved. The high microbial activity pit mud culture solution provided by the invention comprises the raw materials of 11wt% of old pit mud, 4.6wt% of bran hydrolysate, 2wt% of monascus liquid, 10.5wt% of yellow water, 5wt% of ethanol and 66.9wt% of purified water. The cellar mud culture solution can improve the quality of cellar mud and the yield and quality of highly aromatic white spirit.

Description

High-microbial-activity pit mud culture solution and preparation method thereof

Technical Field

The invention belongs to the technical field of pit mud culture solutions, and particularly relates to a high-microbial-activity pit mud culture solution and a preparation method thereof.

Background

The key reason for brewing good wine is that organic acids, alcohols, esters, aldehydes, carbon, nitrogen and other organic matters and inorganic matters produced by fermented grains are soaked and permeated into pit mud in the long-term continuous brewing fermentation process, and some anaerobic functional bacteria and facultative functional bacteria related to aroma production are gradually enriched, cultured, naturally eliminated and domesticated to form a microbial system of the strong aromatic white wine.

Caproic acid bacteria are very important acid-producing microorganisms in the production of strong aromatic Chinese spirits, and caproic acid produced by the metabolism of caproic acid bacteria and alcohol produced by fermentation of Daqu produce ethyl caproate, which is the main fragrant component of the strong aromatic Daqu liquor. The caproic acid bacteria culture solution can be widely applied to cellar filling, cellar pool maintenance, artificial cellar mud culture, esterification liquid production and the like of the strong aromatic Daqu liquor so as to improve and enhance the quality of the strong aromatic Daqu liquor. However, both the current research and the practical application show that the quality improvement of the strong aromatic Chinese spirits is not expected by adding single caproic acid bacteria culture into the cellar. The reason is that the environment in the pit is originally a complex microbial ecology, and the inoculation of a single strain is difficult to be rapidly integrated and play a role.

Disclosure of Invention

The invention aims to solve the technical problem that the quality of the Luzhou-flavor liquor is not improved by adding single caproic acid bacteria into a pit and has an expected effect in the prior art, and provides a high-microbial-activity pit mud culture solution and a preparation method thereof.

In order to realize the purpose, the following technical scheme is adopted:

the high microbial activity pit mud culture solution provided by the invention comprises the raw materials of 11wt% of old pit mud, 4.6wt% of bran hydrolysate, 2wt% of monascus liquid, 10.5wt% of yellow water, 5wt% of ethanol and 66.9wt% of purified water.

Preferably, the old cellar mud is white spirit cellar mud for more than ten years, the water content is between 40% and 50%, the pH is between 5 and 6.5, the content of ethyl caproate in the produced wine is more than 2.5g/L, and the content of ethyl acetate is more than 2.0 g/L.

Preferably, the bran hydrolysate is prepared by the following method: uniformly stirring 36-38% of hydrochloric acid, purified water and bran according to a mass ratio of 5.

Preferably, the monascus esterification capacity of the monascus bacterial liquid is 50 mg/g.100 h.

Preferably, the yellow water is the yellow water produced by the old cellar mud sampling cellar.

The invention also provides a preparation method of the high microbial activity pit mud culture solution, which comprises the following steps:

taking the components according to the proportion, heating purified water to more than 90 ℃, naturally airing to 40 ℃, adding monascus liquid, yellow water and ethanol, uniformly stirring, adding bran hydrolysate at 37-39 ℃, uniformly stirring and mixing, adding old pit mud at 35-37 ℃, uniformly stirring, sealing, and culturing at 37.5 ℃ for more than 30 days to obtain the high-microbial-activity pit mud culture solution.

Preferably, the old cellar mud is kneaded into a suspension before being added into the old cellar mud.

Preferably, the sealing temperature is 35 to 37 ℃.

Preferably, the stirring is performed once every morning and afternoon during the 30-day culture at 37.5 ℃.

Compared with the prior art, the invention has the following beneficial effects:

the pit mud culture solution disclosed by the invention adopts the optimal proportion and the optimal process, and is beneficial to improving the quality of pit mud and the content and activity of beneficial microorganisms in a pit and maintaining the water content of pit mud to delay the aging of pit mud when the pit mud is manufactured and the pit is maintained.

The pit mud culture solution can greatly increase the generation of ethyl caproate with main fragrance in the strong aromatic Chinese spirits, improve the excellent quality of the strong aromatic Chinese spirits, enrich the effect of the spirits and endow the Chinese spirits with tasty and refreshing flavor.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

In fig. 1, a is a DGGE spectrum of pit mud after curing with the pit mud culture solution of comparative example 1, and b is a DGGE spectrum of pit mud before oxygen curing.

FIG. 2 shows the results of a single-factor experiment of the addition of old pit mud to caproic acid bacteria.

FIG. 3 shows the result of single factor experiment of adding amount of yellow water to caproic acid bacteria.

FIG. 4 shows the results of single-factor experiments on caproic acid bacteria by adding bran hydrolysate.

FIG. 5 shows the results of a single-factor experiment of the culture temperature on caproic acid bacteria.

Detailed Description

For a further understanding of the invention, the following description of the preferred embodiments of the invention is provided, but it is to be understood that the description is only intended to illustrate further features and advantages of the invention, and not to limit the scope of the claims.

The aged pit mud is preferably white wine pit mud for over ten years, the water content is between 40 and 50 percent, and the pH value is between 5 and 6.5; the content of ethyl caproate in the produced wine is more than 2.5g/L, and the content of ethyl acetate is more than 2.0g/L; sensory: the color is black; the hand feeling is wet and smooth; smells hydrogen sulfide odor and the fragrance of ethyl caproate.

The bran hydrolysate is prepared by the following method: uniformly stirring hydrochloric acid, purified water and bran according to a mass ratio of 5.

The monascus esterifying capacity of the monascus ruber liquid is 50 mg/g-100 h.

The yellow water is preferably the yellow water produced by the old pit mud sampling pit.

The preparation method of the pit mud culture solution with high microbial activity comprises the following steps:

taking the components according to the proportion, heating purified water to more than 90 ℃, naturally airing to 40 ℃, adding monascus liquid, yellow water and ethanol, uniformly stirring, adding bran hydrolysate at 37-39 ℃, uniformly stirring, adding aged pit mud which is kneaded into turbid liquid at 35-37 ℃, uniformly stirring, sealing at 35-37 ℃, culturing for more than 30 days at 37.5 ℃, and respectively stirring once every morning and afternoon during the process to obtain the high-microbial-activity pit mud culture solution.

The determination process of the raw material proportion and the culture condition of the high microbial activity pit mud culture solution comprises the steps of taking raw materials consisting of 10wt% of old pit mud, 5wt% of bran hydrolysate, 2wt% of monascus liquid, 8wt% of yellow water, 5wt% of ethanol and 70wt% of purified water as basic proportion, and taking the raw materials as basic culture conditions at 35 ℃; tests prove that the pit mud culture solution has the advantages that the caproic acid content is 5.453g/L, the acetic acid content is 4.506g/L, and the butyric acid content is 5.254g/L, and sufficient precursor substances are supplied for generating ethyl caproate and ethyl butyrate (the ethyl caproate and the ethyl acetate are used as main fragrance in the strong aromatic white spirit and form unique style characteristics of strong pit fragrance, sweet taste, good taste and the like of the strong aromatic white spirit together with a proper amount of ethyl butyrate), the pit mud culture solution is helpful for improving the quality of pit mud, stabilizing the flavor of the strong aromatic white spirit and playing an important role in improving the good yield of the strong aromatic white spirit, the pit mud culture solution is used for maintaining an aged pit pool and a new pit pool, the water content and the water permeability of pit mud can be maintained, the content of various fragrant substances in the strong aromatic white spirit can be coordinated, the yield and the quality of the strong aromatic white spirit can be improved, and the DGE number of the pit mud culture solution can be obviously increased after two rounds of the pit mud culture solution through experimental verification, the experiment proves that the variety and the number of strains are increased, and the important significance of the pit mud maintenance of the pit mud protection solution is realized in the two rounds of the pit maintenance, the method is determined by adopting a single-factor test and an orthogonal test.

And (3) determining a value taking point of each influencing factor when the maximum value of the caproic acid value produced by the pit mud culture solution formula is determined through a single-factor test. Four single-factor researches are carried out, wherein the four single-factor researches are respectively the addition amount of the aged pit mud, the addition amount of yellow water, the addition amount of bran hydrolysate and the change of the culture temperature, and the experimental results are shown in figures 2-5. As can be seen from FIGS. 2-5, the caproic acid bacteria production value reaches a maximum of 5.5g/L when the old cellar mud is added in an amount of 11wt%, and the caproic acid production value begins to decrease when the old cellar mud is added in an amount of less than or more than 11 wt%; when the addition amount of the yellow water is 7-10 wt%, the caproic acid production value is gradually increased, and when the addition amount of the yellow water is 10wt%, the caproic acid production value reaches the peak, is 5.5g/L, and exceeds 10wt%, the caproic acid production value begins to be reduced; the addition amount of the bran hydrolysate at the early stage is gradually increased, the caproic acid production value at the later stage is gradually reduced, the production value is unchanged when the content is 4.5wt% -5.0wt%, the optimal addition amount is between the two, and further research needs to be carried out in an orthogonal test; the caproic acid yield reaches the highest value when the culture temperature is 37 ℃; the caproic acid production is reduced below or above 37 ℃.

The rough range of each factor when the pit mud culture solution produces more caproic acid is preliminarily determined through a single-factor test, and the optimal value of each factor when the caproic acid production value is maximum is determined through an orthogonal test (four-factor three-level, 3.1 version of orthogonal test assistant); the factors and levels of the orthogonality test are shown in table 1, and the results are shown in table 2.

TABLE 1 factors and levels of orthogonal experiments

In the column (b)	1	2	3	4
					Factors of the fact	Yellow water (wt%)	Laojiao mud (wt%)	Bran hydrolysate (wt%)	Culture temperature (. Degree. C.)
1	9.5	10.5	4.6	36.5
					2	9.5	11	4.75	37
3	9.5	11.5	4.9	37.5
					4	10	10.5	4.75	37.5
5	10	11	4.9	36.5
					6	10	11.5	4.6	37
7	10.5	10.5	4.9	37
					8	10.5	11	4.6	37.5
9	10.5	11.5	4.75	37.5

Table 2 results of orthogonal experiments

As can be seen from Table 2, 9 experiments are carried out, the caproic acid production value varies from 4.872 g/L to 6.277g/L, the optimal factor ratio is A3B2C1D3 of experiment 8, the caproic acid production value is 6.277g/L, and the extreme difference analysis can obtain that the biggest influence on the caproic acid production value in the four factors of the experiment is the yellow water addition amount, the influence factors are respectively A > B > C > D from large to small, the caproic acid production value of the basic formula is 5.453g/L, and the caproic acid production value of the A3B2C1D3 is 6.277g/L. Therefore, the optimal formula of the pit mud culture solution is A3B2C1D3, namely: 11wt% of aged pit mud, 4.6wt% of bran hydrolysate, 2wt% of monascus liquid, 10.5wt% of yellow water, 5wt% of ethanol and 66.9% of purified water; culturing at 37.5 deg.C for more than 30 days.

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail with reference to the following embodiments.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.

The present invention is further illustrated by the following examples.

Comparative example 1

The pit mud culture solution is prepared from 10wt% of old pit mud, 5wt% of bran hydrolysate, 2wt% of monascus liquid, 8wt% of yellow water, 5wt% of ethanol and 70wt% of purified water;

wherein the old cellar mud is 15 years old white spirit cellar mud, the water content is 45%, and the pH value is 6.0; the content of ethyl caproate in the produced wine is 3.0g/L, and the content of ethyl acetate in the produced wine is 2.0g/L; sensory: the color is black; the hand feeling is wet and smooth; smells the odor of hydrogen sulfide and the fragrance of ethyl caproate;

the bran hydrolysate is prepared by the following method: uniformly stirring 37% of hydrochloric acid, purified water and bran according to a mass ratio of 5;

the monascus esterifying power of the monascus liquid is 50 mg/g.100 h;

the yellow water is the yellow water produced by the old pit mud sampling pit.

The preparation method of the pit mud culture solution comprises the following steps:

taking the components according to the proportion, heating purified water to over 90 ℃, naturally airing to 40 ℃, adding monascus liquid, yellow water and ethanol, uniformly stirring, adding bran hydrolysate at 37-39 ℃, uniformly stirring and mixing, adding aged pit mud which is kneaded into suspension at 35-37 ℃, uniformly stirring, sealing at 35-37 ℃, culturing for 32 days at 35 ℃, stirring once in the morning and afternoon every day, and stirring for 30min every time to obtain pit mud culture solution.

Example 1

The high microbial activity pit mud culture solution comprises the raw materials of 11wt% of old pit mud, 4.6wt% of bran hydrolysate, 2wt% of monascus liquid, 10.5wt% of yellow water, 5wt% of ethanol and 66.9% of purified water;

wherein the old cellar mud is 15 years old white spirit cellar mud, the water content is 45%, and the pH value is 6.0; the content of ethyl caproate in the produced wine is 3.0g/L, and the content of ethyl acetate is 2.0g/L; sensory: the color is black; the hand feeling is moist and oily; smells the odor of hydrogen sulfide and the fragrance of ethyl caproate;

the monascus esterifying power of the monascus liquid is 50 mg/g-100 h;

the yellow water is the yellow water produced by the old pit mud sampling pit.

taking the components according to the proportion, heating purified water to more than 90 ℃, naturally airing to 40 ℃, adding monascus liquid, yellow water and ethanol, uniformly stirring, adding bran hydrolysate at 37-39 ℃, uniformly stirring, adding aged pit mud which is kneaded into turbid liquid at 35-37 ℃, uniformly stirring, sealing at 35-37 ℃, culturing for 32 days at 37.5 ℃, stirring in the morning and afternoon every day, and stirring for 30min each time to obtain the high-microbial-activity pit mud culture solution.

And (3) detecting the performance of the pit mud culture solution prepared in the comparative example 1.

1.1 adding 2mL of pit mud culture solution into 10mL of alcohol (analytically pure), filtering with filter paper, filtering the filtrate with an inlet membrane (Jinteng brand, nylon No. 66, pore diameter 0.45 μm), and analyzing 4-5mL of filtrate with gas chromatography. The test results are shown in table 1.

TABLE 1 pit mud culture solution Total analysis data (g/L)

Numbering	Ethyl acetate	Butyric acid ethyl ester	Hexanoic acid ethyl ester	Lactic acid ethyl ester	Acetic acid	Butyric acid	Hexanoic acid
								Comparative example 1	0.176	0.058	0.137	0.354	4.506	5.254	5.453

As can be seen from Table 1, the pit mud culture solution prepared in comparative example 1 had a caproic acid content of 5.453g/L, an acetic acid content of 4.506g/L and a butyric acid content of 5.254g/L.

1.2 after the fermented grains in the cellar are discharged from the cellar and cleaned, spraying 10L of cellar mud culture solution onto the cellar mud on the wall of the cellar, adding the fermented grains, and starting a new fermentation period, wherein the fermentation period is about 70 days. After two fermentation periods, the cellar mud of taking this cellar for storing things pond pool wall carries out the physical and chemical analysis to carry out the contrast with the physical and chemical analysis data of cellar for storing things mud before the maintenance, the data of physical and chemical analysis data have: water, PH, available potassium, available phosphorus, ammoniacal nitrogen humus. The results are shown in Table 2.

TABLE 2 data of pit mud culture solution before and after pit maintenance

As can be seen from Table 2, the pit mud culture solution is used for maintaining the pit, and the quality change of the pit mud before and after the maintenance is analyzed: the pit mud water content before curing is 45.75%, the pit mud water content after curing is 40.94%, and the pit mud water content after normal curing is 40-50%, which indicates that the pit water content before and after curing is a normal value. The pH of the pit mud is increased from 4.42 to 6.76 before and after maintenance, and the pit mud is weakly acidic. The content of available potassium, available phosphorus and ammonia nitrogen is greatly increased; the content of humus changes smoothly. The pit mud culture solution has obvious effect on improving the quality of the pit.

1.3 extracting DNA of the pit mud cured in the step 1.2 by using a soil kit (EZUP column type soil DNA extraction kit of biological engineering (Shanghai) Co., ltd.), performing PCR amplification on the pit mud by referring to the specification of the soil kit according to the extraction method, wherein the PCR amplification conditions are shown in Table 3, and after a 5 mu LPCR amplification product is taken and is subjected to 3% agarose gel electrophoresis detection to verify that the DNA is extracted, the PCR amplification is successful, and strips are free of pollution, the DNA is used for subsequent DGGE analysis.

TABLE 3PCR amplification conditions

1.4 before the experiment, the gel glass plate is cleaned and dried, and is arranged on a gel rack according to the gel thickness. Preparing 40% acrylamide gel (acrylamide/bis) according to Table 4, and preparing a deforming agent according to Table 5; adding TEMED18 μ L and 10 APS (ammonium persulfate) 80 μ L into 50% and 60% of denaturant respectively before preparing for injecting glue, mixing, injecting glue, rotating the disc at constant speed during injecting glue, allowing the syringe needle to cling to the two glass plates, slowly inserting the comb after injecting, putting the syringe needle into clear water, rotating in reverse direction, cleaning the syringe and the needle tube to prevent the gel from coagulating in the tube, and removing the comb after the gel is completely coagulated (about 4 h). Adding the prepared 1 XTAE buffer solution into the 'fill' position of the gel electrophoresis tank, covering the lid, opening the gel electrophoresis switch, turning on the 'heat' and 'pump', setting the temperature, and raising the temperature of the buffer solution to 60 ℃. After reaching the temperature, the gel plate is placed in an electrophoresis tank, the PCR amplification product in 1.3 is added into a lane, a voltage-stabilized power supply is connected, the voltage is firstly adjusted to 20v, electrophoresis is carried out for 20min (the lane is washed), then the voltage is adjusted to 80v, and electrophoresis is carried out for 12h. Turning off the voltage transformer and the gel heater, placing the gel in deionized water containing gel stain in the dark for 1h, then washing the gel for 5min, and finally placing the gel in a gel imaging system for imaging observation, wherein the result is shown in figure 1.

TABLE 4 acrylamide gel formulation at 40%

TABLE 5 formulation of denaturants

Gel concentration	50％	60％
			Formamide	20mL	24mL
Urea	21g	25.2g
			40％acrylamide/bis	20mL	20mL
50×TAEbuffer	2mL	2mL

As can be seen from FIG. 1, after two curing cycles of the pit by the pit mud culture solution, the number of bands of DGGE is obviously increased, and the types and the number of strains are increased. Can show that the pit mud maintenance liquid has important significance for the maintenance of the pit.

The pit mud culture solutions of example 1 and comparative example 1 were examined.

After fermented grains in the pit are discharged from the pit and cleaned, 10L of pit mud culture solution of comparative example 1 and example 1 is sprinkled on pit mud on the wall of the pit, then esterification solution (physical and chemical analysis and quality evaluation are shown in tables 1 and 2) is added to start a new fermentation period, one fermentation period is about 70 days, and after three fermentation periods, raw wine in each fermentation period is taken for physical and chemical analysis and quality evaluation. The results are shown in tables 6 and 7.

Table 6 analysis of raw wine data of pit mud broth culture of example 1 and comparative example 1

TABLE 7 evaluation comments on wine base cultured from esterified liquid and pit mud culture liquid of example 1

In conclusion, in the comparative example 1, the pit mud culture solution is determined to play an important role in maintaining and improving the quality of the pit through gas-phase analysis of the pit mud culture solution, physicochemical data of pit mud before and after maintenance and microbial map change. The pit mud culture solution of the comparative example 1 is rich in rich microbial flora, so that the enrichment of various microorganisms in the pit mud is realized, the microorganisms can rapidly grow in the strong-flavor pit environment, and the micro-ecological environment in the pit is coordinated. The quality of the pit mud is greatly improved. When the daily pit mud is maintained, the prepared pit mud culture solution is added, so that the water content and the water permeability of the pit mud can be kept, the content of various flavor substances in the strong aromatic Chinese spirits is coordinated, and the yield and the quality of the strong aromatic Chinese spirits are improved.

The component ratio and the cultivation temperature of embodiment 1 make cellar mud culture solution technology realize the optimal ratio, add cellar mud culture solution when making cellar mud and maintenance cellar for storing things pond and help promoting cellar mud quality and cellar for storing things pond in useful microorganism's content and activity. The water content of the pit mud is kept, the aging of the pit mud is delayed, the generation of ethyl caproate with main fragrance in the strong aromatic Chinese spirits can be greatly increased, and the excellent quality of the strong aromatic Chinese spirits is improved. The wine body function is enriched, and the white wine is endowed with a refreshing flavor.

It should be understood that the above-described embodiments are merely examples for clarity of description and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. It need not be, and cannot be exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims

1. The high microbial activity pit mud culture solution is characterized in that the raw materials comprise 11wt% of old pit mud, 4.6wt% of bran hydrolysate, 2wt% of monascus liquid, 10.5wt% of yellow water, 5wt% of ethanol and 66.9wt% of purified water.

2. The cellar mud culture solution with high microbial activity as claimed in claim 1, wherein the old cellar mud is white spirit cellar mud for more than ten years, the water content is between 40% and 50%, the pH is between 5 and 6.5, the content of ethyl hexanoate in the produced wine is more than 2.5g/L, and the content of ethyl acetate in the produced wine is more than 2.0 g/L.

3. The pit mud culture solution with high microbial activity as claimed in claim 1, wherein the bran hydrolysate is prepared by the following method: uniformly stirring 36-38% of hydrochloric acid, purified water and bran according to a mass ratio of 5.

4. The high microbial activity pit mud culture solution of claim 1, characterized in that the monascus esterifying ability of the monascus ruber liquid is 50 mg/g-100 h.

5. The high microbial activity pit mud culture solution of claim 1, wherein the yellow water is produced by the old pit mud sampling pit.

6. The method for preparing the pit mud culture solution with high microbial activity as claimed in any one of claims 1 to 5, which is characterized by comprising the following steps:

taking the components according to the proportion, heating purified water to above 90 ℃, naturally airing to 40 ℃, adding monascus liquid, yellow water and ethanol, uniformly stirring, adding bran hydrolysate at 37-39 ℃, uniformly stirring and mixing, adding old pit mud at 35-37 ℃, uniformly stirring, sealing, and culturing at 37.5 ℃ for more than 30 days to obtain the high-microbial-activity pit mud culture solution.

7. The method for preparing cellar mud culture solution with high microbial activity as claimed in claim 6, wherein the old cellar mud is kneaded into suspension before being added.

8. The method for preparing the pit mud culture solution with high microbial activity as claimed in claim 6, wherein the sealing temperature is 35-37 ℃.

9. The method for preparing cellar mud culture fluid with high microbial activity as claimed in claim 6, wherein stirring is performed once in the morning and in the afternoon of each day during the 30-day culture at 37.5 ℃.