阅读说明：本技术 一种高产四甲基吡嗪的菌株及其制备方法 (Bacterial strain for high yield of tetramethylpyrazine and preparation method thereof ) 是由 江东材 朱和琴 彭礼群 罗陟 于 2021-07-23 设计创作，主要内容包括：本发明公开了一种高产四甲基吡嗪的菌株及其制备方法,其中,所述高产四甲基吡嗪的菌株为地衣芽孢杆菌,分类命名为Bacillus licheniformis xf-4-1,保藏编号为CCTCC NO:M 2021664,形态为棒状,细胞直径(0.8～1.0)μm×(1.5～2.0)μm,菌落颜色为乳白色,菌落边缘呈不规则状,有黏液,革兰氏阳性。本发明高产四甲基吡嗪的菌株能够应用于柔雅香型白酒的生产,得到的白酒中乙偶姻和四甲基吡嗪含量分别为268.1mg/L和5.6mg/L；另外本发明方法得到的菌株生长旺盛、无污染,且能够解决菌株在培养过程中出现的自溶现象。(The invention discloses a bacterial strain for high yield of tetramethylpyrazine and a preparation method thereof, wherein the bacterial strain for high yield of tetramethylpyrazine is Bacillus licheniformis, the bacterial strain is classified and named as Bacillus licheniformis xf-4-1, the preservation number is CCTCC NO: M2021664, the shape is rod-shaped, the cell diameter is (0.8-1.0) Mumx (1.5-2.0) Mum, the colony color is milky white, the colony edge is irregular, mucus exists, and gram is positive. The bacterial strain for high yield of the tetramethylpyrazine can be applied to production of the soft and elegant flavor type white spirit, and the contents of acetoin and tetramethylpyrazine in the obtained white spirit are 268.1mg/L and 5.6mg/L respectively; in addition, the strain obtained by the method of the invention has vigorous growth and no pollution, and can solve the autolysis phenomenon of the strain in the culture process.)

1. The bacterial strain for high yield of tetramethylpyrazine is characterized in that Bacillus licheniformis is classified and named as Bacillus licheniformis xf-4-1, and the preservation number is CCTCC NO: M2021664.

2. The tetramethylpyrazine-highly yielding strain according to claim 1, wherein the bacteriological characteristics of the Bacillus licheniformis strain are: the form is a rod, and the cell diameter is (0.8 to 1.0) μm x (1.5 to 2.0) μm.

3. The method for preparing the tetramethylpyrazine-highly-producing strain according to claim 1 or 2, wherein the enhanced Daqu is prepared after the precursor acetoin is accumulated by microbial fermentation to obtain the acetoin-highly-producing strain;

the method for fermenting the microorganisms comprises the following steps:

inoculating the first-stage seed culture solution into a screening culture medium for culture, leaching and distilling to obtain fermented grains containing precursor acetoin.

4. The method for preparing the strain according to claim 3, wherein the primary seed culture solution is made up to 1L by distilled water, and comprises: 8-15 g/L of peptone, 3-8 g/L of yeast powder, 8-15 g/L of NaCl and 6.8-7.5 of pH value;

preferably 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl and 7.0 of pH value.

5. The method for producing a strain according to claim 3, wherein the screening medium is wheat flour having a moisture content of 30 to 50%, and further wheat flour having a moisture content of 30 to 35%.

6. The method for producing the strain according to claim 3, wherein the culture conditions are: 20-60 ℃ for 28-32 days;

the leaching reagent is 40-90% of absolute ethyl alcohol, the leaching time is 20-30 hours, and further is 60% of absolute ethyl alcohol, and the leaching time is 24 hours;

the fermented grains are: the mass-to-volume ratio of the absolute ethyl alcohol is 1: 1-3, and the preferred mass-to-volume ratio is 1: 2.

7. The method for preparing the strain according to claim 3, wherein the method for preparing the enhanced yeast for making hard liquor comprises the following steps:

inoculating the high-yield acetoin strain to a secondary seed culture medium for culture, and mixing with wheat flour to obtain the acetoin strain.

8. The method for preparing the strain according to claim 7, wherein the secondary seed culture medium is made up to 1L by distilled water, and comprises: peptone 20-40 g/L, yeast powder 5-15 g/L, glucose 40-60 g/L, diammonium hydrogen phosphate 20-40 g/L, pH value 6.8-7.2,

preferably 30g/L of peptone, 10g/L of yeast powder, 50g/L of glucose and 30g/L of diammonium hydrogen phosphate, and the pH value is 7.0.

9. The method for preparing the strain according to claim 7, wherein the culture conditions are 25-32 ℃ for 2-4 days, preferably 30 ℃ for 3 days;

during mixing, the volume ratio of the secondary seed culture medium after the strain culture to the mixed water is 1: 500; the mass ratio of the secondary seed culture medium after the strain culture to the wheat flour after being mixed with water is 0.1-2 per mill, and preferably 1 per mill.

10. Use of the tetramethylpyrazine-highly yielding strain according to claim 1 or 2 in wine production.

Technical Field

The invention relates to the technical field of biology, and particularly relates to a bacterial strain for high yield of tetramethylpyrazine and a preparation method thereof.

Background

The elegant and soft flavor type white spirit is a nationwide pioneer white spirit product of Symphonic city Syfu wine industry GmbH in Sichuan province, has the characteristics of colorless transparency, harmonious thick sauce, elegant fragrance, mellowness, sweetness, plump wine body, clean and refreshing aftertaste, and typical multi-grain and fragrant style, and is deeply loved by consumers since the product is on the market. Researches show that the traditional Chinese fermented food white spirit contains a certain amount of pyrazine substances, has aromaticity, and has the effects of expanding blood vessels, improving blood circulation and protecting liver, wherein the content of tetramethylpyrazine is the highest.

Tetramethylpyrazine (2, 3, 5, 6-tetramethylpyrazine) is a common heterocyclic nitrogen-containing compound, has the fragrance of roasted peanuts, hazelnuts and cocoa, is commonly used for blending essence of roasted foods, cold drinks, meat, dairy products, cigarettes and the like, and has been proved to have pharmacological action for treating cardiovascular and cerebrovascular diseases and preventive action on cisplatin-induced oxidative stress, apoptosis and nephrotoxicity by being used as a main active alkaloid component of rhizomes of a Chinese medicinal material Ligusticum wallichii (Ligusticum wallichi) except for application as a food flavor additive. With the progress of pharmacological research, the calcium ion antagonist is widely applied to clinic as a novel calcium ion antagonist, is a bulk drug for circulation, central nervous system, respiration and other system related diseases, and is widely applied to treatment of cardiovascular and cerebrovascular diseases, respiratory system diseases, glomerular diseases and the like. The tetramethylpyrazine in the white spirit is mainly generated in starter propagation and fermentation and is brought into the white spirit by distillation, so that the white spirit has the functions of expanding blood vessels, improving microcirculation and inhibiting platelet accumulation, and the white spirit in China has a health function, and the precursor substance of the white spirit is acetoin.

Acetoin, a chemical name of which is 3-hydroxy-2-butanone and also a name of which is 3-hydroxy butanone, diacetyl or methyl acetyl methanol, is a metabolite of physiological significance of a plurality of microorganisms, and the application of the acetoin as a precursor of tetramethylpyrazine in the production of tetramethylpyrazine through microbial fermentation has been proved by a plurality of researchers. The method of adding the precursor 3-hydroxy-2-butanone by external source is successfully applied to the improvement of the yield of tetramethylpyrazine produced by fermentation. However, the degree of utilization of the added precursor by microorganisms is low, and the utilization rate of the precursor is less than 1% even when pyrazine is produced in a high amount. And a large amount of exogenously added precursor 3-hydroxy-2-butanone is added into the culture medium, so that the culture medium is toxic to microbial cells and can cause disturbance of microbial metabolic balance.

Therefore, if a strain capable of improving the utilization rate of exogenous acetoin and realizing high yield of tetramethylpyrazine can be produced, the strain has important significance for the flavor and the economic value of the white spirit.

Disclosure of Invention

The invention aims to provide a bacterial strain for high yield of tetramethylpyrazine and a preparation method thereof, the bacterial strain effectively improves the utilization rate of acetoin and the yield of TTMP, and can be applied to production of elegant and fragrant white spirit.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention aims to provide a bacterial strain for high-yield tetramethylpyrazine production, wherein Bacillus licheniformis of the Bacillus licheniformis is classified and named as Bacillus licheniformis xf-4-1 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2021664, the preservation address of No. 299 in the Wuchang district of Wuhan city, Hubei province and the preservation date of No. 2021, 6 months and 2 days.

Further, the bacteriological characteristics of the bacillus licheniformis strain are: the shape is a rod, and the cell diameter is (0.8-1.0) μm x (1.5-2.0) μm; the colony color is milk white, the colony edge is irregular, and the colony has mucus and is gram positive.

The bacillus licheniformis has the functions of acidity and aroma enhancement on the white spirit, and can improve the style and flavor of the white spirit.

The second purpose of the invention is to provide a preparation method of the bacterial strain for high yield of tetramethylpyrazine, which comprises the following steps: carrying out microbial fermentation to accumulate precursor acetoin, and preparing enhanced yeast after obtaining a high-yield acetoin strain;

the method for fermenting the microorganisms comprises the following steps:

inoculating the first-stage seed culture solution into a screening culture medium for culture, leaching and distilling to obtain fermented grains containing precursor acetoin.

Further, the primary seed culture solution is constant volume to 1L by distilled water, and comprises: 8-15 g/L of peptone, 3-8 g/L of yeast powder, 8-15 g/L of NaCl and 6.8-7.5 of pH value;

preferably 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl and 7.0 of pH value.

Further, the screening medium is wheat flour with the water content of 30-50%, and further is wheat flour with the water content of 30-35%;

further, the wheat flour is obtained by sieving with a 20-mesh sieve.

The traditional screening method adopts laboratory culture medium for optimized screening, and the screening culture medium disclosed by the invention adopts the wheat starter as a matrix for screening, so that the screening culture medium is more suitable for production practice.

Further, the culture conditions in the screening medium were: 20-60 ℃ for 28-32 days;

the leaching reagent is 40-90% of absolute ethyl alcohol, the leaching time is 20-30 hours, and further is 60% of absolute ethyl alcohol, and the leaching time is 24 hours;

the fermented grains are: the mass-to-volume ratio of the absolute ethyl alcohol is 1: 1-3, and the preferred mass-to-volume ratio is 1: 2.

The 60% absolute ethyl alcohol is prepared by diluting absolute ethyl alcohol to 60% by mass concentration; the mass-to-volume ratio of 1:2 represents 100g of fermented grains and 200ml of absolute ethyl alcohol.

Further, the preparation method of the reinforced Daqu comprises the following steps:

inoculating the high-yield acetoin strain to a secondary seed culture medium for culture, and mixing with wheat flour to obtain the acetoin strain.

Further, the secondary seed culture medium is constant volume to 1L by distilled water, and comprises: peptone 20-40 g/L, yeast powder 5-15 g/L, glucose 40-60 g/L, diammonium hydrogen phosphate 20-40 g/L, pH value 6.8-7.2,

preferably 30g/L of peptone, 10g/L of yeast powder, 50g/L of glucose and 30g/L of diammonium hydrogen phosphate, and the pH value is 7.0.

Further, the culture condition is that the culture is carried out for 2-4 days at 25-32 ℃, preferably for 3 days at 30 ℃;

when mixing, the volume ratio of the secondary seed culture medium after the strain culture to water is 1: 500; the mass ratio of the secondary seed culture medium after the strain culture to the wheat flour after being mixed with water is 0.1-2 per mill, and preferably 1 per mill.

The invention also provides application of the bacterial strain for producing the tetramethylpyrazine in high yield in wine production.

The bacterial strain for high yield of tetramethylpyrazine can be applied to wine production, and also can be applied to food, food additives and medicines.

The invention has the beneficial effects that:

the contents of acetoin and tetramethylpyrazine in the soft-type white spirit prepared by the bacillus licheniformis strain for high yield of tetramethylpyrazine provided by the invention are 268.1mg/L and 5.6mg/L respectively, the contents are improved by 49.4% and 51.8% compared with those of a control cellar pool, the wine yield of the soft-type white spirit and the soft-type white spirit are basically consistent, no obvious influence is caused on a Daqu microflora and the final wine body style, and the healthy flavor substances of the soft-type white spirit are promoted and improved.

Drawings

FIG. 1 is a DGGE profile of the population of test and blank curves (1: control curve 2: test curve);

FIG. 2 is a graph showing the temperature variation trend of the fermentation process of the test pit and the comparative pit.

Detailed Description

The technical solutions of the present invention are described clearly and completely below, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The data of the examples of the present invention are mean values.

Material

The strain is as follows: the strains were isolated from the koji and deposited after 16SrDNA identification, among which b.licheniformis strain 9, b.subtiliss strain 2 (table 3).

The main reagents are as follows: tetramethylpyrazine standard sample (purity is more than or equal to 98.0%) and trihydroxybutanone (purity is more than or equal to 98.0%): a Chinese standard reagent net; other reagents are all domestic analytical purifiers.

Activating a culture medium: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl and the pH value of 7.0, adding 2% agar into the culture medium, sterilizing at 0.1MPa for 20min, and using the culture medium for plate activation.

First-order seed culture solution: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 7.0 of pH value and 20min of sterilization under 0.1 MPa.

Acetoin screening medium: mixing the pure wheat flour until the water content is 30-35%, and sterilizing for 20min at 0.1 MPa.

Secondary seed culture medium: 30g/L of peptone, 10g/L of yeast powder, 50g/L of glucose, 30g/L of diammonium hydrogen phosphate, pH value of 7.0, adding glucose after independent sterilization, and sterilizing the rest components for 20min under 0.1 MPa.

EXAMPLE 1 high acetoin Strain screening

Inoculating the first-class seed culture solution into an acetoin screening culture medium according to the inoculation amount of 1 per mill, culturing at different temperatures for different time, and then adding 60% absolute ethyl alcohol into the culture medium according to the ratio of fermented grains: the method comprises the steps of extracting absolute ethyl alcohol in a ratio of 1:2(100g of fermented grains and 200ml of absolute ethyl alcohol) for 24 hours, distilling in a water bath, and researching the influence of different temperatures and culture times on the content of acetoin, wherein the results are shown in tables 1-3.

TABLE 1 influence of different temperatures, incubation times on the acetoin content (mg/L)

TABLE 2 first order seed liquid growth

+: growth (10)⁴)，Better growth (10)⁶) (ii) a +++: grow well (10)⁸) (ii) a -: slow growth or autolysis.

The results in table 1 show that in a wheat flour screening culture medium, acetoin is greatly enriched after being cultured for 25-30 days at a growth temperature of 20-60 ℃, and when the acetoin is cultured for 30 days at 30 ℃, the strain just reaches an optimal growth state, the strain grows unaged, the acetoin content reaches a maximum value, and then the acetoin content is reduced; in Table 2, the temperature is increased, so that the germination growth period and the vigorous growth period of the strain can be advanced, but the strain is aged more quickly, the autolysis phenomenon of the strain can also occur after the liquid culture medium is cultured for 3 days at the temperature of more than 45 ℃, the autolysis phenomenon is obvious when the temperature is higher, and the strain grows best after being cultured for 72 hours at the temperature of 30 ℃; therefore, the invention takes 30 days of culture at 30 ℃ as the optimal culture condition for the first-class seed culture.

Inoculating the first-class seed culture solution into an acetoin screening culture medium according to the inoculation amount of 1 per mill, culturing for 30 days at 30 ℃ in the same period of the production process of a koji room, and adding 60% absolute ethyl alcohol according to the ratio of koji grains: extracting with absolute ethanol at a ratio of 1:2(100g fermented grains, 200ml absolute ethanol) for 24h, distilling in water bath, and detecting the content of acetoin, as shown in Table 3.

TABLE 3 preliminary screening results for strains

As shown in Table 3, the acetoin content of the screened Bacillus licheniformis9 strain and Bacillus subtilis2 strain is more than 1000mg/L, but the yield of the Bacillus licheniformis xf-4-1 reaches 3284 mg/L.

A physiological and biochemical identification experiment shows that the bacillus licheniformis xf-4-1 strain obtained through the experiment has typical bacillus characteristics, the cell diameter is (0.8-1.0) Mumx (1.5-2.0) Mum, the color is milky white, the edges of bacterial colonies are not integrated, mucus exists, the diameter of the bacterial colonies after 48 hours of culture is 1-2 cm, and the bacterial colonies are gram-positive and rod-shaped.

The Bacillus licheniformis xf-4-1 strain is preserved in China Center for Type Culture Collection (CCTCC) xf-4-1 at 2021, 6 months and 2 days, the preservation number is M2021664, and the preservation address is Wuhan city, Wuchang, Hubei, 299 No. eight.

Example 2 preparation of enhanced Daqu

(1) Effect of different culture conditions of secondary seeds on the growth of the strain

The screened xf-4-1 bacterial strains with high acetoin yield are gradually expanded to a secondary seed culture medium, after the bacterial strains are cultured for different time under different temperature conditions, the bacterial strains are diluted by 500 times and inoculated to a wheat flour culture medium according to the proportion of 1 per mill, fermented grains are prepared after uniform mixing, the influence of different culture conditions on the growth of the bacterial strains is researched, and the results are shown in table 4.

TABLE 4 Effect of different culture conditions on the growth of the strains of the second seed

In Table 4, the strains grew best when cultured at 30 ℃ for 72 h.

(2) Influence of different inoculation ratios on Daqu acetoin content

The screened xf-4-1 bacterial strain with high acetoin yield is gradually expanded to a secondary seed culture medium, and after the bacterial strain is cultured for 3 days at the temperature of 30 ℃, the bacterial concentration reaches 5.8 multiplied by 10⁷cfu/mL, diluting by 500 times, inoculating wheat flour culture medium according to different proportions, finding the optimal inoculation proportion, and uniformly mixing to obtain the fermented grains. 8 chambers were selected as the test group and 8 chambers were selected as the control group, and the results are shown in Table 5.

TABLE 5 Effect of different inoculation ratios on the target products

In Table 5, the maximum yield of acetoin was 3235.5mg/L when the addition rate was 1 ‰.

(3) Effect of different inoculation ratios on fermentation of koji embryo

As can be seen from Table 6, when the amount of the added strain is controlled within 1 ‰, the fermented grain fermentation process and the final appearance of the finished Daqu are not affected. Preliminary analysis on the microflora shows that the addition of 1 per mill has no substantial influence on the Daqu microflora, and the strip 20 in the graph of FIG. 1 shows that the brightness of B.licheniformis is improved to a certain extent before and after strengthening, which shows that the strengthening process has a certain effect on the enrichment of the strain.

TABLE 6 influence of different inoculation ratios on fermentation of koji

The screened xf-4-1 bacterial strain with high acetoin yield is gradually expanded to a secondary seed culture medium, and after the bacterial strain is cultured for 3 days at the temperature of 30 ℃, the bacterial concentration reaches 5.8 multiplied by 10⁷cfu/mL, diluting 500 times, inoculating the bacterial liquid into a wheat flour culture medium according to an inoculation ratio of 1 ‰, uniformly mixing to obtain fermented grains, and comparing the quality with that of Daqu in a control group, the results are shown in tables 7 and 8.

TABLE 7 sensory evaluation of Daqu

TABLE 8 analysis of physical and chemical indexes of Daqu

As can be seen from tables 7 and 8, the physical and chemical indexes of the Daqu are basically equivalent to those of the control group and the test group, and the exogenous microorganisms added in the invention have no obvious influence on the physical and chemical indexes of the Daqu.

(4) The influence of the reinforced Daqu on the physicochemical index of fermented grains

The control group and the test group have the same parameter control of fermented grains entering the cellar: the usage amount of the yeast is 20% of the usage amount of the grain, the cellar-entering water content is controlled to be 55-56%, the cellar-entering acidity is controlled to be 1.8, and the results are shown in table 9, table 10 and figure 2 by comparing and analyzing the fermentation process, the fermentation end, the fermented grain fermentation temperature and the physicochemical indexes after 90 days of storage.

TABLE 9 analysis of physical and chemical indexes of the use of koji after 90 days of storage

TABLE 10 analysis of physical and chemical indexes of fermented grains

As can be seen from table 9, there was no significant difference in Daqu acidity, glycation power, liquefaction power, and fermentation power in the test group after 90 days of storage compared to the control group; in the figure 2, the cellar entry temperature is controlled to be about 21 ℃, the top temperature is reached 20 days after the cellar entry, the fermentation period is 75 days, the variation trends of 'front slow, middle straight and rear slow falling' are met, the temperature variation trends of the two are basically the same, and the fermentation process is normal. The fermented grain detection results from table 10 show that each index of fermented grains indicates that the fermentation result is normal, and the differences of the detection results of the test pit and the control pit are not obvious; the results show that the reinforced Daqu and the non-reinforced Daqu have no obvious influence on the lees claim fermentation process and final physical and chemical indexes.

(5) Effect of intensified Daqu on liquor yield

TABLE 11 analysis of the main flavor Components of the wine body

In Table 11, the ethyl acetate, ethyl butyrate and ethyl lactate in the control cell were slightly higher than in the test cell, and the ethyl hexanoate was the opposite. The contents of acetoin and tetramethylpyrazine in the test pit are 286.1mg/L and 5.6mg/L respectively, which are improved by 49.4 percent and 51.8 percent compared with the reference pit. The wine yield of the test cellar is higher than that of the comparative cellar, which shows that the strengthened Daqu can improve the wine yield and the flavor quality thereof.

The influence of 9 Bacillus licheniformis strains and 2 Bacillus subtilis strains selected in example 1 on the alcohol yield was examined, and the results are shown in Table 12.

TABLE 12 liquor yield of different strains

As can be seen from the table above, the screened Bacillus licheniformis xf-4-1 has the highest wine yield.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.