A bacillus strain and application thereof in increasing content of 4-vinylguaiacol in liquor brewing process

Abstract

The application discloses a bacillus strain and application thereof in increasing the content of 4-vinyl guaiacol in the liquor brewing process, the classification name of the bacillus strain is Bacillus sp.B100, has been preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO:M20221674, and the preservation date is October 26, 2022.The application prepares intensified Daqu by using the bacillus strain Bacillus sp.B100, in the fermentation process, the naturally existing ferulic acid in fermented grains is converted into 4-vinyl guaiacol, the content of 4-vinyl guaiacol in liquor is increased, and the health quality of the liquor is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of brewing microorganisms, and particularly relates to a bacillus strain and application thereof in increasing the content of 4-vinylguaiacol in the process of liquor brewing. BACKGROUND

[0002] Chinese liquor is one of the six major distilled liquors in the world, with unique brewing process and profound cultural heritage, and is unique in the world. Chinese liquor mainly contains 98%-99% ethanol and water, and 1%-2% flavor components, which determines the aroma type and taste of the liquor. In recent years, with the proposal of the national development strategy of "healthy China", the concept of healthy and comfortable drinking has been deeply rooted in people's minds. The research on Chinese liquor is from analyzing its components to finding the source, and finding the key fermentation microorganisms, so as to realize the metabolic control of the whole fermentation system. Bacillus is one of the important microorganisms affecting the quality of liquor in the process of Chinese liquor brewing, and has great significance for improving the quality of liquor.

[0003] 4-vinylguaiacol (4-VG) is a special volatile component with baking aroma, nutty aroma and spicy aroma, and its sensory threshold is low, which is 0.3mg / L. It is not only one of the main aroma components in liquor, but also a medical synthesis intermediate with health function. Ferulic acid is a natural substance in fermented grains and wheat, which is the precursor of 4-VG. Due to its high boiling point, it cannot be effectively distilled, and finally becomes discarded waste. At present, the related reports on the production of 4-VG by natural fermentation of microorganisms are as follows: such as patent CN201510748054.0, which uses bacillus licheniformis 71722 for biological fermentation with ferulic acid substrate. When the addition amount of ferulic acid is 100mM, 55.37mM 4-VG is obtained by fermentation; patent CN201310413429.9 proposes that the liquid seed liquid is inoculated into the liquid fermentation medium, and then xylanase and ferulic acid esterase are added in turn, and the fermentation liquid containing 4-vinylguaiacol is prepared. The fermentation liquid is filtered, extracted, and phase-separated to prepare the extraction liquid containing 4-vinylguaiacol, and the extraction liquid is vacuum evaporated to prepare the 4-vinylguaiacol product. Both of them increase the production cost and the cumbersome process in the process of liquor brewing. SUMMARY

[0004] The present application provides a bacillus strain and application thereof in increasing the content of 4-vinylguaiacol in the process of liquor brewing.

[0005] The application does not introduce foreign strains in the self-microbial system, excavates the discovered Bacillus sp. B100, can utilize the naturally existing ferulic acid in the five grains of the fermented grains after the strengthened starter is made, improves the utilization rate of the ferulic acid in the fermented grains, realizes the high added value utilization of the fermented grains, and improves the content of the healthy component 4-vinyl guaiacol in the liquor body, and the method does not add extra and has no food safety risk.

[0006] The Bacillus sp. B100 strain is obtained from the high-temperature Daqu of Gujinggong liquor, is classified and named as Bacillus sp. B100, has been preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC NO: M 20221674, and a preservation date of October 26, 2022.

[0007] The application of the Bacillus sp. B100 is to prepare the strengthened Daqu, utilize the naturally existing ferulic acid in the fermented grains in the fermentation process, convert the ferulic acid into 4-vinyl guaiacol, and improve the content of 4-vinyl guaiacol in the liquor.

[0008] The Bacillus sp. B100 provided in the application has the ability of high production of 4-vinyl guaiacol. After the Bacillus sp. B100 is made into the strengthened Daqu, the Bacillus sp. B100 is allowed to participate in the fermentation process of the fermented grains, consumes the naturally existing ferulic acid in the fermented grains, converts the ferulic acid into 4-vinyl guaiacol, and the 4-vinyl guaiacol is taken out with the liquor in the distillation link.

[0009] Specifically includes the following steps:

[0010] (1) a single Bacillus sp. B100 strain is picked from a slope test tube, and is cultured in LB liquid medium (commercially available, the same below) for 20-24 hours;

[0011] (2) the bacterial suspension obtained in step 1 is inoculated into a seed tank for secondary culture at an inoculation amount of 5%-10%, the culture medium is LB liquid medium, and after 1-2 days of culture at 37 DEG C, the bacterial suspension is transferred into a fermentation tank for tertiary culture at an inoculation amount of 5%-10%, and after 2-3 days of culture at 37 DEG C, the culture medium formula for the tertiary culture is as follows: 2%-3% of crushed sorghum is boiled in water for 30 minutes, and then cooled to room temperature; the obtained bacterial suspension is inoculated on bran, and after airing, the strengthened Daqu is obtained.

[0012] (3) the strengthened Daqu is added when the fermented grains are put into a pool for fermentation, and the fermentation lasts for 45-60 days.

[0013] Further, in step (3), the addition amount of the strengthened Daqu is 0.1%-0.5% of the production grain amount.

[0014] The Bacillus sp. B100 has the advantages that after being made into the strong Daqu, the utilization rate of the ferulic acid in the fermented grains is improved, resource waste is avoided, and the content of the healthy component 4-vinyl guaiacol in the liquor is improved. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a Bacillus sp. B100 colony photo (left) and a bacterial body gram staining micrograph (right).

[0016] Figure 2 It is a 4-vinyl guaiacol GC-MS spectrum. DETAILED DESCRIPTION

[0017] The present application is described below through specific embodiments. Unless specifically stated, the technical means used in the present application are methods known to those skilled in the art, and in addition, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art belong to the scope of protection of the present application.

[0018] Example 1: Isolation and identification of Bacillus sp. B100

[0019] 1. Take medium-high temperature Daqu (Anhui Gujinggong Liquor Co., Ltd.), crush and weigh 10 g, into 90 mL of normal saline, shake and mix, then take the supernatant for stepwise dilution to 10 -5 , 10 -5 mL of the dilution liquid is coated on a nutrient agar plate and cultured in a 37℃ incubator for 1 day. After the single colony is streaked and cultured, the purified strain is obtained by repeating twice.

[0020] 2. The colony shows rough surface and opaque, and is dirty white on the plate. It is determined as gram-positive bacteria under a microscope (see Figure 1 ).

[0021] 3. Strain identification. The purified strain is identified by 16S rDNA. The sequencing result is compared by BLAST on GenBank, and it is determined that the strain is Bacillus. The strain has been preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20221674.

[0022] Example 2: Bacillus fermentation to produce 4-vinyl guaiacol

[0023] 1. The activated Bacillus is picked and cultured in a commercially available LB liquid medium at 37℃ and 160 rpm for 24 h;

[0024] 2. Transfer to fermentation medium at 2% inoculation amount, cultivate at 37℃ for 72h. The fermentation medium consists of bran 100g / L, glucose 10g / L, distilled water to volume.

[0025] 3. Take the fermentation broth cultivated for 72h in step 2, mix with equal volume of 10% ethanol, dilute 100 times, then perform quantitative analysis by GC-MS. The detection conditions are: HS-SPME extraction temperature is 50℃; GC conditions: the chromatographic column is DB-wax (60m x 0.25mm x 0.25μm); the gas chromatography injection port temperature is 250℃, the carrier gas is He, the flow rate is 1.6mL / min, no split injection; the chromatographic program temperature rising conditions are: 40℃ for 2min, then increase the temperature to 180℃ at a rate of 4℃ / min, then increase the temperature to 230℃ at a rate of 6℃ / min, keep for 27min; MS conditions: the transfer line temperature is 230℃, the ionization mode is EI source, the electron energy is 70eV, the ion source temperature is 230℃. The internal standard substance used is 2,6-dimethyl phenol. Figure 2 The spectrum is that of 2,6-dimethyl phenol and 4-vinyl guaiacol. The final detection of 4-VG content in the fermentation broth is 375mg / L.

[0026] Example 3: Analysis of volatile substances produced by the strain

[0027] Take 8ml of the fermentation broth in Example 2, and perform qualitative analysis of the fermentation broth by HS-SPME-GC-MS.

[0028] The GC-MS analysis method is the same as in Example 2, and Table 1 is the 7 kinds of volatile components qualitatively analyzed in the fermentation broth.

[0029] Table 1. Volatile components in the fermentation broth detected by HS-SPME-GC-MS

[0030]

[0031] Example 4: Production of fortified Daqu

[0032] 1. Pick a single Bacillus sp. B100 strain from the slant test tube, cultivate in LB liquid medium (purchased) for 20-24h;

[0033] 2. Transfer the bacterial suspension obtained in step 1 to the seed tank for secondary culture at 5%-10% inoculation amount, the medium formula is LB liquid medium, cultivate at 37℃ for 1-2 days, then transfer to the fermentation tank for tertiary culture at 5%-10% inoculation amount, cultivate at 37℃ for 2-3 days, the medium is 2%-3% crushed sorghum, which is boiled in water for 30min, then used after cooling to room temperature. The obtained bacterial suspension is inoculated onto bran, after airing, fortified Daqu is obtained.

[0034] Example 5: Production application

[0035] According to the intensified Daqu production method in Example 4, 0.5% of intensified Daqu was added to the production grain, and 3 pits were selected, of which 2 were used as the test group, and the control group was not added with intensified Daqu, and the normal pit fermentation was carried out for 60 days. After the fermentation was completed, the production liquor samples were sampled and analyzed, and the volatile component analysis of the liquor samples was the same as in Example 2. The quantitative analysis of 4-vinyl guaiacol in the obtained liquor samples is shown in Table 2 below. The utilization of ferulic acid in the pit is shown in Table 3.

[0036] Table 2. Determination of volatile component content in liquor samples by HS-SPME combined with GC-MS

[0037]

[0038] Table 3. Ferulic acid content before and after fermentation of pit

[0039]

Claims

1. A Bacillus strain, characterized in that: The classification of the Bacillus strains is named Bacillus sp. B100 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221674 and deposit date of October 26, 2022.

2. The application of the Bacillus strain according to claim 1, characterized in that: With the Bacillus strain Bacillus sp. B100 is used to prepare fortified koji. During the fermentation process, ferulic acid naturally present in the mash is converted into 4-vinylguaiacol, thereby increasing the 4-vinylguaiacol content in baijiu. Includes the following steps: (1) Pick a single one from the slanted test tube Bacillus strain B100 was cultured in LB liquid medium for 20-24 hours. (2) The bacterial suspension obtained in step (1) is inoculated into a seed tank at an inoculation rate of 5% to 10% for secondary culture. LB liquid medium is used as the culture medium. After the secondary culture is completed, the bacterial suspension is transferred to a fermenter at an inoculation rate of 5% to 10% for tertiary culture. The obtained bacterial suspension is inoculated onto wheat bran and then spread out to dry to obtain enhanced Daqu. (3) Add strong Daqu when fermenting the mash in the fermentation tank, and ferment for 45-60 days; In step (3), the amount of Daqu (a type of starter culture) added is 0.1% to 0.5% of the mass of the grain used in production.

3. The application according to claim 2, characterized in that: The secondary culture temperature is 37℃, and the culture time is 1-2 days.

4. The application according to claim 2, characterized in that: The tertiary culture temperature is 37℃, and the culture time is 2-3 days.

5. The application according to claim 2, characterized in that: The culture medium for tertiary culture is formulated as follows: 2%~3% crushed sorghum, boiled in water for 30 minutes, and then cooled to room temperature before use.

Images