CN-121450547-B - Bacillus licheniformis and application thereof in acetoin and tetramethylpyrazine production

Abstract

The invention relates to the technical field of microbial fermentation and bioengineering, in particular to bacillus licheniformis and application thereof in acetoin and tetramethylpyrazine production. According to the invention, bacillus licheniformis with higher acetoin yield is screened from a large number of strains. The acetoin yield of the strain fermented for 36 hours can reach 98g/L, which is far higher than that of other existing natural screening strains, and the yield of white spirit brewed by using the strain is improved by 11%, the content of tetramethylpyrazine in the white spirit is improved by 57%, and the quality of the white spirit is obviously improved.

Inventors

• ZHANG YAN
• YIN XIANGXIANG
• XIAO MINGHUA
• LIAO BEI
• WU YEXU
• LI ZHAOFEI
• Mu Jizhe

Assignees

• 安琪酵母股份有限公司

Dates

Publication Date

20260505

Application Date

20251231

Claims (15)

1. 1. Bacillus licheniformis (Bacillus licheniformis) is characterized in that the preservation number is CCTCC NO: M20251822.
2. 2. The microbial inoculum is characterized by comprising bacillus licheniformis with the preservation number of CCTCC NO: M20251822 and/or bacillus licheniformis with the preservation number of CCTCC NO: M20251822.
3. 3. The method for preparing the microbial inoculum of claim 2, which is characterized by comprising the following steps: S1, inoculating and culturing bacillus licheniformis with the preservation number of CCTCC NO: M20251822 to obtain seed liquid; s2, carrying out expansion fermentation culture on the seed liquid to obtain the microbial inoculum.
4. 4. The preparation method of the fermentation medium according to claim 3, wherein the culture medium for the enlarged fermentation culture comprises, by mass, 1% -8% of glucose, 0.02% -0.05% of magnesium sulfate heptahydrate, 1.5% -2.5% of FM888, 0.05% -0.1% of tri-ammonium citrate, 0.45% -0.65% of sodium acetate, 5% -8% of dipotassium phosphate trihydrate, 0.001% -0.003% of ferrous sulfate heptahydrate, 0.0010% -0.0014% of zinc sulfate heptahydrate, 0.0001% -0.0004% of manganese sulfate monohydrate and 6.5% -7.0 of pH.
5. 5. The method according to claim 3, wherein the conditions for the expanded fermentation culture include a ventilation ratio of 1.5vvm, a pot pressure of 0.05MPa, a stirring speed of 180-200 r/min, and a culture temperature of 34-37 ℃.
6. 6. A distiller's yeast, which is characterized by comprising the microbial inoculum and the substrate according to claim 2.
7. 7. The distiller's yeast of claim 6, wherein the substrate comprises at least one of wheat, bran, corn, sorghum, rice flour, rice hulls.
8. 8. The distiller's yeast of claim 6, wherein the distiller's yeast is a bran koji prepared from bran and the microbial inoculum of claim 2 in a mass ratio of 1:1.
9. 9. The method for preparing distiller's yeast according to any one of claims 6-8, characterized by mixing the microbial inoculum and the substrate according to claim 2, drying, and stirring.
10. 10. Use of the bacillus licheniformis of claim 1, the microbial inoculum of claim 2 or the distiller's yeast of any of claims 6-8 in any of the following: (1) Brewing white spirit; (2) Acetoin and/or tetramethylpyrazine are produced by fermentation.
11. 11. The use according to claim 10, wherein the white spirit is Maotai-flavor Daqu spirit.
12. 12. The use according to claim 10, wherein the bacillus licheniformis increases the acetoin and/or tetramethylpyrazine content of white spirit.
13. 13. The preparation method of the Maotai-flavor Daqu liquor is characterized by comprising the steps of inoculating bacillus licheniformis as claimed in claim 1, the microbial inoculum as claimed in claim 2 or the distiller's yeast as claimed in any one of claims 6-8 to fermented grains, and fermenting in a cellar.
14. 14. The method according to claim 13, wherein the fermented grains are cooled after steaming the wine for the 7 th round.
15. 15. The preparation method of claim 14, wherein the temperature of the fermented grains after cooling is 32-34 ℃.

Description

Bacillus licheniformis and application thereof in acetoin and tetramethylpyrazine production Technical Field The invention relates to the technical field of microbial fermentation and bioengineering, in particular to bacillus licheniformis and application thereof in acetoin and tetramethylpyrazine production. Background Acetoin is an important food flavor substance and an industrial intermediate, and has wide application prospect in the fields of food, medicine, chemical industry and the like. It naturally occurs in a variety of fermented foods and imparts a unique creamy aroma to the product and is therefore often used as a flavour enhancer in the food industry. Tetramethyl pyrazine (Tetramethylpyrazine, TMP for short, also called ligustrazine) is an important flavor active substance and functional component in white spirit, has certain physiological activity, and can be used for adding health attributes to the product. The microbial fermentation method is an important way for producing acetoin, has the advantages of wide raw material sources, mild reaction conditions, environmental friendliness and the like, and accords with the development trend of green production. At present, research has reported that bacillus licheniformis can be used for fermenting and producing acetoin, and for example, chinese patent CN105969703B discloses bacillus licheniformis and application thereof in acetoin fermentation and production. However, the strain disclosed by the patent has obvious limitations that on one hand, the yield of acetoin is lower, the yield of fermentation is only 5.6g/L for 36 hours, and the requirements of industrial production on yield and efficiency are difficult to meet, and on the other hand, the existing fermentation system based on bacillus licheniformis is only focused on acetoin single products, does not relate to synthesis and accumulation of tetramethylpyrazine, cannot realize the cooperative production of two high-value substances, and is especially not suitable for the scenes of brewing white spirit, compound fermentation food and the like which need to simultaneously consider flavor and functional properties. Therefore, development of a microbial strain and a fermentation technology capable of synchronously improving acetoin yield and realizing synthesis of tetramethylpyrazine becomes a key problem to be broken through at present. Disclosure of Invention In view of this, the present invention provides bacillus licheniformis and its use in acetoin and tetramethylpyrazine production. Aims at solving the problems that the existing strain can not simultaneously produce acetoin and tetramethylpyrazine with high yield, and the production cost is high. The invention provides bacillus licheniformis (Bacillus licheniformis) with a preservation number of CCTCC NO: M20251822. According to the invention, single bacterial colony is separated from distiller's yeast, and a high-yield acetoin strain X-11 with the acetoin yield remarkably higher than that of other strains is obtained by screening a large number of strains by utilizing a color reaction based on acetoin characteristics. The strain X-11 was identified as a novel strain of Bacillus (Bacillus) and designated Bacillus licheniformis (Bacillus licheniformis) X-11 by morphological and 16S rRNA gene sequence analysis. The strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20251822. The invention also provides a microbial inoculum, which comprises bacillus licheniformis with the preservation number of CCTCC NO: M20251822 and/or fermentation liquor, culture, lysate or extract of bacillus licheniformis with the preservation number of CCTCC NO: M20251822 and the like. The preparation formulation of the microbial inoculum comprises water aqua, emulsion or powder. The invention also provides a preparation method of the microbial inoculum, which comprises the following steps: S1, inoculating and culturing bacillus licheniformis with the preservation number of CCTCC NO: M20251822 to obtain seed liquid; s2, carrying out expansion fermentation culture on the seed liquid to obtain the microbial inoculum. In some embodiments, the culture medium for the enlarged fermentation culture comprises, by mass, 1% -8% of glucose, 0.02% -0.05% of magnesium sulfate heptahydrate, 1.5% -2.5% of FM888, 0.05% -0.1% of tri-ammonium citrate, 0.45% -0.65% of sodium acetate, 5% -8% of dipotassium phosphate trihydrate, 0.001% -0.003% of ferrous sulfate heptahydrate, 0.0010% -0.0014% of zinc sulfate heptahydrate, 0.0001% -0.0004% of manganese sulfate monohydrate and 6.5% -7.0 of pH. Wherein the components with the mass percentages are added based on the mass of water, namely the percentages of the components are the percentages of the mass of each component in the mass of water. In some embodiments, the culture medium for the enlarged fermentation culture comprises the following components of glucose 8%, magnesium sulfate heptahyd