CN116555124A - Bacillus Daqu and application thereof - Google Patents
Bacillus Daqu and application thereof Download PDFInfo
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- CN116555124A CN116555124A CN202310697236.4A CN202310697236A CN116555124A CN 116555124 A CN116555124 A CN 116555124A CN 202310697236 A CN202310697236 A CN 202310697236A CN 116555124 A CN116555124 A CN 116555124A
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- bacillus
- daqu
- producing
- trimethyl pyrazine
- pyrazine
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 38
- IAEGWXHKWJGQAZ-UHFFFAOYSA-N trimethylpyrazine Chemical compound CC1=CN=C(C)C(C)=N1 IAEGWXHKWJGQAZ-UHFFFAOYSA-N 0.000 claims abstract description 58
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000004321 preservation Methods 0.000 claims abstract description 13
- 150000003216 pyrazines Chemical class 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 21
- 238000012258 culturing Methods 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 13
- 238000010563 solid-state fermentation Methods 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 8
- 241000992035 Scopulibacillus daqui Species 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000012880 LB liquid culture medium Substances 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 241000209140 Triticum Species 0.000 claims description 3
- 235000021307 Triticum Nutrition 0.000 claims description 3
- 239000000796 flavoring agent Substances 0.000 claims description 3
- 235000019634 flavors Nutrition 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000012137 tryptone Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 230000000813 microbial effect Effects 0.000 claims description 2
- 238000009736 wetting Methods 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 6
- 244000005700 microbiome Species 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 5
- 239000011435 rock Substances 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 239000007858 starting material Substances 0.000 abstract description 3
- 238000013048 microbiological method Methods 0.000 abstract description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 244000063299 Bacillus subtilis Species 0.000 description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- -1 pyrazine compound Chemical class 0.000 description 4
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 description 3
- KYQCOXFCLRTKLS-UHFFFAOYSA-N Pyrazine Natural products C1=CN=CC=N1 KYQCOXFCLRTKLS-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- MOMFXATYAINJML-UHFFFAOYSA-N 2-Acetylthiazole Chemical compound CC(=O)C1=NC=CS1 MOMFXATYAINJML-UHFFFAOYSA-N 0.000 description 2
- ROWKJAVDOGWPAT-UHFFFAOYSA-N Acetoin Chemical compound CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 244000299461 Theobroma cacao Species 0.000 description 2
- 235000009470 Theobroma cacao Nutrition 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 238000001319 headspace solid-phase micro-extraction Methods 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- SJWFXCIHNDVPSH-UHFFFAOYSA-N octan-2-ol Chemical compound CCCCCCC(C)O SJWFXCIHNDVPSH-UHFFFAOYSA-N 0.000 description 2
- DTUQWGWMVIHBKE-UHFFFAOYSA-N phenylacetaldehyde Chemical compound O=CCC1=CC=CC=C1 DTUQWGWMVIHBKE-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 235000013599 spices Nutrition 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 235000020097 white wine Nutrition 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 235000006468 Thea sinensis Nutrition 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 235000020279 black tea Nutrition 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- OWBTYPJTUOEWEK-UHFFFAOYSA-N butane-2,3-diol Chemical compound CC(O)C(C)O OWBTYPJTUOEWEK-UHFFFAOYSA-N 0.000 description 1
- 235000010354 butylated hydroxytoluene Nutrition 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 108010079058 casein hydrolysate Proteins 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000003988 headspace gas chromatography Methods 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 235000014571 nuts Nutrition 0.000 description 1
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 229940100595 phenylacetaldehyde Drugs 0.000 description 1
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 1
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 235000011962 puddings Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 235000014214 soft drink Nutrition 0.000 description 1
- 238000002470 solid-phase micro-extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 235000014101 wine Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
- C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to high-temperature-resistant Daqu rock bacillus capable of producing 2,3, 5-trimethyl pyrazine and application thereof. Aiming at the problem of few microorganisms capable of producing pyrazine compounds in the prior report, the invention provides a novel high-temperature-resistant Bacillus Daqu rock bacillus capable of producing 2,3, 5-trimethyl pyrazine, and the preservation number is CGMCC No.26367. The bacillus Daqu LZLJ2-3 has high temperature resistance, has the capacity of producing 2,3, 5-trimethyl pyrazine by fermentation, can be used for preparing starter for brewing white spirit, and enriches the baking aroma substances of the white spirit body; meanwhile, the method has short fermentation period and low cost, provides possibility for preparing the 2,3, 5-trimethyl pyrazine by a microbiological method, can be used as a research object of recombinant organisms, and has important significance for promoting the industrialized production of the 2,3, 5-trimethyl pyrazine.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to high-temperature-resistant Daqu rock bacillus capable of producing 2,3, 5-trimethyl pyrazine and application thereof.
Background
The pyrazine compound is an important intermediate for synthesizing medicines and pesticides, and is also used as edible essence. 2,3, 5-trimethyl pyrazine is a high-grade edible spice developed in recent years, has rich aroma of cocoa beans, nuts, roasted potatoes and roasted peanuts, is naturally present in food such as roasted barley, black tea, cheese, cocoa products, coffee, rum and the like, is an important essence and tobacco essence raw material, and can be directly used for calling various foods such as bread, pudding, soft drinks and the like in the food industry.
The prior researches show that the pyrazine compound is a substance basis of white spirit showing baking fragrance. Few reports have been made about microorganisms capable of producing 2,3, 5-trimethylpyrazine, such as Guo Chengshuan et al (Guo Chengshuan, ouyang Puyue, GC/MS analysis of volatile flavor components produced by fermentation of Bacillus subtilis E20 [ J ]. Chinese brewing, 2010 (9): 3 ]) found that Bacillus subtilis E20 solid fermentation metabolites were primarily phenylacetaldehyde, 4-methyl 2, 6-di-tert-butyl-phenol, indole, a-furanmethanol, 2,3, 5-trimethylpyrazine, 2-acetylthiazole, and the like. Liang Huizhen et al (Liang Huizhen, lu Yan, liu Zheng, et al; screening and application of Bacillus subtilis for high yield of pyrazines in high temperature Daqu [ J ]. Chinese brewing, 2022,41 (1): 7.) screening high yield of pyrazines from high temperature Daqu by high temperature culture and solid state fermentation, and detecting metabolites of the strains by headspace solid phase microextraction and gas chromatography-mass spectrometry, wherein the screened strains are Bacillus sorafei, bacillus subtilis, bacillus licheniformis, bacillus cereus and Bacillus amyloliquefaciens, and the solid state fermentation has the capability of producing 2,3, 5-trimethylpyrazine, which is up to 130.28 mug/kg.
It has been found that thermotolerant bacteria can metabolize 3-hydroxy-2-butanone, 2, 3-butanediol, pyrazines and other fragrant substances, and can produce various enzymes such as amylase and protease, thereby promoting various biochemical reactions. The flavor of the white wine is closely related to heat-resistant bacteria, and the typical style and characteristics of the white wine are important manifestations of heat-resistant function bacteria in Daqu. Therefore, the obtained strain which is resistant to high temperature and has the function of producing 2,3, 5-trimethyl pyrazine has important significance for the research of the white spirit starter propagation process.
Disclosure of Invention
Aiming at the problem that microorganisms capable of producing pyrazine compounds in the prior report are fewer, the invention provides a novel high-temperature-resistant Bacillus Daqu rock bacillus capable of producing 2,3, 5-trimethyl pyrazine and application thereof.
The invention firstly provides a strain of Bacillus Daqu (Scopulibacillus daqui) for producing 2,3, 5-trimethyl pyrazine, and the preservation number is CGMCC No.26367.
The preservation time is 2022, 12 months and 30 days, the preservation unit is China general microbiological culture Collection center (CGMCC), address: the institute of microbiology, national institute of sciences, no. 3, north chen west way 1, region of korea, beijing city, postal code: 100101. the saccharomycete is screened in distiller's yeast and has the name: bacillus Daquus LZLJ2-3 (Scopulibacillus daqui).
Wherein, the 16S rDNA sequence of the bacillus Daqu is shown in SEQ ID NO: 1:
ATTGTCACCTTCGGCGGCTGGCTCCAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGGATTCACCGCGGCATGCTGATCCGCGATTACTAGCAATTCCGGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGGGAGTGGCTTTATGGGATTCGCTTAACCTCGCGGTCTCGCAGCCCTTTGTACCACCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCCGAAGGGGAAAGCCCTATCTCTAGGGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGTGTTGACTTCAGCACTAAGGGGTTGGACCCCCCTAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGGCCAGAGAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTCCTGCACTCAAGCTCCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAGCCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCACAGGGCATCATTTCCTATGAACCTATTCTCTCTAAAACAGAGTT。
the invention also provides a microbial agent which takes the bacillus Daqu as an active ingredient.
The invention also provides application of the bacillus Daqu in producing 2,3, 5-trimethyl pyrazine.
The invention also provides application of the bacillus Daqu in the field of white spirit brewing.
The invention also provides application of the bacillus Daqu in improving the content of pyrazine compounds in fermentation products or essence and spice.
Specifically, in the application, the pyrazine compound is prepared by adopting the following fermentation process, which comprises the following steps: inoculating the bacillus Daqu into LB liquid culture medium to prepare seed liquid, inoculating the seed liquid into solid state fermentation culture medium, fermenting and culturing.
Wherein, the culture conditions of the bacillus Daqu seed liquid are as follows: shake culturing at 50-60 deg.c and 120r/min for 18-24 hr.
The formula of the LB liquid medium is as follows: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, and steam sterilization at 115℃for 20min.
Wherein, the seed liquid is inoculated in the solid state fermentation culture medium with an inoculum size of 5-10v/v%.
Wherein the solid state fermentation medium is prepared by the following method: wetting wheat with 10wt% pure water for 20h, adding 40wt% water, wet pulverizing, packaging, and autoclaving at 121deg.C for 20min.
Wherein, the fermentation culture conditions are as follows: culturing at 50deg.C for 1d, culturing at 55deg.C for 1d, and culturing at 60deg.C for 1d.
Further, by adopting the fermentation process, the content of 2,3, 5-trimethyl pyrazine produced by the bacillus Daqu is 0.362mg/kg.
The beneficial effects are that: the invention separates and screens out a strain of Bacillus Daqu (Scopulibacillus daqui) capable of producing 2,3, 5-trimethyl pyrazine from Luzhou Laojiao medium temperature Daqu, the preservation number is CGMCC No.26367, and the strain is named as Bacillus Daqu LZLJ2-3. The bacillus Daqu LZLJ2-3 provided by the invention is high temperature resistant, grows at 37-65 ℃, has an optimal culture temperature of 50-55 ℃, and can be suitable for a high-temperature fermentation process in the preparation of medium-high temperature Daqu. The preparation method has the capability of producing 2,3, 5-trimethyl pyrazine by fermentation, can be used for preparing starter for brewing white spirit, and enriches the baking fragrance substances of the white spirit body; meanwhile, the method has short fermentation period and low cost, provides possibility for preparing the 2,3, 5-trimethyl pyrazine by a microbiological method, can be used as a research object of recombinant organisms, and has important significance for promoting the industrialized production of the 2,3, 5-trimethyl pyrazine.
Drawings
FIG. 1 is a colony morphology of B.Daquus of example 1;
FIG. 2 is a mass spectrum of 2,3, 5-trimethylpyrazine GC-MS molecular fragments produced by Bacillus Daqu in example 2;
FIG. 3 is a mass spectrum of GC-MS molecular fragments of 2,3, 5-trimethylpyrazine standard.
The invention provides a Daqu rock bacillus (Scopulibacillus daqui) for producing 2,3, 5-trimethyl pyrazine, which is preserved in 2022 in 12 months and 30 days, wherein the preservation unit is China general microbiological culture Collection center (CGMCC), address: the institute of microbiology, national institute of sciences, no. 3, north chen west way 1, region of korea, beijing city, postal code: 100101. the preservation number is CGMCC No.26367. The classification was designated as Bacillus Daqu (Scopulibacillus daqui).
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The medium formulation referred to in the examples:
R2A solid medium: yeast extract powder 0.5g/L, peptone 0.5g/L, casein hydrolysate 0.5g/L, glucose 0.5g/L, soluble starch 0.5g/L, dipotassium hydrogen phosphate 0.3g/L, anhydrous magnesium sulfate 0.024g/L, sodium pyruvate 0.3g/L, agar 15.0g/L, pH 7.2+ -0.2, and high pressure steam sterilization at 115 ℃ for 20 minutes.
LB liquid medium: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, and steam sterilization at 115℃for 20 minutes.
Solid state fermentation medium: soaking wheat grains with 10wt% pure water for 20 hr, adding 40wt% water, wet pulverizing with a crusher, packaging in 250mL triangular flask, 100 g/bottle, and autoclaving at 121deg.C for 20min.
Example 1: isolation and purification of strains
(1) The separation method comprises the following steps: taking 20g of luzhou-temperature Daqu sample in a luzhou-state wine, putting the luzhou-temperature Daqu sample into a conical flask filled with 180mL of sterile distilled water, and oscillating for 10min by a constant-temperature shaking table to fully scatter and mix the sample uniformly. Taking 1mL of sample suspension, diluting to 10 by adopting a double ratio dilution method -2 ～10 -7 100 mu L of each gradient of diluent is sucked and uniformly coated on a R2A solid culture medium plate, two plates are prepared in parallel, inverted and placed in a 50 ℃ incubator for culturing for 36-48 hours, and timely observation is performed.
(2) And (3) scribing and purifying: and taking out the plate with the colonies, picking single colonies with different colony morphologies, and carrying out secondary streaking until all the single colonies are purified. Colonies on the R2A solid medium were moist and translucent white with smooth surface as shown in FIG. 1.
(3) And (3) strain preservation: the single colony of each strain after purification is picked into 5mL LB liquid medium, placed at 50 ℃ for 24h, 1mL of bacterial liquid is sucked into a bacteria-preserving tube, 0.5mL of 60% sterile glycerol solution is added, resuspended and placed at-80 ℃ for preservation.
Example 2: 2,3, 5-trimethyl pyrazine production capability detection
(1) Preparing a bacterial liquid to be tested:
after the glycerol preservation tube of the strain obtained by screening is dissolved, the strain is respectively inoculated into LB liquid culture medium, and is cultured for 24 hours at 50 ℃, and after three generations of activation, the strain is inoculated into 50mL of LB liquid culture medium according to the inoculum size of 2% (volume ratio) for 24 hours, so as to obtain the bacterial liquid to be tested.
(2) HS-SPME/GC-MS method for detecting 2,3, 5-trimethylpyrazine:
the headspace solid-phase microextraction/gas chromatography-mass spectrometer technique is applied: taking supernatant, adding the supernatant into a headspace bottle, adding saturated NaCl solution, taking 0.822mg/ml of 2-octanol as an internal standard substance, carrying out heat preservation and balance on the prepared sample at 60 ℃ for 5min, extracting the sample at 60 ℃ for 50min by using a 50/30 mu m DVB/CAR/PDMS extraction head, and desorbing the sample at 250 ℃ for 5min at a GC sample inlet after the extraction is finished. And (5) matching the compound search result with an NIST standard spectrum library, and confirming that the similarity reaches more than 80% as a target compound. The culture solution without bacteria is used as a blank control group to calculate the content of each volatile substance.
GC-MS detection chromatographic conditions:
gas chromatography conditions: HP-INNOWAX column (60 m. Times.0.25 mm. Times.0.25 μm); heating program: the initial temperature is 40 ℃ for 5min, the temperature is increased to 100 ℃ at 4 ℃/min, the temperature is increased to 230 ℃ at 6 ℃/min, the temperature is maintained for 10min, and the carrier gas is high-purity helium (1.0 mL/min); the temperature of the sample inlet is 250 ℃, and the flow is not split.
Mass spectrometry conditions: electron ionization source with electron energy of 70eV; electron multiplier voltage 350V; the ion source temperature is 230 ℃; the temperature of the transmission line is 250 ℃; the mass range is 40-450 m/z.
By the detection method, as shown in figures 2 and 3, a strain with stronger capacity of producing 2,3, 5-trimethyl pyrazine is screened from the separated strains, the content of the produced 2,3, 5-trimethyl pyrazine is 0.143mg/L, and the strain is selected for further research.
Example 3: morphological and molecular identification of bacteria
And (3) amplifying and culturing the target strain, taking fresh bacterial liquid in the logarithmic growth phase, centrifugally collecting bacterial cells, and extracting genome DNA by using a bacterial genome extraction kit. The full-length sequence of the 16S rDNA is amplified by adopting a bacterial universal primer 27F/1492R, and the method is concretely as follows:
SEQ ID NO:2：27F(5′-AGAGTTTGATCCTGGCTCAG-3′)
SEQ ID NO:3：1492R(5′-GGTTACCTTGTTACGACTT-3′)
(1) reaction system (50. Mu.L)
(2) Reaction procedure
The PCR reaction of the "denaturation-annealing-extension" was performed 35 times in the above procedure, and the PCR product was checked by electrophoresis on a 1.0% agarose gel at a voltage of about 11V/cm for 20min.
The purification of PCR products was performed as described in the Shanghai Biotechnology Co small amount of gel recovery PCR product purification kit, and sequencing was performed by Shanghai Biotechnology Co.
The gene sequence of the 16S rDNA fragment obtained by sequencing is subjected to BLAST comparison through NCBI, strain species information is determined according to strain morphology characteristics, the strain is identified as Bacillus Daqu (Scopulibacillus daqui), the 16S rDNA is shown as SEQ ID NO. 1 and named as Bacillus Daqu LZLJ2-3, and the strain is preserved in China general microbiological culture collection center (CGMCC) at 12 months 30 of 2022, and the preservation number is CGMCC No.26367.
Example 4: solid state fermentation of Bacillus Daqu LZLJ2-3
After the obtained LZLJ2-3 glycerol storage tube of the Bacillus Daqu is dissolved, the obtained tube is inoculated into 10mL of LB liquid medium with liquid loading capacity according to 1% of inoculum size, and after 20h of culture at 50 ℃, the culture medium is transferred to 150mL of LB liquid medium with liquid loading capacity according to 1% of inoculum size, and seed liquid is prepared based on 1d of shaking culture at 120r/min at 50 ℃. Inoculating into solid fermentation culture medium according to 5% inoculum size, culturing at 50deg.C for 1d, culturing at 55deg.C for 1d, and culturing at 60deg.C for 1d with solid culture medium containing LB liquid culture medium as blank. The sample after fermentation is taken, and the detection method is the same as the volatile component determination method in the example 2, and the detection result shows that 2,3, 5-trimethyl pyrazine produced by solid state fermentation of the Bacillus Daqu LZLJ2-3 is 0.362mg/kg.
It is to be noted that the particular features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in any one or more embodiments. Furthermore, the various embodiments described in this specification, as well as the features of the various embodiments, can be combined and combined by one skilled in the art without contradiction.
Claims (10)
1. Bacillus Daqu (Scopulibacillus daqui), which is characterized in that: the preservation number is CGMCC No.26367.
2. A microbial agent is characterized in that: a method for preparing a composition comprising the Bacillus Daqu as claimed in claim 1.
3. The use of the bacillus Daqu in producing 2,3, 5-trimethyl pyrazine according to claim 1.
4. The use of the bacillus Daqu in the field of white spirit brewing according to claim 1.
5. The use of the bacillus Daqu in increasing the content of pyrazines in a fermentation product or flavor.
6. Use according to any one of claims 3-5, characterized in that: the 2,3, 5-trimethyl pyrazine is prepared by adopting the following fermentation process, which comprises the following steps: inoculating the bacillus Daqu into LB liquid culture medium to prepare seed liquid, inoculating the seed liquid into solid state fermentation culture medium, fermenting and culturing.
7. The use according to claim 6, characterized in that: the culture condition of the seed liquid of the Bacillus Daqu is 50-60 ℃ and 120r/min shaking culture is carried out for 18-24 h.
8. The use according to claim 6, characterized in that: the formula of the LB liquid medium is as follows: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, and steam sterilization at 115℃for 20min.
9. The use according to claim 6, characterized in that: the inoculation amount of the seed liquid to the solid state fermentation culture medium is 5-10v/v%; the fermentation culture conditions are as follows: culturing at 50deg.C for 1d, culturing at 55deg.C for 1d, and culturing at 60deg.C for 1d.
10. The use according to claim 6, characterized in that: the solid state fermentation medium is prepared by the following method: wetting wheat with 10wt% pure water for 20h, adding 40wt% water, wet pulverizing, packaging, and autoclaving at 121deg.C for 20min.
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