CN114606152B - Bacillus bailii, microbial agent and application thereof - Google Patents
Bacillus bailii, microbial agent and application thereof Download PDFInfo
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- CN114606152B CN114606152B CN202011427019.6A CN202011427019A CN114606152B CN 114606152 B CN114606152 B CN 114606152B CN 202011427019 A CN202011427019 A CN 202011427019A CN 114606152 B CN114606152 B CN 114606152B
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 61
- 230000000813 microbial effect Effects 0.000 title claims abstract description 18
- 239000000052 vinegar Substances 0.000 claims abstract description 99
- 235000021419 vinegar Nutrition 0.000 claims abstract description 99
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 18
- 238000004321 preservation Methods 0.000 claims abstract description 13
- 125000003118 aryl group Chemical group 0.000 claims abstract description 11
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 62
- 239000007788 liquid Substances 0.000 claims description 42
- 230000004151 fermentation Effects 0.000 claims description 35
- 238000000855 fermentation Methods 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 14
- 235000013405 beer Nutrition 0.000 claims description 13
- 238000011081 inoculation Methods 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 13
- 235000007164 Oryza sativa Nutrition 0.000 claims description 12
- 235000009566 rice Nutrition 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 11
- 235000013339 cereals Nutrition 0.000 claims description 10
- 239000002054 inoculum Substances 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 102000004139 alpha-Amylases Human genes 0.000 claims description 5
- 108090000637 alpha-Amylases Proteins 0.000 claims description 5
- 229940024171 alpha-amylase Drugs 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 229940088598 enzyme Drugs 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
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- 108010080698 Peptones Proteins 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
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- 238000012258 culturing Methods 0.000 claims description 4
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000004310 lactic acid Substances 0.000 description 6
- 235000014655 lactic acid Nutrition 0.000 description 6
- 239000002994 raw material Substances 0.000 description 6
- 239000012267 brine Substances 0.000 description 5
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 5
- 235000014101 wine Nutrition 0.000 description 5
- 229960003624 creatine Drugs 0.000 description 4
- 239000006046 creatine Substances 0.000 description 4
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- 239000003205 fragrance Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 201000009240 nasopharyngitis Diseases 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 241001249117 Bacillus mojavensis Species 0.000 description 3
- 238000002479 acid--base titration Methods 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 239000002068 microbial inoculum Substances 0.000 description 3
- 239000003223 protective agent Substances 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 2
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- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
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- 244000017020 Ipomoea batatas Species 0.000 description 1
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- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
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- 241000235342 Saccharomycetes Species 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 150000003792 acetoin derivatives Chemical class 0.000 description 1
- ODHCTXKNWHHXJC-UHFFFAOYSA-N acide pyroglutamique Natural products OC(=O)C1CCC(=O)N1 ODHCTXKNWHHXJC-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
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- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- 230000002354 daily effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
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- 230000002708 enhancing effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 235000021552 granulated sugar Nutrition 0.000 description 1
- 235000019674 grape juice Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000019991 rice wine Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
- C12J1/00—Vinegar; Preparation or purification thereof
- C12J1/04—Vinegar; Preparation or purification thereof from alcohol
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Distillation Of Fermentation Liquor, Processing Of Alcohols, Vinegar And Beer (AREA)
Abstract
The invention discloses bacillus beliae, a microbial agent and application thereof, wherein the bacillus beliae is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 6 months and 10 days in 2020, the preservation registration number is CGMCC NO.20063, and the application of the bacillus beliae and the microbial agent is provided. The bacillus beijerinckii is obtained by separating and screening the vinegar grains in the Zhenjiang aromatic vinegar production process, and is relatively suitable for the vinegar brewing environment; through the use of the strain, the content of acetoin and ligustrazine in the vinegar can be obviously improved, the flavor of the product is improved, and the quality of the product is improved.
Description
Technical Field
The invention relates to bacillus and microbial agent and application thereof, in particular to bacillus bailii and microbial agent and application thereof.
Background
The production and consumption of vinegar in the world is mainly focused on two areas: the European traditional wine production area, mainly Italy and France, and the China-Korean three-kingdom in southeast Asia. The western national table vinegar is mainly brewed by taking grape juice as a main raw material through a liquid submerged fermentation process, and the fermentation is mainly performed by taking acetic acid bacteria as main materials, so that the product has relatively single flavor substance and a single taste. In eastern countries such as China and Korea, vinegar is mainly brewed by taking grains with high starch content such as glutinous rice, sorghum or sweet potato as main raw materials, bran and wheat bran as auxiliary materials and solid fermentation processes of various microorganisms. The main acid of the solid brewing vinegar is acetic acid, organic acid such as lactic acid, succinic acid, pyroglutamic acid and the like, amino acid such as glutamic acid, alanine, lysine and the like, and a plurality of volatile flavor compounds, and the substances endow the vinegar with rich taste and coordinated aroma. In addition, the microorganism participating in fermentation can also produce functional factors such as ligustrazine, flavone, polyphenol and the like, and the health care function of the vinegar is given to a certain extent.
Acetoin, also known as 3-hydroxy-2-butanone, is a food flavor additive, has very rich cheese flavor and ester flavor, gives people pleasant feelings at reasonable concentration, and is a national regulatory food additive (GB 2760-86) which can be used. In addition, acetoin is also a precursor of ligustrazine which is an important functional component of vinegar, and ligustrazine can be generated by the reaction of acetoin and amino compounds, so that the ligustrazine not only has the fragrance of baked nuts, but also has the health-care functions of protecting heart and cerebral vessels, resisting oxidization and the like, and the health-care value of the vinegar is related to the content of the ligustrazine, so that the ligustrazine can be used as a characteristic index for identifying the quality of the vinegar.
At present, many strains for producing acetoin are researched and reported, and mainly include lactobacillus, acetic acid bacteria, saccharomycetes, bacillus and the like. Bacillus belicus is a new species of Bacillus, and research on the Bacillus is also increasing, but the research focus is mainly on plant growth, disease and pest resistance, disease resistance of an induction system and the like.
The invention relates to a preparation method of a high-yield acetoin and flavored bacillus mojavensis direct vat set starter and application thereof in the production of Shanxi aged vinegar, and discloses a method for enhancing the content of ligustrazine in the Shanxi aged vinegar by using the flavored bacillus mojavensis CGMCC 16910 direct vat set starter in the production of Shanxi aged vinegar. The acetoin yield of the flavored bacillus mojavensis CGMCC 16910 utilized by the method is 45.63g/L, and the content of ligustrazine in the finally produced new vinegar is 266.47 mug/mL.
Disclosure of Invention
The invention aims to: the invention provides bacillus belicus capable of improving the content of acetoin and ligustrazine in vinegar, the invention provides a microbial agent, the invention provides the application of bacillus belicus and the microbial agent in the food field, the invention provides the application of bacillus belicus and the microbial agent in vinegar brewing, and the invention provides a fifth object of a vinegar brewing method for improving the flavor and the quality of vinegar products.
The technical scheme is as follows: the bacillus beleiensis (Bacillus velezensis) is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), the preservation date is 6 months and 10 days in 2020, and the preservation registration number is CGMCC NO.20063.
Further, the microbial agent of the present invention comprises bacillus bailii. The bacillus bailii and the microbial agent are applied to the field of food.
The bacillus bailii or microbial agent is applied to vinegar brewing. Preferably, the vinegar is aromatic vinegar.
The brewing method of the table vinegar comprises the following steps:
(1) Preparing strain seed liquid by bacillus bailii;
(2) Fermenting the strain seed liquid to obtain the edible vinegar.
In the step (1), the preparation of the strain seed liquid comprises the preparation of a primary seed liquid and the preparation of a secondary seed liquid.
The preparation of the first-stage seed liquid specifically comprises the following steps: according to the inoculation amount of 1% -10%, inoculating the purified bacillus beijerinckii bacterial liquid in logarithmic growth phase into a culture medium, and culturing at 30-37 ℃ to obtain first-stage seed liquid.
The preparation of the secondary seed liquid specifically comprises the following steps: heating water to 90-95deg.C by liquid fermentation method, adding rice flour and high temperature alpha-amylase, and maintaining the temperature to obtain mash; then the temperature of the mash is reduced to 45-55 ℃, and saccharifying enzyme is added for heat preservation, thus obtaining saccharified liquid; adding 0.5% -2% peptone and 0.5% -2% yeast powder into the saccharified solution, and sterilizing; cooling to 30-37 ℃, inoculating the first-stage seed liquid according to the inoculum size of 1-10%, ventilating and stirring, and performing pressure maintaining fermentation at 30-37 ℃ to obtain the second-stage seed liquid.
The step (2) specifically comprises the following steps: mixing and stirring the beer mash, bran and bran, inoculating acetic acid bacteria according to the inoculation amount of 1-10% of the weight of the beer mash, inoculating secondary seed liquid of bacillus bailii according to the inoculation amount of 1-5% of the weight of the beer mash, and carrying out seed-carrying and solid layered fermentation; sealing fermented grains, spraying vinegar and decocting vinegar.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
(1) The bacillus beljalis which is more suitable for the brewing environment of the table vinegar is obtained by separating and screening from vinegar grains in the production process of the Zhenjiang aromatic vinegar, and can obviously improve the content of acetoin and ligustrazine in the table vinegar, improve the flavor of the product and improve the quality of the product when applied to the field of table vinegar brewing;
(2) The strain has high acetoin yield activity in vinegar brewing, wherein the acetoin content can reach 23.16g/L, and the ligustrazine content can reach 362.52mg/L;
(3) The strain is applied to vinegar brewing, and the flavor and taste of the product are obviously better than those of a control group;
(4) The strain can also be prepared into biological bacteria, and the strain can be prepared into bacterial liquid or biological bacteria for vinegar brewing, so that the flavor and the product quality are improved.
Drawings
FIG. 1 is a colony morphology of Bacillus bailii HSCY3015 of the invention;
FIG. 2 is a graph of total acid variation;
FIG. 3 is another graph of total acid variation.
Detailed Description
The technical scheme of the invention is further described below by referring to examples.
In the embodiment of the invention, the high-temperature alpha-amylase and the saccharifying enzyme are all from commercial sources.
Example 1 application of Bacillus bailii HSCY3015 in Zhenjiang aromatic vinegar brewing
1. Expansion culture of strains
Preparing primary seed liquid: according to the inoculation amount of 1% (v/v), the purified bacillus bailii HSCY3015 bacterial liquid in the logarithmic growth phase is inoculated into MRS culture medium, and the temperature is 30 ℃, and the rotating speed is 180r/min for 20h.
Preparing a secondary seed solution: selecting a 50L liquid fermentation tank, adding 35L of water, stirring at a speed of 120r/min, heating water to 90 ℃, adding 10kg of ground rice obtained by crushing and sieving with a 100-mesh sieve, adding 2 ten thousand U/mL of high-temperature alpha-amylase with an addition amount of 3.0mL, and preserving heat for 35min to obtain mash; then the temperature of the mash is reduced to 45 ℃, 5 ten thousand U/g saccharifying enzyme is added, the adding amount is 0.50g, and the temperature is kept for 35min, so as to prepare saccharifying liquid; adding 0.5% (w/v) peptone and 0.5% (w/v) yeast powder into the saccharified solution, and sterilizing at 115deg.C for 20min; cooling to 30deg.C, inoculating first-stage seed solution according to 1% inoculum size, adjusting ventilation to 0.1vvm, stirring at 120r/min, fermenting at 30deg.C for 20 hr under pressure maintaining, and viable count reaching 10 8 cfu/mL. A colony morphology of Bacillus belicus grown on solid medium is shown in FIG. 1.
2. Inoculating and fermenting vinegar grains
Control group 1:
selecting 500kg vat, adding 132kg of wine mash, 45kg of bran and 22kg of bran, fully and uniformly stirring the raw materials, inoculating acetic acid bacteria with CICC (common cold) and bacterial strain preservation number of CICC 20001, wherein the inoculum size is 1% of the weight of the wine mash, and carrying out seed carrying and solid layering fermentation according to the Zhenjiang aromatic vinegar brewing process until the fermentation is finished.
Bacillus beijerinus group was added:
selecting 500kg vat, adding 132kg of beer, 45kg of bran and 22kg of bran, fully and uniformly stirring the materials, inoculating acetic acid bacteria with CICC (common cold) and bacterial strain preservation number of CICC 20001, wherein the inoculation amount is 1% of the weight of the beer, inoculating second-level seed liquid of bacillus bailii with the inoculation amount of 1% of the weight of the beer, and carrying out seed carrying and solid layering fermentation on the beer according to the Zhenjiang aromatic vinegar brewing process until the fermentation is finished.
3. Sealing fermented grains
And compacting the mature vinegar grains to seal the grains, wherein the sealing period is 7 days. Control group 1 procedure was consistent with example 1.
4. Vinegar for treating stranguria
Adding clear water into the fermented grains after sealing, soaking, adding stir-fried rice color accounting for 8.0% of the weight of the fermentation raw material and salt accounting for 1.5% of the weight of the fermentation raw material, and pouring after soaking to obtain raw vinegar. Control group 1 procedure was consistent with example 1.
5. Vinegar and filling
Decocting raw vinegar at 100deg.C for 30min, and adding 2% white granulated sugar. Hot filling the mature vinegar liquid, wherein the filling temperature is not lower than 70 ℃, and the finished product is obtained after the filling is finished. Control group 1 procedure was consistent with example 1.
6. Total acid content detection
The bottom vinegar-halogen is taken daily from the beginning of fermentation on day 5 to the end of fermentation on day 19 for detection. The total acid is measured by acid-base titration based on acetic acid. At the end of fermentation, the total acid content of the Bacillus belicus group added was 8.16g/100mL, the control group 1 was 7.60g/100mL, and the total acid was raised by 7.4% as compared with the control group 1, and the total acid content change curves of the example 1 and the control group 1 are shown in FIG. 2.
7. Organic acid content detection
The content of 7 organic acids in the fermented vinegar brine was analyzed by HPLC, and the results are shown in Table 1. After fermentation, the organic acid in the vinegar brine mainly comprises acetic acid and lactic acid, the contents of which in the embodiment 1 are 56.85 mg/mL and 14.52mg/mL respectively, and the contents are improved by 15.71% and 17.19% respectively compared with the control group 1.
TABLE 1 organic acid content of Bacillus bailii HSCY3015 in Zhenjiang aromatic vinegar brewing
8. Detection of content of acetoin and ligustrazine in raw vinegar
The method for measuring the acetoin content by adopting the creatine colorimetric method comprises the following steps of: a series of acetoin standard solutions with the concentration of 25, 50, 75, 100, 125, 175 and 225mg/L are accurately prepared. 100 mu L of standard solution is respectively taken, 2.4 m deionized water, 0.5mL creatine solution (0.5%, w/v), 0.5mL L-naphthol solution (5%, w/v) and 0.5mL sodium hydroxide solution (10%, w/v) are added, evenly mixed, after water bath at 30 ℃ for 1h, the OD of the solution is measured 520nm . On the abscissa, the acetoin concentration, OD 520nm And drawing a standard curve for the ordinate. Taking 100 mu L of diluted sample, and obtaining sample OD according to a standard curve determination method 520nm And calculating the acetoin content by using a standard curve. The content of ligustrazine is measured by adopting an HPLC method, and the specific method comprises the following steps: a series of ligustrazine standard solutions with the concentration of 5, 10, 25, 50 and 100mg/L are accurately prepared, and a standard curve is obtained by an HPLC method. Accurately measuring 10mL of vinegar, regulating pH to be more than 8 by using sodium hydroxide, extracting the vinegar twice by using 10mL of chloroform, combining the two chloroform phases, extracting the chloroform phases twice by using 9mL of dilute hydrochloric acid solution (0.2 mol/L), combining the water phases, and then fixing the volume to 10mL by using dilute hydrochloric acid, wherein the obtained solution is used for obtaining the peak area of ligustrazine according to a standard curve determination method, and calculating the content of ligustrazine according to the standard curve. The detection results of acetoin and ligustrazine content in raw vinegar are shown in table 2.
TABLE 2 acetoin and ligustrazine content of Bacillus bailii HSCY3015 in Zhenjiang aromatic vinegar brewing
| Group of | Acetoin (g/L) | Ligustrazine (mg/L) |
| Example 1 | 21.89 | 216.23 |
| Control group 1 | 9.61 | 35.38 |
9. Sensory index analysis of finished vinegar
Table 3 shows the sensory index scoring results of the vinegar products of example 1 and control 1. The flavor and taste of the finished vinegar of example 1 are significantly better than those of control group 1, indicating that the addition of bacillus belicus during the acetic acid fermentation stage significantly improves the product quality.
TABLE 3 sensory index analysis of finished Vinegar from Bacillus bailii HSCY3015 in Zhenjiang aromatic Vinegar brewing
| Group of | Color (10 min) | Posture (10 min) | Fragrance (10 min) | Taste (10 min) |
| Example 1 | 9.0 | 9.1 | 9.6 | 9.2 |
| Control group 1 | 8.8 | 9.0 | 7.0 | 6.4 |
Example 2 application of Bacillus bailii HSCY3015 in Shanxi mature vinegar
1. Expansion culture of strains
Preparing primary seed liquid: according to the inoculation amount of 10% (v/v), the purified bacillus bailii HSCY3015 bacterial liquid in the logarithmic growth phase is inoculated into MRS culture medium, and the temperature is 37 ℃ and the rotating speed is 200r/min for culturing for 36h.
Preparing a secondary seed solution: selecting a 50L liquid fermentation tank, adding 35L of water, stirring at a speed of 150r/min, heating water to 95 ℃, adding 12kg of ground rice powder obtained by crushing and sieving with a 100-mesh sieve, adding 2 ten thousand U/mL of high-temperature alpha-amylase with an addition amount of 3.5mL, and preserving heat for 40min to obtain mash; then the temperature of the mash is reduced to 55 ℃, 5 ten thousand U/g saccharifying enzyme is added, the adding amount is 1.0g, and the temperature is kept for 40min, so as to prepare saccharifying liquid; adding 2.0% (w/v) peptone and 2.0% (w/v) yeast powder into the saccharified solution, sterilizing at 115deg.C for 20min, cooling to 37deg.C, inoculating first-stage seed solution according to 10% (v/v) inoculum size, adjusting aeration rate to 0.3vvm, stirring at 150r/min, and fermenting at 37deg.C for 36 hr until viable count reaches 10 8 cfu/mL。
2. Inoculating and fermenting vinegar grains
Control group 2:
selecting 500kg vat, adding 150kg of wine mash, 50kg of bran and 33kg of bran, fully and uniformly stirring the raw materials, inoculating acetic acid bacteria with CICC (common cold) and bacterial strain preservation number of CICC 20001, wherein the inoculation amount is 10% of the weight of the wine mash, and fermenting, fumigating and spraying vinegar according to the solid state fermentation process of Shanxi mature vinegar.
Bacillus beijerinus group was added:
selecting 500kg vat, adding 150kg of beer, 50kg of bran and 33kg of bran, fully and uniformly stirring the materials, inoculating acetic acid bacteria with CICC (common cold) and bacterial strain preservation number of CICC 20001, wherein the inoculation amount is 10% of the weight of the beer, inoculating second-stage seed liquid of bacillus beijerinckii with the inoculation amount of 5% of the weight of the beer, and fermenting, fumigating and spraying vinegar according to a solid state fermentation process of Shanxi mature vinegar.
3. Total acid content
The bottom vinegar-halogen is taken every day from the beginning of fermentation on day 3 to the end of fermentation on day 12 for detection. The total acid is measured in terms of acetic acid by acid-base titration. At the end of fermentation, the total acid content of the Bacillus belicus group was 5.42g/100mL, the control group 2 was 4.90g/100mL, and the total acid was increased by 10.61% as compared with the control group 2, and the total acid content change curves of the example 2 and the control group 2 are shown in FIG. 3.
4. Organic acid content
The content of 7 organic acids in the fermented vinegar brine was analyzed by HPLC, and the results are shown in Table 4. After fermentation, the organic acid in the vinegar brine mainly comprises acetic acid and lactic acid, the contents of the acetic acid and the lactic acid in the vinegar brine are 29.73 mg/mL and 11.36mg/mL respectively in the example 2, and the contents are improved by 14.83% and 17.84% respectively compared with the control group 2.
TABLE 4 organic acid content of Bacillus bailii HSCY3015 in Shanxi mature vinegar brewing
5. Detection of content of acetoin and ligustrazine in raw vinegar
The content of acetoin was measured by creatine colorimetric method and the content of ligustrazine was measured by HPLC method, and the results are shown in Table 5.
TABLE 5 acetoin and ligustrazine content of Bacillus bailii HSCY3015 in Shanxi aged vinegar brewing
| Group of | Acetoin (g/L) | Ligustrazine (mg/L) |
| Example 2 | 23.16 | 362.52 |
| Control group 2 | 8.43 | 95.89 |
6. Sensory index analysis of finished vinegar
Table 6 shows the sensory index scoring results of the vinegar products of example 2 and control group 2. The flavor and taste of the finished vinegar of example 2 are significantly better than control group 2, indicating that the addition of bacillus belicus during the acetic acid fermentation stage significantly improves the product quality.
Table 6 sensory index analysis of finished vinegar in Shanxi aged vinegar brewing by bacillus bailii HSCY3015
| Group of | Color (10 min) | Posture (10 min) | Fragrance (10 min) | Taste (10 min) |
| Example 2 | 9.5 | 9.2 | 9.6 | 9.7 |
| Control group 2 | 9.3 | 9.0 | 7.1 | 6.5 |
Example 3 application of Bacillus bailii HSCY3015 in liquid rice vinegar brewing
1. Preparation of bacillus bailii microbial inoculum
Inoculating Bacillus bailii into MRS culture medium according to 10% inoculum size, culturing at 40deg.C and 220: 220 r/min for 48 hr, centrifuging to collect thallus, mixing sterile protectant and thallus uniformly, placing in 45 deg.C incubator, heat shock for 8s, and spray drying at low temperature to obtain bacterial agent with living bacterial count of about 10 8 cfu/g microbial inoculum, and the storage period under the condition of cool and dry condition is 1 year.
Preparation of a sterility protective agent: the preparation method of the protective agent comprises the following steps: a: 10g of skimmed milk powder, 100mL of distilled water, and sterilizing at 115 ℃ for 15min; b: 0.5g of glycerin, 2g of sorbitol, 1g of xylitol, 1g of maltodextrin and 100mL of distilled water, and sterilizing for 15min at 121 ℃; and (3) respectively sterilizing the A and the B, cooling to room temperature, and mixing to obtain the protective agent.
Substrate spray drying process parameters: the air outlet temperature is 55 ℃, the air inlet temperature is 120 ℃, the drying time is 5 minutes, and the moisture content is less than or equal to 5 percent.
2. Inoculating fermentation
2 500L fermenters were selected as example 3 and control group 3, 100L of rice wine with an alcohol content of 6-8% vol was added, acetic acid bacteria purchased from CICC and having a strain preservation number of CICC 20001 were inoculated according to an inoculum size of 10%, acetic acid fermentation was performed at a temperature of 30 ℃, a ventilation amount of 0.34vvm, and a stirring speed of 200 r/min. When the alcoholic strength was reduced to 3% vol or less, 5g of activated Bacillus belicus was added to the fermenter of example 3, and acetic acid fermentation was continued without adding to the fermenter of control group 3. The activation method comprises the following steps: 1g of microbial inoculum is dissolved in 10mL of sterile water and activated for 30min at 35 ℃ for later use.
3. Physical and chemical index detection
After the fermentation, the total acid, lactic acid and acetoin contents of the brewed rice vinegar were respectively detected, as shown in Table 7. The total acid is measured by acid-base titration based on acetic acid. The lactic acid adopts a high performance liquid chromatography method, and refers to the method in annex B of the Zhenjiang aromatic vinegar which is a geographic marking product of GB/T18623-2011. The acetoin content is determined by creatine colorimetric method. The results are shown in Table 7.
Table 7 physical and chemical index detection of Bacillus bailii HSCY3015 in rice vinegar brewing
4. Sensory index analysis of finished vinegar
The sensory index scoring results of the rice vinegar products of example 3 and control 3 are shown in Table 8. The taste score of the finished vinegar of example 3 is obviously better than that of the control group 3, which shows that the addition of bacillus beijerinckii in the acetic acid fermentation stage obviously contributes to the taste of the rice vinegar and obviously improves the quality of the product.
Table 8 sensory index analysis of finished vinegar in rice vinegar brewing by bacillus bailii HSCY3015
| Group of | Color (10 min) | Posture (10 min) | Fragrance (10 min) | Taste (10 min) |
| Example 3 | 9.7 | 9.5 | 9.8 | 9.6 |
| Control group 3 | 9.6 | 9.5 | 6.9 | 7.1 |
Sequence listing
<110> Jiangsu Hengshun Vinegar industry Co., ltd
<120> Bacillus bailii, microbial agent and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1419
<212> DNA/RNA
<213> Bacillus bailii (bacillus velezensis)
<400> 1
catgcaagtc gagcggacag atgggagctt gctccctgat gttagcggcg gacgggtgag 60
taacacgtgg gtaacctgcc tgtaagactg ggataactcc gggaaaccgg ggctaatacc 120
ggatggttgt ttgaaccgca tggttcagac ataaaaggtg gcttcggcta ccacttacag 180
atggacccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc gacgatgcgt 240
agccgacctg agagggtgat cggccacact gggactgaga cacggcccag actcctacgg 300
gaggcagcag tagggaatct tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga 360
gtgatgaagg ttttcggatc gtaaagctct gttgttaggg aagaacaagt gccgttcaaa 420
tagggcggca ccttgacggt acctaaccag aaagccacgg ctaactacgt gccagcagcc 480
gcggtaatac gtaggtggca agcgttgtcc ggaattattg ggcgtaaagg gctcgcaggc 540
ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg ggagggtcat tggaaactgg 600
ggaacttgag tgcagaagag gagagtggaa ttccacgtgt agcggtgaaa tgcgtagaga 660
tgtggaggaa caccagtggc gaaggcgact ctctggtctg taactgacgc tgaggagcga 720
aagcgtgggg agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgatgagtgc 780
taagtgttag ggggtttccg ccccttagtg ctgcagctaa cgcattaagc actccgcctg 840
gggagtacgg tcgcaagact gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 900
agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac atcctctgac 960
aatcctagag ataggacgtc cccttcgggg gcagagtgac aggtggtgca tggttgtcgt 1020
cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgatcttagt 1080
tgccagcatt cagttgggca ctctaaggtg actgccggtg acaaaccgga ggaaggtggg 1140
gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta caatggacag 1200
aacaaagggc agcgaaaccg cgaggttaag ccaatcccac aaatctgttc tcagttcgga 1260
tcgcagtctg caactcgact gcgtgaagct ggaatcgcta gtaatcgcgg atcagcatgc 1320
cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga gagtttgtaa 1380
cacccgaagt cggtgaggta accttttagg agccagccg 1419
Claims (10)
1. A bacillus beleiensis strain, characterized in that: the bacillus belicus is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) with a preservation date of 2020, 6 and 10 days, and a preservation registration book with a number of CGMCC NO.20063.
2. A microbial agent is characterized in that: the microbial agent comprises bacillus belicus according to claim 1.
3. Use of bacillus belicus according to claim 1 or the microbial agent according to claim 2 in the field of food.
4. Use of bacillus belicus according to claim 1 or the microbial agent according to claim 2 in vinegar brewing.
5. The use of bacillus belgium or microbial agents according to claim 4, in vinegar brewing, characterized in that: the edible vinegar is aromatic vinegar.
6. A brewing method of table vinegar is characterized by comprising the following steps:
(1) Preparing a strain seed solution using the bacillus belicus of claim 1;
(2) Fermenting the strain seed liquid to obtain the edible vinegar.
7. The method for brewing vinegar according to claim 6, wherein: in the step (1), the preparation of the strain seed liquid comprises the preparation of a primary seed liquid and the preparation of a secondary seed liquid.
8. The method for producing vinegar according to claim 7, wherein: the preparation of the first-level seed liquid specifically comprises the following steps: according to the inoculation amount of 1% -10%, inoculating the purified bacillus beijerinckii bacterial liquid in logarithmic growth phase into a culture medium, and culturing at 30-37 ℃ to obtain first-stage seed liquid.
9. The method for producing vinegar according to claim 7, wherein: the preparation of the secondary seed liquid specifically comprises the following steps: heating water to 90-95deg.C by liquid fermentation method, adding rice flour and high temperature alpha-amylase, and maintaining the temperature to obtain mash; then the temperature of the mash is reduced to 45-55 ℃, and saccharifying enzyme is added for heat preservation, thus obtaining saccharified liquid; adding 0.5% -2% of peptone and 0.5% -2% of yeast powder into the saccharification liquid, and sterilizing; cooling to 30-37 ℃, inoculating the first-stage seed liquid according to the inoculum size of 1-10%, ventilating and stirring, and performing pressure maintaining fermentation at 30-37 ℃ to obtain the second-stage seed liquid.
10. The method for brewing vinegar according to claim 9, wherein the step (2) specifically comprises the steps of: mixing and stirring the beer mash, bran and bran, inoculating acetic acid bacteria according to the inoculation amount of 1-10% of the weight of the beer mash, inoculating secondary seed liquid of bacillus bailii according to the inoculation amount of 1-5% of the weight of the beer mash, and carrying out seed-carrying and solid layered fermentation; sealing fermented grains, spraying vinegar and decocting vinegar.
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