CN103865833A - Preparation method of thermophilic bacillus subtilis microbial inoculum - Google Patents
Preparation method of thermophilic bacillus subtilis microbial inoculum Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 42
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 19
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 19
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 18
- 235000003434 Sesamum indicum Nutrition 0.000 claims abstract description 12
- 239000000796 flavoring agent Substances 0.000 claims abstract description 12
- 235000019634 flavors Nutrition 0.000 claims abstract description 12
- 238000000855 fermentation Methods 0.000 claims abstract description 11
- 230000004151 fermentation Effects 0.000 claims abstract description 11
- 241000894006 Bacteria Species 0.000 claims description 16
- 235000015097 nutrients Nutrition 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000003795 chemical substances by application Substances 0.000 claims description 14
- 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 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 10
- 235000015278 beef Nutrition 0.000 claims description 10
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 239000012533 medium component Substances 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 229940066779 peptones Drugs 0.000 claims description 10
- 230000000813 microbial effect Effects 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 5
- 238000009631 Broth culture Methods 0.000 claims description 5
- 241000726221 Gemma Species 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 239000006916 nutrient agar Substances 0.000 claims description 5
- 235000016709 nutrition Nutrition 0.000 claims description 5
- 230000001681 protective effect Effects 0.000 claims description 5
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 3
- 244000000231 Sesamum indicum Species 0.000 claims 1
- 238000005728 strengthening Methods 0.000 claims 1
- 241000207961 Sesamum Species 0.000 abstract description 11
- 108091005804 Peptidases Proteins 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 6
- 239000004365 Protease Substances 0.000 abstract description 4
- 230000007062 hydrolysis Effects 0.000 abstract description 3
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 208000012788 shakes Diseases 0.000 abstract 1
- 239000007858 starting material Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 22
- 102000035195 Peptidases Human genes 0.000 description 9
- 230000012010 growth Effects 0.000 description 7
- 238000000034 method Methods 0.000 description 4
- 235000019833 protease Nutrition 0.000 description 4
- 108090000145 Bacillolysin Proteins 0.000 description 3
- 102000035092 Neutral proteases Human genes 0.000 description 3
- 108091005507 Neutral proteases Proteins 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000010200 folin Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
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- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a preparation method of a thermophilic bacillus subtilis microbial inoculum. Bacillus subtilis are separated from high temperature koji fermented by sesame flavor liquor and have the characteristics of resisting high temperature and generating high protease hydrolysis activity and the like. By using the preparation method, the microbial inoculum is prepared through slope seeds, cultured by a primary shake flask and re-cultured by secondary expansion; the prepared microbial inoculum is very suitable for the thermophilic fermentation process of the sesame flavor liquor; the sesame flavor liquor has very high potential use values in high temperature starter propagation and brewing.
Description
Technical field
The present invention relates to preparation method and the characteristic thereof of bacillus subtilis microbial agent, be specifically related to the high temperature tolerance of this bacillus subtilis microbial agent and produce proteinase activity.
Background technology
Subtilis be distilled spirit with sesame flavour high temperature koji and brewage in critical function microorganism.The subtilis CGMCC 8087 (=CICC 15073) that is located away from the high-temperature daqu of distilled spirit with sesame flavour fermentation can tolerate higher temperature (55 ℃ of well-growns, and 55 ℃ of general subtilises can not grow), can produce higher hydrolysis vigor neutral protease.In the fermenting processs such as high temperature koji, the thermophilic protease that this bacterium produces can produce free amino acid by degrade proteins, for the fermenting processs such as brewed spirit provide important precursor substance.Therefore, be applicable to very much the thermophilic fermentation process of distilled spirit with sesame flavour by cultivating step by step the bacillus subtilis microbial agent of preparation, there is very high potential use value in distilled spirit with sesame flavour high temperature koji with in brewageing.
Summary of the invention
The object of the invention is to: a kind of preparation method of thermophilic bacillus subtilis microbial agent is provided, and this microbial inoculum can withstand higher temperatures, and the ability of higher protease hydrolysis vigor.By inclined-plane seed activation, primary seed solution is cultivated, secondary expands cultivation again and vacuum lyophilization has prepared this bacillus subtilis microbial agent, the thermophilic fermentation process that is applicable to very much distilled spirit with sesame flavour, has very high potential use value in distilled spirit with sesame flavour high temperature koji with in brewageing.
Technical solution of the present invention is the preparation of this bacillus subtilis microbial agent.
1) subtilis withered grass subspecies
bacillus subtilis subsp. subtilisbacterial strain: be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on August 30th, 2013, be numbered CGMCC 8087 (=CICC 15073) (product heat resistant proteases), the Genebank accession number of its 16S rDNA sequence is KF742413; This bacterium bacterium colony is oyster white, circle, and surface irregularity, neat in edge, opaque, compared with thickness, easily picking.Bacterium colony size is (0.3-0.4) cm × (0.3-0.4) cm, and colony growth is rapid.Micro-Microscopic observation, thalline is elongated rod shape, single in the majority, has minority two connecting rods, three connecting rods, chain phenomenon.The growth temperature range of this bacterium is 25-55 ℃ (general subtilis 55 ℃ can not grow).
2) preparation of inclined-plane seed: subtilis CGMCC 8087 (=CICC 15073) is inoculated in nutrient agar slant medium, cultivates at 50 ℃ 24 hours, prepare the inclined-plane seed of thermophilic subtilis; Medium component used: 10.0 grams of peptones, 3.0 grams of beef powders, 5.0 grams, sodium-chlor, 1.0 grams of glucose, 20 grams of agar powders, 1000 milliliters of distilled water, pH 7.0;
3) preparation of primary seed solution: get a ring test tube slant seed and be transferred to liquid nutritional broth culture, at 220 revs/min, cultivate the preparation of carrying out primary seed solution for 24 hours under 50 ℃ of conditions, obtain one-level nutrient solution; Wherein, medium component used: 10.0 grams of peptones, 3.0 grams of beef powders, 5.0 grams, sodium-chlor, 1.0 grams of glucose, 1000 milliliters of distilled water, pH 7.0;
4) preparation of secondary nutrient solution: first order seed bacteria suspension is linked into (water content is 50%) in bran mass by 2% inoculum size, and at 220 revs/min, 45-55 ℃ of condition bottom fermentation cultivated the preparation of carrying out secondary nutrient solution for 24 hours.
5) preparation of microbial inoculum: collect secondary and cultivate the bacillus subtilis strain obtaining, prepare gemma bacillus agent by vacuum lyophilization (12% skimmed milk is as protective material).
Embodiment
Embodiment 1: prepare subtilis CGMCC 8087 (=CICC 15073) microbial inoculum according to following steps
1) preparation of inclined-plane seed: subtilis CGMCC 8087 (=CICC 15073) is inoculated in nutrient agar slant medium, cultivates at 50 ℃ 24 hours, prepare the inclined-plane seed of thermophilic subtilis; Medium component used: 10.0 grams of peptones, 3.0 grams of beef powders, 5.0 grams, sodium-chlor, 1.0 grams of glucose, 20 grams of agar powders, 1000 milliliters of distilled water, pH 7.0;
2) preparation of primary seed solution: get a ring test tube slant seed and be transferred to liquid nutritional broth culture, at 220 revs/min, cultivate the preparation of carrying out primary seed solution for 24 hours under 50 ℃ of conditions, obtain one-level nutrient solution; Wherein, medium component used: 10.0 grams of peptones, 3.0 grams of beef powders, 5.0 grams, sodium-chlor, 1.0 grams of glucose, 1000 milliliters of distilled water, pH 7.0;
3) preparation of secondary nutrient solution: first order seed bacteria suspension is linked into (water content is 50%) in bran mass by 2% inoculum size, and at 220 revs/min, 45 ℃ of condition bottom fermentations are cultivated the preparation of carrying out secondary nutrient solution for 24 hours.
4) preparation of microbial inoculum: collect secondary and cultivate the bacillus subtilis strain obtaining, prepare gemma bacillus agent by vacuum lyophilization (12% skimmed milk is as protective material).
Embodiment 2: prepare subtilis CGMCC 8087 (=CICC 15073) microbial inoculum according to following steps
1) preparation of inclined-plane seed: subtilis CGMCC 8087 (=CICC 15073) is inoculated in nutrient agar slant medium, cultivates at 50 ℃ 24 hours, prepare the inclined-plane seed of thermophilic subtilis; Medium component used: 10.0 grams of peptones, 3.0 grams of beef powders, 5.0 grams, sodium-chlor, 1.0 grams of glucose, 20 grams of agar powders, 1000 milliliters of distilled water, pH 7.0;
2) preparation of primary seed solution: get a ring test tube slant seed and be transferred to liquid nutritional broth culture, at 220 revs/min, cultivate the preparation of carrying out primary seed solution for 24 hours under 50 ℃ of conditions, obtain one-level nutrient solution; Wherein, medium component used: 10.0 grams of peptones, 3.0 grams of beef powders, 5.0 grams, sodium-chlor, 1.0 grams of glucose, 1000 milliliters of distilled water, pH 7.0;
3) preparation of secondary nutrient solution: first order seed bacteria suspension is linked into (water content is 50%) in bran mass by 2% inoculum size, and at 220 revs/min, 50 ℃ of condition bottom fermentations are cultivated the preparation of carrying out secondary nutrient solution for 24 hours.
4) preparation of microbial inoculum: collect secondary and cultivate the bacillus subtilis strain obtaining, prepare gemma bacillus agent by vacuum lyophilization (12% skimmed milk is as protective material).
Embodiment 3: prepare subtilis CGMCC 8087 (=CICC 15073) microbial inoculum according to following steps
1) preparation of inclined-plane seed: subtilis CGMCC 8087 (=CICC 15073) is inoculated in nutrient agar slant medium, cultivates at 50 ℃ 24 hours, prepare the inclined-plane seed of thermophilic subtilis; Medium component used: 10.0 grams of peptones, 3.0 grams of beef powders, 5.0 grams, sodium-chlor, 1.0 grams of glucose, 20 grams of agar powders, 1000 milliliters of distilled water, pH 7.0;
2) preparation of primary seed solution: get a ring test tube slant seed and be transferred to liquid nutritional broth culture, at 220 revs/min, cultivate the preparation of carrying out primary seed solution for 24 hours under 50 ℃ of conditions, obtain one-level nutrient solution; Wherein, medium component used: 10.0 grams of peptones, 3.0 grams of beef powders, 5.0 grams, sodium-chlor, 1.0 grams of glucose, 1000 milliliters of distilled water, pH 7.0;
3) preparation of secondary nutrient solution: first order seed bacteria suspension is linked into (water content is 50%) in bran mass by 2% inoculum size, and at 220 revs/min, 55 ℃ of condition bottom fermentations are cultivated the preparation of carrying out secondary nutrient solution for 24 hours.
4) preparation of microbial inoculum: collect secondary and cultivate the bacillus subtilis strain obtaining, prepare gemma bacillus agent by vacuum lyophilization (12% skimmed milk is as protective material).
In embodiment 1-3, after differing temps secondary is cultivated, compared with the subtilis of laboratory, measure the viable bacteria colony number of cultivating as shown in table 1.
Viable bacteria colony number under table 1 differing temps culture condition
Note: "-" represents not growth
Table 1 shows: the optimum temperuture of bacterial strain CGMCC 8087 (=CICC 15073) growth is 50 ℃, but it can be 55 ℃ of normal growths (general subtilis 55 ℃ can not grow).And by relatively can finding out of the viable count before and after dry, it is practicable that vacuum freeze-drying method is prepared microbial inoculum.
In embodiment 1-2, after differing temps secondary is cultivated, detect neutral protease vigor, as shown in table 2.
Table 2: bacterial strain CGMCC 8087 under condition of different temperatures (=CICC 15073) produces proteinase activity comparison
Table 2 shows: the proteolytic enzyme optimum temperuture that bacterial strain CGMCC 8087 (=CICC 15073) produces is 50 ℃, and maximum enzyme vigor is 141.8U/g.Note: proteinase activity measuring method: 1mL casein is placed in vitro, preheating 5min in 40 ℃ of water-baths, then adds the bacteria culture fluid through the 1mL of same preheating, is accurately incubated 10min, add immediately 2mL 0.4mol/L trichoroacetic acid(TCA) with enzymolysis reaction, centrifugal after continuation insulation 20min.Get filtrate 1mL, add 0.4mol/L sodium carbonate 5mL and Folin reagent 1mL, shake up, different mensuration in temperature water-bath measured optical density(OD) (OD) after insulation colour developing 20min at 680nm wavelength place.Blank solution is measured the same, just before adding casein, first adds 2mL 0.4mol/L trichoroacetic acid(TCA), makes enzyme deactivation, and then adds casein.Proteinase activity calculation formula is:
the corresponding tyrosine concentration of the final liquid of sample that A1-is drawn by typical curve; The extension rate of n1-sample; The volume (mL) of the volumetric flask that V1-dissolution sample is used; The cumulative volume (mL) of 4-reaction reagent; The quality (g) of m4-sample; 10-reaction times 10min.
By comparing the preparation method of the thermophilic bacillus subtilis microbial agent that under condition of different temperatures, (45 ℃ 50 ℃ and 55 ℃) are cultivated, we can find out that the optimum growth temperature of subtilis CGMCC 8087 (=CICC 15073) is 50 ℃, the viable bacteria number maximum (1.6 × 10 obtaining after cultivating for 24 hours
10the viable bacteria number of the microbial inoculum cfu), preparing can reach 1.6 × 10
10cfu.This bacterium of what is more important can be at 55 ℃ of normal growths, and the microbial inoculum preparing can reach 5 × 10
8cfu, and general subtilis can not grow at 55 ℃.The activity the highest (141.8U/g) of the neutral protease that this bacterium produces, other that separate from high-temperature daqu far above laboratory produce the bacterial strain of proteolytic enzyme.The preparation method of the vacuum lyophilization that in addition, we adopt also can obtain enough microbial inoculums.Therefore, the microbial inoculum of being prepared by thermophilic subtilis CGMCC 8087 (=CICC 15073) is applicable to the thermophilic fermentation process of distilled spirit with sesame flavour very much, has very high potential use value in distilled spirit with sesame flavour high temperature koji with in brewageing.
Claims (2)
1. a preparation method for thermophilic bacillus subtilis microbial agent, is characterized in that: using thermophilic subtilis CGMCC 8087 (=CICC 15073) as strengthening bacterial strain.
2. preparation method as claimed in claim 1, is characterized in that: comprise the steps:
1) bacillus subtilis strain: subtilis CGMCC 8087 (=CICC 15073) is located away from the high-temperature daqu of distilled spirit with sesame flavour fermentation, be preserved in Chinese common micro-organisms DSMZ, be numbered CGMCC 8087 (=CICC 15073);
2) preparation of inclined-plane seed: subtilis CGMCC 8087 (=CICC 15073) is inoculated in nutrient agar slant medium, cultivates at 50 ℃ 24 hours, prepare the inclined-plane seed of thermophilic subtilis; Medium component used: 10.0 grams of peptones, 3.0 grams of beef powders, 5.0 grams, sodium-chlor, 1.0 grams of glucose, 20 grams of agar powders, 1000 milliliters of distilled water, pH 7.0;
3) preparation of primary seed solution: get a ring test tube slant seed and be transferred to liquid nutritional broth culture, at 220 revs/min, cultivate the preparation of carrying out primary seed solution for 24 hours under 50 ℃ of conditions, obtain one-level nutrient solution; Wherein, medium component used: 10.0 grams of peptones, 3.0 grams of beef powders, 5.0 grams, sodium-chlor, 1.0 grams of glucose, 1000 milliliters of distilled water, pH 7.0;
4) preparation of secondary nutrient solution: first order seed bacteria suspension is linked into (water content is 50%) in bran mass by 2% inoculum size, and at 220 revs/min, 45-55 ℃ of condition bottom fermentation cultivated the preparation of carrying out secondary nutrient solution for 24 hours;
5) preparation of microbial inoculum: collect secondary and cultivate the bacillus subtilis strain obtaining, prepare gemma bacillus agent by vacuum lyophilization (12% skimmed milk is as protective material).
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105685479A (en) * | 2016-03-09 | 2016-06-22 | 广东海洋大学 | Preparation method of fermented soybean meal |
| CN105746890A (en) * | 2016-03-09 | 2016-07-13 | 广东海洋大学 | Method of utilizing fermentation of aquatic product protein and plant feed protein to prepare fish meal replacing protein |
| CN107475159A (en) * | 2017-09-19 | 2017-12-15 | 湖北白云边酒业股份有限公司 | Bacillus subtilis and its application in Sauce flavor white wine |
| CN110408557A (en) * | 2019-06-17 | 2019-11-05 | 常州大学 | A kind of bacillus subtilis that improves soil phosphatase activity and preparation method thereof |
| CN110734903A (en) * | 2019-11-18 | 2020-01-31 | 山东隆科特酶制剂有限公司 | method for producing high-temperature resistant neutral protease |
| CN110760466A (en) * | 2019-11-18 | 2020-02-07 | 山东隆科特酶制剂有限公司 | Bacillus subtilis for producing high-temperature-resistant neutral protease and application thereof |
| CN110846176A (en) * | 2019-12-03 | 2020-02-28 | 江苏洋河酒厂股份有限公司 | Method for producing peptide koji from white spirit |
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| CN102618474A (en) * | 2012-04-10 | 2012-08-01 | 江苏今世缘酒业股份有限公司 | Bacillus subtilis and separate culture method for same |
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Cited By (9)
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| CN105685479A (en) * | 2016-03-09 | 2016-06-22 | 广东海洋大学 | Preparation method of fermented soybean meal |
| CN105746890A (en) * | 2016-03-09 | 2016-07-13 | 广东海洋大学 | Method of utilizing fermentation of aquatic product protein and plant feed protein to prepare fish meal replacing protein |
| CN107475159A (en) * | 2017-09-19 | 2017-12-15 | 湖北白云边酒业股份有限公司 | Bacillus subtilis and its application in Sauce flavor white wine |
| CN110408557A (en) * | 2019-06-17 | 2019-11-05 | 常州大学 | A kind of bacillus subtilis that improves soil phosphatase activity and preparation method thereof |
| CN110734903A (en) * | 2019-11-18 | 2020-01-31 | 山东隆科特酶制剂有限公司 | method for producing high-temperature resistant neutral protease |
| CN110760466A (en) * | 2019-11-18 | 2020-02-07 | 山东隆科特酶制剂有限公司 | Bacillus subtilis for producing high-temperature-resistant neutral protease and application thereof |
| CN110760466B (en) * | 2019-11-18 | 2021-04-20 | 山东隆科特酶制剂有限公司 | Bacillus subtilis for producing high-temperature-resistant neutral protease and application thereof |
| CN110734903B (en) * | 2019-11-18 | 2021-06-04 | 山东隆科特酶制剂有限公司 | Method for producing high-temperature-resistant neutral protease |
| CN110846176A (en) * | 2019-12-03 | 2020-02-28 | 江苏洋河酒厂股份有限公司 | Method for producing peptide koji from white spirit |
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Application publication date: 20140618 |