CN102618474B - Bacillus subtilis and separate culture method for same - Google Patents
Bacillus subtilis and separate culture method for same Download PDFInfo
- Publication number
- CN102618474B CN102618474B CN 201210101364 CN201210101364A CN102618474B CN 102618474 B CN102618474 B CN 102618474B CN 201210101364 CN201210101364 CN 201210101364 CN 201210101364 A CN201210101364 A CN 201210101364A CN 102618474 B CN102618474 B CN 102618474B
- Authority
- CN
- China
- Prior art keywords
- subtilis
- bacillus subtilis
- genus bacillus
- medium
- same
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to bacillus subtilis and a separate culture method for the same. The bacillus subtilis is separated from high-temperature yeast manufactured by Jin Shi Yuan bioengineering company, and after performance comparison tests, the bacillus subtilis has the advantages of high temperature resistance, fine pyrazine compound productivity, high adaptability, easiness in culture and the like, and the quality and the yield of sesame wine can be improved.
Description
Technical field
The present invention relates to the isolation cultivation method of bacterium and bacterium, be specifically related to a kind of subtilis and isolation cultivation method thereof.
Background technology
Distilled spirit with sesame flavour is raw material with grain, Daqu, wheat bran etc. are saccharifying ferment, utilize nature microorganism open type fermented, mould, yeast, bacterium synergy, incorporate the fragrant build up process of sauce, formation has the liquor of individual style, and mixing was applied to wine brewing production after general distilled spirit with sesame flavour wheat bran adopted many bacterial classifications pure cultures such as bacterium, yeast, mould, by actual production as can be known, the performance of microbial strains has significant effects to the quality of sesame-flavor wine.
Summary of the invention
The objective of the invention is to: a kind of subtilis and isolation cultivation method thereof are provided, obtain subtilis high temperature resistant, that product pyrazine compounds ability is better, adaptive faculty is strong by this isolation cultivation method, improve sesame cordiale quality and yield.
Technical solution of the present invention is: this subtilis B3 Bacillus subtilis bacterial strain is that separation screening obtains from the high temperature song, in on November 29th, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, bacterium numbering is: CGMCC No. 5512.
Wherein, the isolation cultivation method of this subtilis may further comprise the steps: get the bent sample 10g of high temperature that this life, the cultivation of edge bio-engineering corporation was made and be inoculated in the 100mL genus bacillus enrichment medium, cultivated 18～24 hours, and got bacteria suspension for 35 ℃; Place 80 ℃ of water-baths to heat 10～20 minutes to kill the thalline that can not form gemma above-mentioned bacteria suspension; The bacteria suspension dilution 10 of subsequently heat treated being crossed
-3, 10
-4, 10
-5, it is in the 90mm sterile petri dish that the sample of each dilution gradient is got 1mL adding diameter, pours among genus bacillus isolation medium 25 mL that melt insulation and mixes, and is inverted in 50-60 ℃ of incubator after waiting to solidify and cultivates 24～48 hours; The bacterium colony of form rule in the picking culture dish, streak culture on the genus bacillus isolation medium, carry out repetitious separation and purification, obtain single purebred bacterium colony, microscopy is defined as adopting 16S rDNA sequence homology analysis behind the rod-shaped bacterium, filter out subtilis, it is standby to be preserved in the nutrient agar medium inclined-plane; The strong bacterial strain called after B3 of the vigor of behind strain identification, getting.
Wherein, genus bacillus enrichment medium: peptone 10g, KH
2PO
41.5g, yeast extract paste 3g, Na
2HPO
42g, starch 3g, MgSO
47H
2O 0.1g, H
2O 1000 mL, 121 ℃ of pH7.8,15 minutes.
Wherein, genus bacillus isolation medium: by genus bacillus enrichment medium and 7
0The Bx malt extract medium, the quality mixing with 1:1 after dissolving forms.
The present invention's separation screening from the high temperature song obtains subtilis, the heat resistance of subtilis is better, 60 ℃ still can keep good performance, and having the ability of producing pyrazine compound preferably, the wheat bran that adopts this bacterial classification to produce is used for the quality percentage that distilled spirit with sesame flavour production can improve self-produced wine.
Embodiment
Further specify technical solution of the present invention below in conjunction with specific embodiment, embodiment just is used for understanding technical scheme, and can not be interpreted as it is restriction to technical solution.
Embodiment 1: according to following steps separation and Culture subtilis
Get the bent sample 10g of high temperature that this life, the cultivation of edge bio-engineering corporation was made and be inoculated in the 100mL genus bacillus enrichment medium, cultivated 18 hours, and got bacteria suspension for 35 ℃; Place 80 ℃ of water-baths to heat 10 minutes to kill the thalline that can not form gemma above-mentioned bacteria suspension; Suitably dilution 10 of the bacteria suspension that heat treated is crossed subsequently
-3, 10
-4, 10
-5, it is in the 90mm sterile petri dish that the sample of each dilution gradient is got 1mL adding diameter, pours among genus bacillus isolation medium 25 mL that melt insulation and mixes, and is inverted in 50 ℃ of incubators after waiting to solidify and cultivates 48 hours; The bacterium colony of form rule in the picking culture dish, streak culture on the genus bacillus isolation medium, carry out repetitious separation and purification, obtain single purebred bacterium colony, microscopy is defined as adopting 16S rDNA sequence homology analysis behind the rod-shaped bacterium, filter out subtilis, it is standby to be preserved in the nutrient agar medium inclined-plane; The strong bacterial strain called after B3 of the vigor of behind strain identification, getting; Wherein, genus bacillus enrichment medium: peptone 10g, KH
2PO
41.5g, yeast extract paste 3g, Na
2HPO
42g, starch 3g, MgSO
47H
2O 0.1g, H
2O 1000 mL, 121 ℃ of pH7.8,15 minutes; Wherein, genus bacillus isolation medium: by genus bacillus enrichment medium and 7
0The Bx malt extract medium, the quality mixing with 1:1 after dissolving forms.
Embodiment 2: according to following steps separation and Culture subtilis
Get the bent sample 10g of high temperature that this life, the cultivation of edge bio-engineering corporation was made and be inoculated in the 100mL genus bacillus enrichment medium, cultivated 21 hours, and got bacteria suspension for 35 ℃; Place 80 ℃ of water-baths to heat 15 minutes to kill the thalline that can not form gemma above-mentioned bacteria suspension; Suitably dilution 10 of the bacteria suspension that heat treated is crossed subsequently
-3, 10
-4, 10
-5, it is in the 90mm sterile petri dish that the sample of each dilution gradient is got 1mL adding diameter, pours among genus bacillus isolation medium 25 mL that melt insulation and mixes, and is inverted in 55 ℃ of incubators after waiting to solidify and cultivates 36 hours; The bacterium colony of form rule in the picking culture dish, streak culture on the genus bacillus isolation medium, carry out repetitious separation and purification, obtain single purebred bacterium colony, microscopy is defined as adopting 16S rDNA sequence homology analysis behind the rod-shaped bacterium, filter out subtilis, it is standby to be preserved in the nutrient agar medium inclined-plane; The strong bacterial strain called after B3 of the vigor of behind strain identification, getting; Wherein, genus bacillus enrichment medium: peptone 10g, KH
2PO
41.5g, yeast extract paste 3g, Na
2HPO
42g, starch 3g, MgSO
47H
2O 0.1g, H
2O 1000 mL, 121 ℃ of pH7.8,15 minutes; Wherein, genus bacillus isolation medium: by genus bacillus enrichment medium and 7
0The Bx malt extract medium, the quality mixing with 1:1 after dissolving forms.
Embodiment 3: according to following steps separation and Culture subtilis
Get the bent sample 10g of high temperature that this life, the cultivation of edge bio-engineering corporation was made and be inoculated in the 100mL genus bacillus enrichment medium, cultivated 24 hours, and got bacteria suspension for 35 ℃; Place 80 ℃ of water-baths to heat 20 minutes to kill the thalline that can not form gemma above-mentioned bacteria suspension; Suitably dilution 10 of the bacteria suspension that heat treated is crossed subsequently
-3, 10
-4, 10
-5, it is in the 90mm sterile petri dish that the sample of each dilution gradient is got 1mL adding diameter, pours among genus bacillus isolation medium 25 mL that melt insulation and mixes, and is inverted in 60 ℃ of incubators after waiting to solidify and cultivates 24 hours; The bacterium colony of form rule in the picking culture dish, streak culture on the genus bacillus isolation medium, carry out repetitious separation and purification, obtain single purebred bacterium colony, microscopy is defined as adopting 16S rDNA sequence homology analysis behind the rod-shaped bacterium, filter out subtilis, it is standby to be preserved in the nutrient agar medium inclined-plane; The strong bacterial strain called after B3 of the vigor of behind strain identification, getting; Wherein, genus bacillus enrichment medium: peptone 10g, KH
2PO
41.5g, yeast extract paste 3g, Na
2HPO
42g, starch 3g, MgSO
47H
2O 0.1g, H
2O 1000 mL, 121 ℃ of pH7.8,15 minutes; Wherein, genus bacillus isolation medium: by genus bacillus enrichment medium and 7
0The Bx malt extract medium, the quality mixing with 1:1 after dissolving forms.
Comparative example 4: bacterial classification is optimized simultaneous test
The bacterial strain (B3) of embodiment 2 and four bacillus subtilis (B1, B12, B13, B16) of laboratory preservation are linked into respectively in the bran mass, and inoculum size is 1%, in 60 ℃ of cultivation 48h, measures its pyrazine compounds content, and is as shown in table 1,
The content of pyrazine compounds in the solid-state cultured products of table 1 different strains
Data results shows: subtilis B
3The pyrazine compounds total amount of producing the highest, especially the content of Tetramethylpyrazine is the highest, according to the correlative study bibliographical information, Tetramethylpyrazine has vasodilation, microcirculation improvement and suppresses effect such as platelet aggregation.
Claims (1)
1. subtilis, it is characterized in that: this subtilis B3 Bacillus subtilis bacterial strain is that separation screening obtains from the high temperature song of edge bio-engineering corporation cultivation making this life, in on November 29th, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, bacterium numbering is: CGMCC No. 5512.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210101364 CN102618474B (en) | 2012-04-10 | 2012-04-10 | Bacillus subtilis and separate culture method for same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210101364 CN102618474B (en) | 2012-04-10 | 2012-04-10 | Bacillus subtilis and separate culture method for same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102618474A CN102618474A (en) | 2012-08-01 |
| CN102618474B true CN102618474B (en) | 2013-08-14 |
Family
ID=46558688
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210101364 Active CN102618474B (en) | 2012-04-10 | 2012-04-10 | Bacillus subtilis and separate culture method for same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102618474B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865833A (en) * | 2013-10-25 | 2014-06-18 | 中国食品发酵工业研究院 | Preparation method of thermophilic bacillus subtilis microbial inoculum |
| CN110042073A (en) * | 2019-04-17 | 2019-07-23 | 四川郎酒股份有限公司 | A method of prevent bacillus from degenerating |
| CN110093291A (en) * | 2019-05-06 | 2019-08-06 | 江苏乾隆江南酒业股份有限公司 | It is a kind of based on brewing functional direction bacillus subtilis distilled spirit with sesame flavour production in application |
| CN110172435B (en) * | 2019-06-06 | 2020-12-29 | 江南大学 | Recombinant bacterium for catalytic synthesis of 2, 5-dimethylpyrazine |
| CN110938576A (en) * | 2019-12-29 | 2020-03-31 | 海南卓越生物有限公司 | Preparation method of efficient spore flora for decontamination of aquaculture |
| CN115386525B (en) * | 2022-10-26 | 2023-01-31 | 中粮营养健康研究院有限公司 | Bacillus subtilis, microbial inoculum, application and method for preparing tetramethylpyrazine |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101078006A (en) * | 2006-05-26 | 2007-11-28 | 上海凯信生物科技有限公司 | Bacillus pumilus capable of highly producing tetramethylpyrazine |
| CN101445786A (en) * | 2008-12-08 | 2009-06-03 | 江南大学 | Bacillus subtilis highly producing tetramethylpyrazine and method thereof for fermentation producing tetramethylpyrazine |
| CN101955980A (en) * | 2010-07-28 | 2011-01-26 | 江南大学 | Method and strain for producing tetramethylpyrazine |
-
2012
- 2012-04-10 CN CN 201210101364 patent/CN102618474B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101078006A (en) * | 2006-05-26 | 2007-11-28 | 上海凯信生物科技有限公司 | Bacillus pumilus capable of highly producing tetramethylpyrazine |
| CN101445786A (en) * | 2008-12-08 | 2009-06-03 | 江南大学 | Bacillus subtilis highly producing tetramethylpyrazine and method thereof for fermentation producing tetramethylpyrazine |
| CN101955980A (en) * | 2010-07-28 | 2011-01-26 | 江南大学 | Method and strain for producing tetramethylpyrazine |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102618474A (en) | 2012-08-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102618474B (en) | Bacillus subtilis and separate culture method for same | |
| CN101555461B (en) | Bacterial strain LT3 producing alkalescence cellulase and breeding method and initial optimization of cellulase production conditions thereof | |
| CN102796673B (en) | Feruloyl esterase production strain and method for producing feruloyl esterase by using same | |
| US11279961B2 (en) | Aspergillus oryzae BLCY-006 strain and application thereof in preparation of galactooligosaccharide | |
| CN106939288B (en) | Application of lactobacillus plantarum SG5 in production of gamma-aminobutyric acid | |
| CN110373359B (en) | Streptomyces albus X-18 and method for producing epsilon-polylysine by using same | |
| CN102634464B (en) | High-temperature-resistant saccharomyces cerevisiae and separating and culturing method thereof | |
| CN110819558B (en) | Pediococcus acidilactici AAF3-3 and application thereof | |
| CN110093285B (en) | Acid-resistant lactobacillus fermentum and application thereof | |
| CN1793315A (en) | Screening for strain of steepletop hickory chick and process for preparing strain thereof | |
| CN104046569B (en) | Aspergillus tubingensis for high-yield production of glucoamylase, alpha-amylase and acidic protease and application thereof | |
| WO2020181765A1 (en) | Geomyces mutant strain and application thereof | |
| CN107267401A (en) | One plant of Mucor bacteria strain and the application on fermentation green brick tea | |
| CN110564580B (en) | Method for producing vinegar containing pyrroloquinoline quinone through microbial co-culture fermentation | |
| CN102634460B (en) | Rhizopus oryzae RH1-5 and separating and culturing method thereof | |
| CN1793316A (en) | Screening for strain of balck vein hickory chick and process for preparing strain thereof | |
| RU2381270C1 (en) | STRAIN OF BACTERIA Clostridium acetobutylicum-PRODUCER OF BUTANOL, ACETONE AND ETHANOL | |
| CN106754486B (en) | Pseudomonas for high-yield trehalose synthase and fermentation enzyme production method thereof | |
| CN117581943A (en) | Fermented product of pepper and pepper processing waste and application thereof | |
| CN103710291B (en) | The method of one strain bacillus megaterium Z2013513 and production phenyl-lactic acid thereof | |
| CN108841743B (en) | Cold region straw rotten bacterial strain and preparation method and application thereof | |
| CN114410523B (en) | Strain combination for preparing black tea fungus and application thereof | |
| CN112410231B (en) | Trichoderma viride new strain and application thereof | |
| CN105821100A (en) | Technology for realizing high-yield polyoxins by continuous fermentation coupled with separation | |
| CN106591176B (en) | Lactobacillus pentosus and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |