CN108102981A - A kind of culture medium for improving clostridium butyricum screening efficiency - Google Patents
A kind of culture medium for improving clostridium butyricum screening efficiency Download PDFInfo
- Publication number
- CN108102981A CN108102981A CN201810123831.6A CN201810123831A CN108102981A CN 108102981 A CN108102981 A CN 108102981A CN 201810123831 A CN201810123831 A CN 201810123831A CN 108102981 A CN108102981 A CN 108102981A
- Authority
- CN
- China
- Prior art keywords
- clostridium butyricum
- screening
- culture medium
- glucose
- clostridium
- 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.)
- Granted
Links
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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of culture mediums for improving clostridium butyricum screening efficiency, belong to technical field of bioengineering.The present invention inhibits the growth of other microorganisms in addition to clostridium butyricum using more glutinous mycin B and n-butyric acie, while clostridium butyricum well-grown on tablet, bacterium colony are high-visible.The present invention provides a kind of clostridium butyricum screening and culturing mediums, intuitively can efficiently screen clostridium butyricum, effectively shorten the screening cycle, reduce workload, improve screening efficiency.
Description
Technical field
The present invention relates to a kind of culture mediums for improving clostridium butyricum screening efficiency, belong to technical field of bioengineering.
Background technology
Clostridium butyricum (Clostridium butyricum, C.butyricum), also known as Miyarisan, butyric acid fusiform gemma bar
Bacterium is Bacillaceae, a kind of production butyric acid of fusobacterium, Gram-positive anaerobic bacteria.It is widely present in animal wastes, soil
In earth, cheese, naturally soured milk.Thalline is in rod-shaped, long 3.0-7.0um, and wide 0.6-1.2um, whole body flagellum can move, accidental to have
Filamentous thalline, the oval spore often containing eccentric or secondary end life, cell membrane contain D, L. diaminopimelic acid, and cell wall sugar is Portugal
Grape sugar.Surface colony form is diameter 1-3mm, rounded or slightly irregular, and colony colour is white or cream-colored.Clostridium butyricum
Aerogenesis in fermentation process, tunning include acetic acid, butyric acid, butanol, amylase, lipase, vitamin etc..Japan thousand in 1933
Leaf medical college doctor Miyai has isolated clostridium butyricum (C.Clostridium MIYAIRI) from excrement for the first time, is subsequently found
It is cultivated in filtrate containing a small amount of aliphatic acid, can inhibit the growth that harmful bacteria in enteron aisle grows and promotes beneficial bacterium, and in
Realization in 1940 is commercially produced.Hereafter, clostridium butyricum be widely used as people's enteritis treatment drug, food additives, veterinary drug and
Feed addictive.
Clostridium butyricum mainly screens, at present, generally using RCM and TSN agar as anaerobic bacteria from enteron aisle and soil
Culture medium is screened, however RCM agar medium poor specificities, while clostridium butyricum is not the dominant bacteria in enteron aisle and soil
Strain, its growth are inhibited be subject to other bacillus and C.perfringens, cause finally screening more difficult, TNS agar
Culture medium can inhibit the growth of clostridium butyricum, and cannot inhibit the growth of C.perfringens, cause the sieve bacterium cycle long, specificity
Not high consequence.
The content of the invention
How efficiently for the problem of quick screening clostridium butyricum, of the invention first purpose is to provide a kind of efficient spy
The strong clostridium butyricum screening and culturing medium of the opposite sex, the culture medium contain following compositions：Dusty yeast, beef extract, peptone, grape
Sugar, soluble starch, sodium chloride, sodium acetate, L-cysteine hydrochloride, n-butyric acie and mostly glutinous mycin B.
In one embodiment of the invention, the culture medium contains per L：Dusty yeast 2-5g, beef extract 5-20g, egg
White peptone 5-20g, glucose 2-10g, soluble starch 0.5-3g, sodium chloride 2-10g, sodium acetate 1-5g, L-cysteine hydrochloric acid
Salt 0.2-1g, n-butyric acie 0.2-1g, agar powder 10-30g.
In one embodiment of the invention, the culture medium contains per L：Yeast extract 3g, powdered beef 10g, pancreas egg
White peptone 10g, glucose 5g, soluble starch 1g, sodium chloride 5g, sodium acetate 3g, L-cysteine hydrochloride 0.5g, agar 20g,
N-butyric acie 0.1-1g, mostly glutinous mycin B 0.02g.
Second object of the present invention is to provide the preparation method of the screening and culturing medium, and preparation process is as described below：Claim
Take dusty yeast, beef extract, peptone, glucose, soluble starch, sodium chloride, sodium acetate, L-cysteine hydrochloride, positive fourth
Acid and agar powder add in distilled water and stir to dissolving, adjust pH to 6.8, it is sterilized that final concentration 0.02-0.1g/L is added in after sterilizing
How glutinous mycin B.
Third object of the present invention is to provide a kind of method of efficiently quick screening clostridium butyricum, and the method is to shuttle
More glutinous mycin B and n-butyric acie are added in bacterium proliferated culture medium, then sample to be screened is coated with into the culture medium, in anaerobic environment
Lower culture extremely obtains single bacterium colony.
In one embodiment of the invention, the culture medium contains per L：Dusty yeast 3g, beef extract 10g, peptone
10g, glucose 5g, soluble starch 1g, sodium chloride 5g, sodium acetate 3g, L-cysteine hydrochloride 0.5g, agar powder 20g.
In one embodiment of the invention, the method before coating, by sample to be screened as 75~90 DEG C
Water-bath is heat-treated 5~10min.
In one embodiment of the invention, the method further include to single bacterium colony carry out microbial morphology analysis or
16S rDNA are identified.
The application of aspect is checked or analyzed in food, biological field clostridium butyricum the present invention also provides the method.
Advantageous effect：The present invention is sticking mycin B and n-butyric acie more by being added in into clostridium proliferated culture medium, by anaerobism
The growth inhibition ratio of inhibition other microorganisms in addition to clostridium butyricum be can be very good after culture up to 76.98%, it is effective to reduce
Workload during sieve bacterium improves screening efficiency, and success rate is increased to 26.67% by 1.22%, and the screening cycle was from 9 days
Shorten into 7 days.
Description of the drawings
Fig. 1 is Chicken intestinal bacteria growthform on clostridium enriched medium；
Fig. 2 is Chicken intestinal bacteria growthform on screening and culturing medium.
Specific embodiment
With reference to embodiment, technical solution of the present invention is described further, but specific embodiment party described herein
Formula is merely to illustrate and explain the present invention, and is not intended to limit the invention.
The culture medium and reagent used in the embodiment of the present invention is as follows：
Clostridium enriched medium (RCM culture mediums)：Yeast extract 3g, powdered beef 10g, tryptone 10g, glucose 5g,
Soluble starch 1g, sodium chloride 5g, sodium acetate 3g, L-cysteine hydrochloride 0.5g, distilled water 1000ml, pH 6.8,121
DEG C sterilizing 15min, agar adds in 20g in solid medium
Screening and culturing medium：Yeast extract 3g, powdered beef 10g, tryptone 10g, glucose 5g, soluble starch 1g, chlorine
Change sodium 5g, sodium acetate 3g, L-cysteine hydrochloride 0.5g, agar 20g, n-butyric acie 0.1-1g, mostly glutinous mycin B0.02g, distillation
7.0,121 DEG C of sterilizing 10min of water 1000ml, pH.
Embodiment 1
3 each 1g of chicken cecal content are taken, mixing takes wherein 1g to add in 9ml PBS buffer solution, is placed in 80 DEG C of water-bath heat
10min is handled to kill non-Bacillus.100uL is taken to be placed in RCM culture mediums 37 DEG C of anaerobism enrichment cultures for 24 hours.
The sample that enrichment culture is obtained is diluted to 10 respectively-2, 10-4, 10-6, 100uL is taken to be coated on RCM culture medium flat plates
On, 37 DEG C of Anaerobic culturel 48h, the bacterial strain for selecting normal growth under anaerobic condition carries out microscopy, to meeting clostridium spy
The single bacterium colony of sign carries out line separation on RCM tablets, after 37 DEG C are cultivated 48h, carries out Gram's staining, selects and meet butyric acid shuttle
The bacterium colony of the features such as bacterium colonial morphology, growth characteristics carries out Physiology and biochemistry identification, using universal primer 27F and 1492R to bacterial strain
Carry out 16S rDNA identifications.The results show that being coated with 18 pieces of tablets altogether, 430 single bacterium colonies are obtained, are reflected by microbial morphology
It is fixed, the microorganism of 430 plants of Bacillus is obtained, is identified through 16S rDNA, obtains 0 plant of purpose bacterial strain.When the screening cycle about 216 is small,
It is 0 to screen success rate.
Embodiment 2
Using the identical mode of operation of embodiment 1 and screening sample, difference lies in put down using the screening and culturing medium of the present invention
Plate replaces RCM culture medium flat plates.
The results show that being coated with 18 pieces of tablets altogether, 135 single bacterium colonies are obtained, are identified by microbial morphology, obtain 135
The microorganism of strain Bacillus, identifies through 16S rDNA, obtains 36 plants of purpose bacterial strains.When the screening cycle about 168 is small, success rate is screened
For 26.67%.
The method of inventor's Application Example 2 has carried out multiple screening experiment, and screening success rate reaches more than 25%, with
Existing screening technique is compared, and method of the invention overcomes purpose bacterium condition of culture in screening process and explores difficulty, easily by miscellaneous
The bottleneck problems such as bacterium interference, greatly shorten the cycle needed for screening.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Claims (9)
1. a kind of clostridium butyricum screening and culturing medium of efficient high specificity, which is characterized in that the culture medium contains following compositions：
Dusty yeast, beef extract, peptone, glucose, soluble starch, sodium chloride, sodium acetate, L-cysteine hydrochloride, n-butyric acie
With more glutinous mycin B.
2. clostridium butyricum screening and culturing medium according to claim 1, which is characterized in that contain per L：Dusty yeast or yeast leaching
Cream 2-5g, powdered beef or beef extract 5-20g, tryptone 5-20g, glucose 2-10g, soluble starch 0.5-3g, sodium chloride
2-10g, sodium acetate 1-5g, L-cysteine hydrochloride 0.2-1g, n-butyric acie 0.2-1g, agar powder 10-30g.
3. clostridium butyricum screening and culturing medium according to claim 2, which is characterized in that contain per L：Yeast extract 3g, ox
Digested tankage 10g, tryptone 10g, glucose 5g, soluble starch 1g, sodium chloride 5g, sodium acetate 3g, L-cysteine hydrochloride
0.5g, agar 20g, n-butyric acie 0.1-1g, mostly glutinous mycin B 0.02g.
A kind of 4. method for preparing clostridium butyricum screening and culturing medium described in Claims 2 or 3, which is characterized in that including walking as follows
Suddenly：Weigh dusty yeast, beef extract, peptone, glucose, soluble starch, sodium chloride, sodium acetate, L-cysteine hydrochloride,
N-butyric acie and agar powder add in distilled water and stir to dissolving, adjust pH to 6.6~7.0, final concentration 0.02- is added in after sterilizing
How glutinous mycin B sterilized 0.1g/L.
A kind of 5. method of efficiently quick screening clostridium butyricum, which is characterized in that added in into clostridium proliferated culture medium how glutinous mould
Plain B and n-butyric acie, then sample to be screened is coated with into the culture medium, the culture extremely acquisition single bacterium colony under anaerobic environment.
6. according to the method described in claim 5, it is characterized in that, the clostridium proliferated culture medium contains per L：Dusty yeast 3g,
Beef extract 10g, peptone 10g, glucose 5g, soluble starch 1g, sodium chloride 5g, sodium acetate 3g, L-cysteine hydrochloride
0.5g, agar powder 20g.
7. method according to claim 5 or 6, which is characterized in that before coating, by sample to be screened as 75~90
DEG C water-bath is heat-treated 5~10min.
8. according to any method of claim 5~8, which is characterized in that carry out microorganism to the single bacterium colony that screening obtains
Morphological analysis or 16 S rDNA identifications.
9. the application of aspect is checked or analyzed in food, biological field clostridium butyricum for any the method for claim 5~8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810123831.6A CN108102981B (en) | 2018-02-07 | 2018-02-07 | Culture medium for improving clostridium butyricum screening efficiency |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810123831.6A CN108102981B (en) | 2018-02-07 | 2018-02-07 | Culture medium for improving clostridium butyricum screening efficiency |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108102981A true CN108102981A (en) | 2018-06-01 |
| CN108102981B CN108102981B (en) | 2020-08-04 |
Family
ID=62221965
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810123831.6A Active CN108102981B (en) | 2018-02-07 | 2018-02-07 | Culture medium for improving clostridium butyricum screening efficiency |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108102981B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763652A (en) * | 2020-09-03 | 2020-10-13 | 山东拓普生物工程有限公司 | Culture medium for separating and counting clostridium butyricum |
| CN112961802A (en) * | 2021-03-04 | 2021-06-15 | 江苏永盛生物科技有限公司 | Probiotics preparation for promoting growth of piglets and improving intestinal tracts of piglets and preparation method of probiotics preparation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000046345A1 (en) * | 1999-02-03 | 2000-08-10 | Alain Rambach | Method for detecting bacteria cultivated in anaerobic condition |
| CN101659980A (en) * | 2009-09-28 | 2010-03-03 | 成都瑞琦科技实业有限责任公司 | Selective coloration culture medium of clostridium perfringens |
-
2018
- 2018-02-07 CN CN201810123831.6A patent/CN108102981B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000046345A1 (en) * | 1999-02-03 | 2000-08-10 | Alain Rambach | Method for detecting bacteria cultivated in anaerobic condition |
| CN101659980A (en) * | 2009-09-28 | 2010-03-03 | 成都瑞琦科技实业有限责任公司 | Selective coloration culture medium of clostridium perfringens |
Non-Patent Citations (4)
| Title |
|---|
| GHODDUSI HB等: "Preliminary study on the isolation of Clostridium butyricum strains from natural sources in the UK and screening the isolates for presence of the type E botulinal toxin gene", 《INT J FOOD MICROBIOL》 * |
| ROBERT A等: "Quantitation of Clostridium perfringens in Foods", 《APPL MICROBIOL》 * |
| 熊元林等: "《微生物学实验》", 31 January 2014, 武汉：华中师范大学出版社 * |
| 王静等: "产气荚膜梭菌选择性显色培养基的研制", 《卫生研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763652A (en) * | 2020-09-03 | 2020-10-13 | 山东拓普生物工程有限公司 | Culture medium for separating and counting clostridium butyricum |
| CN111763652B (en) * | 2020-09-03 | 2020-11-20 | 山东拓普生物工程有限公司 | Culture medium for separating and counting clostridium butyricum |
| CN112961802A (en) * | 2021-03-04 | 2021-06-15 | 江苏永盛生物科技有限公司 | Probiotics preparation for promoting growth of piglets and improving intestinal tracts of piglets and preparation method of probiotics preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108102981B (en) | 2020-08-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104974966B (en) | A kind of bacillus coagulans and its fermentation process in high density and dry bacterium powder preparation method | |
| CN106047759B (en) | One plant of Pediococcus pentosaceus and its application | |
| Khardziani et al. | Elucidation of Bacillus subtilis KATMIRA 1933 potential for spore production in submerged fermentation of plant raw materials | |
| MX2013015363A (en) | Process for making chitin derivatives. | |
| CN109161498B (en) | Bacillus subtilis M406 and application thereof in preparation of bacteriocin and cellulase | |
| CN102660461A (en) | Microbial preparation for shortening tobacco fermentation period and application of microbial preparation | |
| CN113652373B (en) | Bacillus and application thereof | |
| CN114107137B (en) | Clostridium butyricum and application and product thereof | |
| CN108102981A (en) | A kind of culture medium for improving clostridium butyricum screening efficiency | |
| KR100264361B1 (en) | Lactobacillus plantarum PMO08 (KFCC-11028) with cholesterol lowering ability | |
| CN103266074B (en) | B.subtilis spores strain and application thereof | |
| Khardziani et al. | Optimization of enhanced probiotic spores production in submerged cultivation of Bacillus amyloliquefaciens B-1895 | |
| KR101854705B1 (en) | Novel strains of Bacillus licheniformis NY1505 producing high amount of α-glucosidase inhibitors | |
| CN106868079A (en) | The method of fermentation aerosporin culture medium and fermenting and producing aerosporin | |
| CN104212745B (en) | Chicken microecological preparation and preparation method thereof | |
| KR100423092B1 (en) | Producing method of microbial cellulose by mixed culture | |
| CN115992065A (en) | Method for preparing animal feed by bioconversion of waste feathers | |
| CN107760724A (en) | A kind of method and the α glucosidase inhibitors that α glucosidase inhibitors are prepared using Paenibacillus polymyxa | |
| CN103960483B (en) | A kind of Liquid-state fermentation production method of the feeding probiotics preparation of main product cellulase | |
| CN114456990A (en) | Preparation method of DDGS (distillers dried grains with soluble) as well as fermentation strain and culture medium thereof | |
| CN115851496B (en) | Low-temperature-resistant biocontrol bacillus and aquatic pathogenic bacteria antagonism application thereof | |
| CN113943667B (en) | Lactobacillus plantarum isolated from camel rumen and application of lactobacillus plantarum in silage | |
| CN109706083A (en) | A kind of Eucommia Ulmoieds that the antibacterial activity after metabolic regulation dramatically increases | |
| LU502072B1 (en) | Preparation method of clostridium butyricum compound microbial agent | |
| CN116286548B (en) | Novel microbacterium strain for degrading fibers and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230625 Address after: Room 117, 1F, Public Training Base, Yueyang High tech Industrial Park, Rongjiawan, Yueyang County, Yueyang, Hunan Province Patentee after: Hunan Daze Nong Biotechnology Co.,Ltd. Address before: No. 1800 road 214122 Jiangsu Lihu Binhu District City of Wuxi Province Patentee before: Jiangnan University |
|
| TR01 | Transfer of patent right |