CN108004184A - A kind of bacillus and its method for producing isovaleric acid - Google Patents
A kind of bacillus and its method for producing isovaleric acid Download PDFInfo
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- CN108004184A CN108004184A CN201810009043.4A CN201810009043A CN108004184A CN 108004184 A CN108004184 A CN 108004184A CN 201810009043 A CN201810009043 A CN 201810009043A CN 108004184 A CN108004184 A CN 108004184A
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- 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
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- 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
- C12N1/02—Separating microorganisms from their culture media
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract
A kind of method the present invention relates to bacillus and its for producing isovaleric acid.The bacillus（Bacillus）It is isolated by selective medium from alcoholization mid-term tobacco leaf, is preserved within 13rd China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.14609 in September in 2017.The bacterial strain can be using tobacco industry discarded object as Material synthesis isovaleric acid, which is colourless thick liquid, then has sweet fruity, and the fragrance of bog bilberry sample after high dilution.The fermentation medium used during the present invention is using bacillus synthesis isovaleric acid is not only of low cost; and a large amount of discarded objects of tobacco industry generation can be handled; environmental protection and rationally utilize resource in terms of suffer from positive effect, have a good application prospect.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of bacillus and its application, more particularly to one plant screening
The bacillus of acquisition and the method using strain fermentation production isovaleric acid.
Background technology
Isovaleric acid is also known as 3 Methylbutanoic acid, colourless thick liquid, and fusing point is -29.3 DEG C, and boiling point is 176.5 DEG C, can with alcohol,
Ether, chloroform etc. dissolve each other.
Isovaleric acid has irritation tapinoma-odour, then has sweet fruity, and the fragrance of bog bilberry sample after high dilution.
Isovaleric acid is naturally stored in valerian oil, vanilla flavour concentrate, hop oil, laurel, spearmint oil etc., and the food used is allowed in China's national standard
With in spices.Isovaleric acid is except to cheese-making and butter essence, the micro preparation for fruit type essence.It can be also used for
Manufacture medicine, spices, flavouring etc..It can be used for baked goods, meat products.Isovaleric acid is urged mainly for the production of calmness at present
Dormancy medicine bromisovalum.But it is more to be used to produce spices.May be used as the isovalerate of spices mainly has isovaleric acid ester, different
Propyl valerate, iso-amyl iso-valeriate, isovaleric acid Mang ox ester, benzyl isovalerate and cinnamyl isovalerate.Rudimentary isovaleric acid esters
Flavorant can be used as, advanced isovaleric acid esters can be used for cosmetics.
The method of industrial production isovaleric acid is to obtain isovaleric acid by isoamyl alcohol or isoamyl are oxidation of aldehydes at present.It is mainly
It is to apply powerful catalyst, using isoamyl alcohol or isopentyl aldehyde as raw material, is made by oxidation reaction, the method not only needs catalyst,
And reactions steps are cumbersome, take longer.The biosynthesis of isovaleric acid is rarely reported, it is probably due to synthesizing the micro- of isovaleric acid
Without excavation, or because of the inefficient of Microbe synthesis isovaleric acid, this biosynthesis for just constraining isovaleric acid exists biology
Industrial application.Therefore how overcome the deficiencies in the prior art, improve microorganism production isovaleric acid on application be current
The problem of biological technical field urgent need to resolve.
The content of the invention
It is an object of the present invention to solve the deficiency of the existing technology and provide a kind of bacillus and its utilize tobacco industry
The method that discarded object produces isovaleric acid, which need not only transform, but also is easily isolated and cultivates, and in addition, use
Fermentation medium it is not only of low cost, but also can handle tobacco industry generation a large amount of discarded objects, in environmental protection and conjunction
Reason suffers from positive effect using resource aspect.
To achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of bacillus（Bacillus）, Chinese microorganism strain preservation conservator is preserved within 13rd in September in 2017
Meeting common micro-organisms center, deposit number CGMCC No. 14609.
The bacillus of the present invention（Bacillus）Preparation method：
1）Separation culture medium and condition of culture：
The LB fluid nutrient mediums of 8 ~ 12 times of dilution：1000mL distilled water；Tryptone 1g；Yeast extract 0.5g；NaCl
1g；PH is naturally, 121 DEG C sterilize 20 minutes；
2）Bacterial strain screening：The agar that 20g/L is added in aforesaid liquid culture medium is prepared into solid medium and is used for bacterial strain screening；
3）Separation method：
The tobacco sample of alcoholization mid-term randomly selects 2g, aseptically shreds, is soaked in sterile saline in 150mL
In, it is 37 DEG C in temperature, under conditions of rotating speed is 180 revs/min, shaken cultivation 60 minutes, is filtered with sterile mono layer gauze, take filter
Liquid, supernatant is abandoned in centrifugation, and is resuspended with 5mL sterile waters, is obtained original bacteria suspension, is taken the 100 original bacterium solutions of μ L to be diluted to 10 respectively0, 10-1, 10-2, 10-3, 10-4, 10-5, and the LB culture mediums that 200 μ L are coated on 8 ~ 12 times of dilution are drawn respectively, it is inverted culture in 37 DEG C
24 it is small when, the bacterium colony cultivated is purified using plate streak, until obtain bacillus single bacterium colony.
Utilize bacillus（Bacillus）The method for producing isovaleric acid：By the bacillus bacteria suspension, according to 5 ~ 10%
Inoculum concentration be seeded in fermentation medium, be placed in shaking table, and 37 DEG C of cultivation and fermentations were extracted through ethyl acetate, obtained after 5 days
Isovaleric acid, is detected using GC-MS.
The fermentation medium is the culture medium using tobacco industry discarded object as nutrients, and component is：Glucose 20g/
L, tobacco industry discarded object 5g/L, pH value 7.0 ~ 7.4.
The tobacco industry discarded object is offal, tobacco fragment, offal and the one of which in cigarette ash rod or several combinations.
The shaking table is 140 revs/min.
The bacillus of the present invention that isovaleric acid is produced using tobacco industry discarded object as raw material（Bacillus）Be from
Refine isolated by selective medium on the tobacco leaf of mid-term.
Bacillus of the present invention is Grain-positive bacillus, there is gemma, produces nearly middle raw ellipticity gemma, sporangiocyst
Expand, the size for observing thalline under the microscope is 0.5 μm of x（1.0~1.5）μm.Somatic cells are in rod-short.Trained in LB solids
Primary surface is supported, thalline forms translucent milky petite, and bacterium colony surface wettability is smooth, neat in edge.The warp of above-mentioned bacterial strains
Universal primer PCR obtains 16SrDNA sequences and the 16SrDNA sequence similarity highests of root bacillus M-5（Attached drawing 1）.
Wherein, above-mentioned PCR sense primers are 27F:5’-agagtttgatcctggctcag-3’（SEQID NO.2）；
Anti-sense primer is 1492R：5’-cggctaccttgttacgactt-3’（SEQID NO.3）.
Present approach provides a kind of new sources of isovaleric acid, bacterial strain of the present invention need not both be transformed, and be easy to point
From and culture, in addition, the fermentation medium used is not only of low cost, and can handle tobacco industry generation it is a large amount of
Discarded object, this suffers from positive effect in terms of resource is utilized in environmental protection and rationally, bacillus provided by the invention and
It is expected to solve the problems, such as that bioanalysis produces isovaleric acid using the method for tobacco industry discarded object production isovaleric acid, can also promote different
The industrialized production of valeric acid and application.
Compared with prior art, the present invention its advantage is：
1）Present invention screening, which obtains bacillus, to be main nutrient source using tobacco industry discarded object, this can handle tobacco
Industry produce a large amount of discarded objects, environmental protection and rationally utilize resource in terms of suffer from positive effect.
2）The bacillus being capable of fermented tobacco trade waste production isovaleric acid.The isovaleric acid is colourless thick liquid,
Then there are sweet fruity, and the fragrance of bog bilberry sample after high dilution.Its one side can be used for produce spices, mainly to
It is also micro to be used for fruit type essence with cheese-making and butter essence, it on the other hand can also be used to produce hypnotic sedative agent bromine isoamyl
Uride.
3）The present invention provides a kind of new way of isovaleric acid synthesis, which need not only transform, Er Qieyi
In separation and culture, this is also expected to solve the problems, such as bioanalysis production isovaleric acid, at the same promote isovaleric acid industrialized production and
It is applied.
Brief description of the drawings
Fig. 1 by the present invention in screening bacillus phylogenetic tree；
Fig. 2 present invention fermentations obtain the mass spectrogram of isovaleric acid；
Bacillus（Bacillus）, China Committee for Culture Collection of Microorganisms to be preserved in September in 2017 within 13rd general
Logical microorganism center, deposit number CGMCC No. 14609, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica.
Embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright scope.In the examples where no specific technique or condition is specified, according to the described technology of document in the art or condition
Or carried out according to product description.Production firm person is not specified in material therefor or instrument, is that can be obtained by buying
Conventional products.
Embodiment 1 produces the separation of isovaleric acid microorganism using tobacco industry discarded object
1. separation culture medium and condition of culture：
10 times of dilution（Or 8 times, or 12 times）LB fluid nutrient mediums：1000mL distilled water；Tryptone 1g；Yeast extract
0.5g；NaCl 1g；PH is naturally, sterilizing；
The sterilising conditions of above-mentioned culture medium sterilize 20 minutes for 121 DEG C.
The agar that 20g/L is added in above-mentioned culture medium is prepared into solid medium and is used for bacterial strain screening.
2. separation method：
The tobacco sample of alcoholization mid-term randomly selects 2g, aseptically shreds, is soaked in sterile saline in 150mL
In, it is 37 DEG C in temperature, under conditions of rotating speed is 180 revs/min, shaken cultivation 60 minutes, is filtered with sterile mono layer gauze, take filter
Liquid, supernatant is abandoned in centrifugation, and is resuspended with 5mL sterile waters, is obtained original bacteria suspension, is taken the 100 original bacterium solutions of μ L to be diluted to 10 respectively0, 10-1, 10-2, 10-3, 10-4, 10-5, and draw 200 μ L respectively and be coated on 10 times of dilution（Or 8 times, or 12 times）LB culture mediums, in
When 37 DEG C of inversion cultures 24 are small, the bacterium colony cultivated is purified using plate streak, until obtaining single bacterium colony.
The strain idenfication of 2 separate microorganism of embodiment
（1）Selected culture medium separated bacterium is incubated in the LB fluid nutrient mediums of 10 times of dilution, 12 it is small when after, obtain phase
Answer the bacteria suspension of bacterial strain.
（2）Take 500 μ L of bacteria suspension, abandon supernatant after centrifugation, extract genomic DNA, using extract genomic DNA as template into
Row PCR (PCR).
Above-mentioned PCR sense primers are 27F:5’-agagtttgatcctggctcag-3’（SEQIDNO.2）；
Anti-sense primer is 1492R：5’-cggctaccttgttacgactt-3’（SEQIDNO.3）.
PCR reaction systems and program are as follows：
PCR amplification system（25μL）：
PCR amplification program：
30 circulations.
（3）5 μ L PCR reaction solutions and DNA maker 2000 are taken respectively, and PCR product verification is carried out using gel electrophoresis.
（4）There to be the PCR product of fluorescent belt appearance in 1600bp or so, send to sequencing company sequencing, sequencing result carries out
After splicing, the BLSAT of NCBI is inputted（https://blast.ncbi.nlm.nih.gov/Blast.cgi）Middle progress sequence ratio
Right, which has 99% similitude with the 16SrDNA sequences with bacillus M-5.The bacterium is that bacillus is
Grain-positive bacillus, there is gemma, produces nearly middle raw ellipticity gemma, and sporangiocyst expands, and observes the size of thalline under the microscope
For 0.5 μm of x（1.0~1.5）μm.Somatic cells are in rod-short.In LB solid culture primary surfaces, thalline forms translucent milky white
Color petite, bacterium colony surface wettability is smooth, neat in edge.The Preliminary Identification strain is root bacillus M-5, the bacterial strain preservation
To China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is：CGMCC No. 14609.Sequencing
As a result as shown in SEQIDNO.1.
Embodiment 3 utilizes tobacco industry discarded object production isovaleric acid
Nutrient media components using tobacco industry discarded object as nutrients is：Glucose 20g/L, tobacco industry discarded object（Offal:It is broken
Piece:Offal is 50:30:20）5g/L, pH value 7.0 ~ 7.4；
Aseptically, bacillus is seeded in sterilized LB fluid nutrient mediums and cultivated, it is 1 to cultivate to OD600.Press
According to 3% inoculum concentration by above-mentioned strain, be inoculated in the tobacco industry discarded object equipped with 50mL sterilizings as the culture medium of nutrients
In the shaking flask of 250mL, and the control group for not connecing bacterial strain is set, in 140 revs/min of shaking table, 37 DEG C of cultures, after cultivating 5 days
Detected using GC-MS.
The qualitative detection of isovaleric acid：Above-mentioned zymotic fluid is taken to be centrifuged 10 minutes through 12000 revs/min, reject precipitation, will ferment
Liquid supernatant is extracted with ethyl acetate, and obtains acetic acid ethyl ester extract, and revolving removes partial solvent, after being removed water with anhydrous sodium sulfate, uses
0.22 μm of membrane filtration, qualitative detection is carried out using GC-MS.Using Innowax capillary chromatographic columns（30m × 0.25mm, 0.25 μ
m.）, 250 DEG C of injector temperature, 230 DEG C of ion source temperature, heating schedule maintains 6 minutes for 40 DEG C, then 3 DEG C/min of programs
100 DEG C are warming up to, 5 DEG C/min of temperature programmings are to 230 DEG C afterwards.Obtain result mass spectrogram as shown in Figure 1, through with java standard library ratio
To for isovaleric acid.
Basic principle, main feature and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Sequence table
<110>Cigarette industry Co., Ltd in Yunnan
<120>A kind of bacillus and its method for producing isovaleric acid
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1402
<212> DNA
<213> Bacillus
<400> 1
cttcggcggt ggctcataaa ggttacctca ccgacttcgg gtgttgcaaa ctctcgtggt 60
gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gatccgcgat 120
tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac tgagaacaga 180
tttatgggat tggctaaacc ttgcggtctt gcagcccttt gttctgtcca ttgtagcacg 240
tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct tcctccggtt 300
tgtcaccggc agtcacctta gagtgcccaa ctgaatgctg gcaactaaga tcaagggttg 360
cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca 420
cctgtcactc tgtccccgaa gggaaagccc tatctctagg gttgtcagag gatgtcaaga 480
cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc 540
ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg agtgcttaat 600
gcgttagctg cagcactaag gggcggaaac cccctaacac ttagcactca tcgtttacgg 660
cgtggactac cagggtatct aatcctgttc gctccccacg ctttcgctcc tcagcgtcag 720
ttacagacca gagagtcgcc ttcgccactg gtgttcctcc acatctctac gcatttcacc 780
gctacacgtg gaattccact ctcctcttct gcactcaagt ttcccagttt ccaatgaccc 840
tccccggttg agccgggggc tttcacatca gacttaagaa accgcctgcg agccctttac 900
gcccaataat tccggacaac gcttgccacc tacgtattac cgcggctgct ggcacgtagt 960
tagccgtggc tttctggtta ggtaccgtca aggtgcgagc agttactctc gcacttgttc 1020
ttccctaaca acagagcttt acgatccgaa aaccttcatc actcacgcgg cgttgctccg 1080
tcagactttc gtccattgcg gaagattccc tactgctgcc tcccgtagga gtctgggccg 1140
tgtctcagtc ccagtgtggc cgatcaccct ctcaggtcgg ctacgcatcg tcgccttggt 1200
gagccattac cccaccaact agctaatgcg ccgcgggtcc atctgtaagt gacagccgaa 1260
accgtctttc atccttgaac catgcggttc aaggaactat ccggtattag ctccggtttc 1320
ccggagttat cccagtctta caggcaggtt acccacgtgt tactcacccg tccgccgcta 1380
acatccggga gcaagctccc tt 1402
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence ()
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence ()
<400> 3
cggctacctt gttacgactt 20
Claims (6)
- A kind of 1. bacillus（Bacillus）, Chinese microorganism strain preservation management committee is preserved within 13rd in September in 2017 Member's meeting common micro-organisms center, deposit number CGMCC No. 14609.
- 2. the bacillus described in claim 1（Bacillus）Preparation method, it is characterised in that comprise the following steps：1）Separation culture medium and condition of culture：The LB fluid nutrient mediums of 8 ~ 12 times of dilution：1000mL distilled water；Tryptone 1g；Yeast extract 0.5g；NaCl 1g；PH is naturally, 121 DEG C sterilize 20 minutes；2）Bacterial strain screening：The agar that 20g/L is added in aforesaid liquid culture medium is prepared into solid medium and is used for bacterial strain screening；3）Separation method：The tobacco sample of alcoholization mid-term randomly selects 2g, aseptically shreds, is soaked in sterile saline in 150mL In, it is 37 DEG C in temperature, under conditions of rotating speed is 180 revs/min, shaken cultivation 60 minutes, is filtered with sterile mono layer gauze, take filter Liquid, supernatant is abandoned in centrifugation, and is resuspended with 5mL sterile waters, is obtained original bacteria suspension, is taken the 100 original bacterium solutions of μ L to be diluted to 10 respectively0, 10-1, 10-2, 10-3, 10-4, 10-5, and the LB culture mediums that 200 μ L are coated on 8 ~ 12 times of dilution are drawn respectively, it is inverted culture in 37 DEG C 24 it is small when, the bacterium colony cultivated is purified using plate streak, until obtain bacillus single bacterium colony.
- 3. the bacillus described in claim 1（Bacillus）Application in isovaleric acid is produced, it is characterised in that by following Carry out：By the bacillus bacteria suspension, it is seeded in fermentation medium, is placed in shaking table according to 5 ~ 10% inoculum concentration, and 37 DEG C culture 5 days after, extracted through ethyl acetate, obtain isovaleric acid.
- 4. bacillus according to claim 3（Bacillus）Application in isovaleric acid is produced, it is characterised in that institute The fermentation medium stated is the culture medium using tobacco industry discarded object as nutrients, its component is glucose 20g/L, tobacco industry Discarded object 5g/L, pH value 7.0 ~ 7.4.
- 5. bacillus according to claim 3（Bacillus）Application in isovaleric acid is produced, it is characterised in that institute The tobacco industry discarded object stated includes offal, tobacco fragment, offal, the one of which in cigarette ash rod or several combinations.
- 6. bacillus according to claim 3（Bacillus）Application in isovaleric acid is produced, it is characterised in that institute The shaking table stated is 140 revs/min.
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Cited By (4)
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| CN109295114A (en) * | 2018-10-29 | 2019-02-01 | 南方医科大学 | Application of the staphylococcus epidermis in synthesis isobutyric acid |
| CN110776412A (en) * | 2019-11-12 | 2020-02-11 | 万华化学集团股份有限公司 | Method for preparing isovaleric acid, ligand, complex and application thereof in catalytic system |
| CN113774072A (en) * | 2021-07-15 | 2021-12-10 | 湖南省农业科学院 | Citrate synthase gene, citrate synthase, application as target and herbicide |
| CN114350540A (en) * | 2021-11-30 | 2022-04-15 | 浙江中烟工业有限责任公司 | Bacillus and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295114A (en) * | 2018-10-29 | 2019-02-01 | 南方医科大学 | Application of the staphylococcus epidermis in synthesis isobutyric acid |
| CN110776412A (en) * | 2019-11-12 | 2020-02-11 | 万华化学集团股份有限公司 | Method for preparing isovaleric acid, ligand, complex and application thereof in catalytic system |
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| CN113774072A (en) * | 2021-07-15 | 2021-12-10 | 湖南省农业科学院 | Citrate synthase gene, citrate synthase, application as target and herbicide |
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| CN114350540B (en) * | 2021-11-30 | 2024-04-09 | 浙江中烟工业有限责任公司 | Bacillus and application thereof |
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| Publication number | Publication date |
|---|---|
| CN108004184B (en) | 2021-01-29 |
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