CN108410775A - One plant height produces farnoquinone（MK-7）Bafillus natto and its application - Google Patents
One plant height produces farnoquinone（MK-7）Bafillus natto and its application Download PDFInfo
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- CN108410775A CN108410775A CN201810392873.XA CN201810392873A CN108410775A CN 108410775 A CN108410775 A CN 108410775A CN 201810392873 A CN201810392873 A CN 201810392873A CN 108410775 A CN108410775 A CN 108410775A
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- natto
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- bacillus subtilis
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- 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
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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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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- C—CHEMISTRY; METALLURGY
- 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
- C12P7/66—Preparation of oxygen-containing organic compounds containing the quinoid structure
Abstract
The invention discloses the bafillus natto of plant height production farnoquinone (MK 7) and its applications, belong to microorganism field.1 A27 of bafillus natto (Bacillus subtilis natto) ND of the present invention are preserved in China typical culture collection center on March 14th, 2018, and preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:M 2018131.The yield of farnoquinone (MK 7) is greatly improved, yield about 70mg/L, and 3 days are only needed to can reach maximum production, substantially reduce fermentation period, time cost is saved, industrial fermentation production is can be applied to and the bacterial strain genetic stability is good, 5 generation of continuous passage, its production farnoquinone (MK 7) yield was basicly stable, same higher level is maintained, it can be as the production bacterial strain further researched and developed.
Description
Technical field
The present invention relates to the bafillus natto of plant height production farnoquinone (MK-7) and its applications, belong to microorganism neck
Domain.
Background technology
Farnoquinone (menaquinone, MK) is a kind of liposoluble vitamin, the naphthoquinones base with phylloquinone bioactivity
The derivative of group, is one of indispensable important vitamin in human body.Farnoquinone is aphthoquinone series compound, yellowish
Color crystal shares 14 kinds of forms according to the length difference of C-3 isoprene side chains on its molecular structure, indicates to refer to MK-n
The number of isoprene unit on side chain), wherein MK-7 (Agua-Mephyton 2) bioactivity is the most notable.
Farnoquinone (MK-7) is mainly based on chemical synthesis at present, but there are precursor raw materials for conventional chemical synthesis
Source limitation, a large amount of isomers of chemical reaction generation, by-product is more, low yield, brings the problems such as environmental pollution, and the dimension synthesized
Raw element K2, isoprene side chains are mostly cis-structures, and activity is relatively low.And it is mostly that fermentation method is made that activity is highest.Therefore, micro-
Biological fermentation process prepares farnoquinone and increasingly receives an acclaim, and the industrialized production of vitamin is carried out using microbe fermentation method
Great advantage be：Production process can be greatly simplified and improve working conditions, reduce environmental pollution while being also beneficial to resource
Exploitation and comprehensive utilization.But the yield of existing strain fermentation production farnoquinone is relatively low, fermentation level is relatively low, from
And cause cost excessively high.This is a main cause of restricted fermentation production farnoquinone development.In addition, current strain fermentation
The fermentation period for producing farnoquinone is also all longer, needs 6 days or more, time cost is larger.Therefore, breeding high-yield bacterial strain is ground
Study carefully strain characteristic and fermentation behavior has important scientific value for improving the production performance of fermenting and producing farnoquinone.
A kind of new and effective new approaches of physical mutagenesis --- helium atmospheric pressure at room plasma mutation breeding technologies are wide at present
The general mutagenic and breeding applied to bacterial strain.Using high-purity helium as working gas, generated under normal temperature and pressure state high-energy it is equal from
Daughter, the high-energy chemistry active particle being rich in can generate bacterial strain the loss of high intensity inhereditary material, and then be opened using cell
Dynamic SOS high serious forgiveness repair mechanisms, generate the mismatch site of wide variety, it is abundant to ultimately form inheritance stability, type
Mutant strain.Therefore it is applied among the mutagenic and breeding of farnoquinone production bacterial strain with important value.
Invention content
Technical problem to be solved by the present invention lies in provide a kind of natto of biofermentation high yield farnoquinone (MK-7)
The mutagenic strain of bacillus (Bacillus subtilis natto), and produce farnoquinone using the strain fermentation
(MK-7) method can reach the requirement of efficiently production farnoquinone (MK-7).
The present invention is to solve above-mentioned technical problem by the following technical programs：
The first purpose of the invention is to provide the bafillus natto (Bacillus of plant height production farnoquinone (MK-7)
Subtilis natto) ND-1-A27, it is preserved in China typical culture collection center, preservation address on March 14th, 2018
For Wuhan, China Wuhan University, deposit number is CCTCC NO:M 2018131.
Second object of the present invention is to provide the bafillus natto ND-1-A27 in fermenting and producing farnoquinone
(MK-7) application in.
In one embodiment of the invention, the application is from bafillus natto ND-1-A27 fermentations 3~6
Farnoquinone (MK-7) is extracted in its obtained zymotic fluid.
Third object of the present invention is to provide the bafillus natto ND-1-A27 fermenting and producing farnoquinones (MK-
7) method, the method are that the cellular liquid culture of above-mentioned bacterial strains is seeded in fermentation with the inoculum concentration of 2~4% (w/w)
In culture medium, at 35~38 DEG C, fermented and cultured 3~6 days under the conditions of stationary culture.
In one embodiment of the invention, the fermentation medium components are 40~60g/L of glycerine；Yeast powder 40~
60g/L；180~200g/L of soy peptone；0.5~0.8g/L of dipotassium hydrogen phosphate.
Fourth object of the present invention is to provide the microbial bacterial agent for including the bafillus natto ND-1-A27.
In one embodiment of the invention, the microbial bacterial agent is solid-state microbial inoculum or liquid microbial inoculum.
Fifth object of the present invention is to provide the food, the medicines that are prepared using the bafillus natto ND-1-A27
Product or health products.
Sixth object of the present invention is to provide the bafillus natto ND-1-A27 in food, drug or health products
Application.
Beneficial effects of the present invention：
The present invention provides a kind of bafillus natto (Bacillus of biofermentation high yield farnoquinone (MK-7)
Subtilis natto) mutagenic strain, and using the strain fermentation production farnoquinone (MK-7) method, significantly carry
The high yield of farnoquinone (MK-7).And since strain growth speed is fast after mutagenesis, growth fermentation period is greatly shortened, is saved
Time cost, can be applied to industrial fermentation production and the bacterial strain genetic stability is good, and 5 generation of continuous passage, it produced vitamin
K2 (MK-7) yield is basicly stable, maintains same higher level, can be as the production bacterial strain further researched and developed.It is raw
Object material preservation
Bafillus natto (Bacillus subtilis natto) ND-1-A27, on March 14th, 2018 is preserved in
State's Type Tissue Collection, preservation address are Wuhan, China Wuhan University, and deposit number is CCTCC NO:M 2018131.
Description of the drawings
Fig. 1 is bafillus natto (Bacillus subtilis natto) ND-1 of the present invention on nutrient agar
Colonial morphology；
Fig. 2 is bafillus natto (the Bacillus subtilis natto) bats of ND-1 under the microscope of the present invention
Take the photograph image；
Fig. 3 is the lethality curve in the present invention；
Fig. 4 is the Yield comparison of mutagenic strain in the present invention；
Fig. 5 is the research of mutagenic strain fermentation period in the present invention；
Fig. 6 is the genetic stability result figure of bafillus natto mutagenic fungi.
Specific implementation mode
The present invention is further described with reference to embodiment, the present invention is not limited only to the embodiment.
Embodiment 1：The separation of initial strains bafillus natto (Bacillus subtilis natto) ND-1 identify with
And the preservation of bacterial strain
(1) separation, screening of bafillus natto (Bacillus subtilis natto) ND-1
The present invention buys natto from market, weighs 1.0-2.0g soil samples in 250mL triangular flasks, with 10-50mL physiology salts
Then aqueous suspension carries out gradient dilution, take 0.1mL dilutions to be coated on nutrient agar panel and cultivated.Under the conditions of 37 DEG C
After cultivating 8h, chooses the preferable single bacterium colony of growing way and carry out fermentation verification.Picking single bacterium colony is inoculated into fresh liquid seeds culture
Base, 37 DEG C, 90-120r/min culture 8h, then trained with the fermentation that the inoculum concentration access liquid amount of 2% (w/w) is 30mL/250mL
It supports base, 37 DEG C, ferment 3-6 days under the conditions of stationary culture, collects zymotic fluid, organic film process with 0.22 μm is extracted through extractant
Afterwards, using high performance liquid chromatography detection farnoquinone (MK-7) production concentration.
(2) identification of bafillus natto (Bacillus subtilis natto) ND-1
After carrying out morphological observation, Physiology and biochemistry identification and the analysis of 16s rDNA strain idenfications to the bacterial strain, ND- is identified
1 is bafillus natto.Specific qualification result is as follows：
Morphological observation：Bacterium colony is canescence, and subcircular, dry tack free is opaque, there is fold, and matt, edge is not only
It slides into decomposite leaf shape, central color is deeper than marginal portion (such as Fig. 1)；To cultivating bacterial strain progress individual morphology observation for 24 hours, bacterium is found
Strain Gram's staining is positive, rod-short, both ends blunt circle (such as Fig. 2).
Physiology and biochemistry is identified：Several physiological and biochemical tests have been carried out respectively to the bacterial strain isolated, including Gram's staining,
Catalase, V-P measurement, V-P cultures, pH measurement, gelatin liquefaction, Starch Hydrolysis, D-Glucose fermentation, D- wood-sugar fermentations, D-
The experiments such as mannose ferment, the growth of NaCl salt tolerants, nitrate reduction.It is compareed with bacillus subtilis to increase in qualification process
The accuracy of qualification result judgement.The characteristics such as the production Nattokinase and synthesis γ-PGA that have in conjunction with it, are further determined as
Bafillus natto.
16s rDNA strain idenfications：Bafillus natto (Bacillus subtilis are extracted by genomic kit
Natto) the full-length genome of ND-1 bacterial strains transfers the 16s rDNA segments of the bacterial strain using universal primer 27F and 1492R, sequencing
As a result in being compared on NCBI.
Embodiment 2：Preliminary screening is carried out to mutagenic strain to ND-1 mutagenesis and using analogue using ARTP
(1) preparation of bacteria suspension
Bafillus natto (Bacillus subtilis natto) ND-1 bacterial strains one on picking nutrient agar
Ring is seeded in the 250mL conical flasks equipped with 30mL seed culture mediums, at 37 DEG C, is placed on shaking table and is turned with 120r/min
Speed culture 8h is centrifuged and thalline is suspended with physiological saline to logarithmic phase.Thalline OD values are suitably diluted to physiological saline to exist
Between 0.8-1.2, metal slide glass is placed in alcolhol burner flame envelope calcination in super-clean bench, sterilized glass plate is put into after cooling
In, it takes bacteria suspension to be uniformly applied to slide glass, without air-drying, carries out mutagenesis.
(2) ARTP mutagenesis
Ultraviolet sterilization is first opened in mutagenesis system operation storehouse, then is put slide glass to mutagenesis system with aseptic nipper and operated storehouse, is adjusted
Knob below microscope carrier, makes slide glass be at flow ports.Setting instrument power is 100W, throughput parameter is 10SLM, with mutagenesis
Time is variable element, and mutation time is respectively 0s, 10s, 20s, 30s, 40s, 50s.Sample treatment finishes, and slide glass is put to dress
Have in the pipe of physiological saline, shakes, natto bacillus subtilis is eluted in liquid, new bacteria suspension is formed.After handling
Bacteria suspension carry out gradient dilution apply tablet, make lethality curve be illustrated in fig. 3 shown below, as can be seen from the figure when mutagenic treatment
Between there are apparent dose-effect relationships between bacterial strain lethality, with the extension of processing time, lethality gradually rises.
(3) resistance screening of analogue
The structure of the farnoquinone precursor substance (DHNA) containing 90mg/L will be coated on after above-mentioned bacteria suspension gradient dilution
On the solid medium of analog 1,4-dihydroxy-2-naphthsaisyuoic acid (HNA), 37 DEG C of incubator overnight incubations are placed in, obtain 31
Single bacterium colony.
Embodiment 3：Utilize the strain fermentation production farnoquinone (MK-7) after mutagenesis preliminary screening
(1) the cellular liquid culture of mutagenic strain is prepared
31 plants of mutagenic strains and initial strains natto gemma bar on the picking base of resistance culture containing analogue respectively
Bacterium (Bacillus subtilis natto) ND-1 is seeded in the 250mL conical flasks equipped with 30mL seed culture mediums, at 37 DEG C
Under, the rotating speed culture 8h on shaking table with 120r/min is placed in logarithmic phase, obtains the cellular liquid culture of mutagenic strain.
(2) ingredient of fermentation medium and proportioning are：
Glycerine 50g/L；Yeast powder 50g/L；Soy peptone 189g/L；Dipotassium hydrogen phosphate 0.6g/L；121 DEG C of high steams
Lower sterilizing 20min.
(3) shake flask fermentation：
The cellular liquid culture of above-mentioned bacterial strains is seeded in the inoculum concentration of 2% (w/w) and is sent out equipped with sterilized 30mL
In the 250mL conical flasks of ferment culture medium, at 37 DEG C, fermented and cultured 6 days, obtain zymotic fluid under the conditions of stationary culture.
(4) product detection：
By above-mentioned zymotic fluid extractant (n-hexane：Isopropanol=2：1) it extracts 3 times, is blown extractant using nitrogen evaporator
It is dry, and redissolved using methanol, then cleaned by 0.22 μm of organic membrane filter, filtrate utilizes high-efficient liquid phase chromatogram technique analysis dimension life
The content of plain K2 (MK-7).It is made to volume analysis figure such as Fig. 4 of initial strains and 31 plants of mutagenic strains.Wherein number 1 is
Initial strains, 2 to 32 bacterial strains obtained for mutagenesis screening.It then obtains No. 27 producing strains of number to protrude, reaches 69.76mg/L, about
It is the 320% of original strain yield.It is named as bafillus natto ND-1-A27, is preserved in on March 14th, 2018
China typical culture collection center, preservation address are Wuhan, China Wuhan University, and deposit number is CCTCC NO:
M2018131。
Embodiment 4：The fermentation period of bacterial strain ND-1-A27 is probed into
Bacterial strain ND-1-A27 through the authenticated high yield farnoquinone (MK-7) of embodiment 3 is subjected to shaking flask continuously culture 6
It, samples, studies the variation of biomass and yield in its fermentation process daily.Following attached drawing 5, then it can be obtained from the figure that, in fermentation two
It when, thalline content has reached highest, and yield terminates to have tended towards stability in third day, therefore fermentation period only needs 3 days.
Embodiment 5：The genetic stability of bacterial strain ND-1-A27 is verified
Bacterial strain ND-1-A27 through the authenticated high yield farnoquinone (MK-7) of embodiment 3 is subjected to shaking flask continuously culture 5
Generation, to detect its genetic stability.Fermentability is measured after shake flask fermentation, is control with well-grown primary bacterial strain.Bacterium
Strain passage fermenting experiment result is illustrated in fig. 6 shown below.As seen from the figure, bacterial strain ND-1-A27 passages have no significant effect fermentation level,
Its produce farnoquinone (MK-7) ability it is basicly stable, maintain same higher level, to show bacterial strain ND-1-A27 have compared with
Good inheritance stability characteristic.
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 can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
Claims (9)
1. a plant height produces the bafillus natto Bacillus subtilis natto ND-1-A27 of farnoquinone (MK-7),
It is characterized in that, the bafillus natto Bacillus subtilis natto ND-1-A27 were in preservation on March 14 in 2018
In China typical culture collection center, preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:M
2018131。
2. bafillus natto Bacillus subtilis natto ND-1-A27 described in claim 1 are tieed up in fermenting and producing
Application in raw element K2 (MK-7).
3. application according to claim 2, which is characterized in that the application is from the bacillus natto to ferment 3~6
Farnoquinone (MK-7) is extracted in its obtained zymotic fluid.
4. bafillus natto Bacillus subtilis natto ND-1-A27 fermenting and producings dimension life described in claim 1
The method of plain K2 (MK-7), which is characterized in that the method is by the cellular liquid culture of above-mentioned bacterial strains with 2~4% (w/w)
Inoculum concentration inoculation in the fermentation medium, at 35~38 DEG C, fermented and cultured 3~6 days under the conditions of stationary culture.
5. according to the method described in claim 4, it is characterized in that, the fermentation medium components are 40~60g/L of glycerine；Ferment
40~60g/L of female powder；180~200g/L of soy peptone；0.5~0.8g/L of dipotassium hydrogen phosphate.
6. a kind of micro- comprising bafillus natto Bacillus subtilis natto ND-1-A27 described in claim 1
Bacteria agent.
7. microbial bacterial agent according to claim 6, which is characterized in that the microbial bacterial agent is solid-state microbial inoculum or liquid
Microbial inoculum.
8. being prepared using bafillus natto Bacillus subtilis natto ND-1-A27 described in claim 1
Food, drug or health products.
9. bafillus natto Bacillus subtilis natto ND-1-A27 described in claim 1 are in food, drug
Or the application in health products.
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Cited By (8)
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| CN110157654A (en) * | 2019-05-21 | 2019-08-23 | 江南大学 | A kind of bafillus natto recombinant bacterium and its construction method and application |
| CN110499345A (en) * | 2019-09-02 | 2019-11-26 | 福建康鸿生物科技有限公司 | A kind of fermentation process of vitamin k 2 (MK-7 type) |
| CN111349639A (en) * | 2020-01-17 | 2020-06-30 | 西宝生物科技（上海）股份有限公司 | High-efficiency biosynthesis vitamin K for improving bacillus natto2(MK-7) method |
| CN112812986A (en) * | 2020-12-21 | 2021-05-18 | 苏州微克生活科技有限公司 | Bacillus subtilis XTK1001 capable of producing natto kinase and vitamin K2 at high yield and application thereof |
| CN113564071A (en) * | 2021-07-16 | 2021-10-29 | 浙江珲达生物科技有限公司 | Bacillus natto for producing menadione-7 and application thereof |
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| CN114231554A (en) * | 2021-12-27 | 2022-03-25 | 安徽工程大学 | Vitamin k2Biosynthesis method of series products |
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| CN110157654A (en) * | 2019-05-21 | 2019-08-23 | 江南大学 | A kind of bafillus natto recombinant bacterium and its construction method and application |
| CN110129234A (en) * | 2019-05-27 | 2019-08-16 | 沈阳农业大学 | The bacillus subtilis strain of high yield Agua-Mephyton 2 through mutagenesis and its application |
| CN110499345A (en) * | 2019-09-02 | 2019-11-26 | 福建康鸿生物科技有限公司 | A kind of fermentation process of vitamin k 2 (MK-7 type) |
| CN110499345B (en) * | 2019-09-02 | 2021-05-28 | 福建康鸿生物科技有限公司 | Fermentation method of vitamin k2(MK-7 type) |
| CN111349639A (en) * | 2020-01-17 | 2020-06-30 | 西宝生物科技（上海）股份有限公司 | High-efficiency biosynthesis vitamin K for improving bacillus natto2(MK-7) method |
| CN112812986A (en) * | 2020-12-21 | 2021-05-18 | 苏州微克生活科技有限公司 | Bacillus subtilis XTK1001 capable of producing natto kinase and vitamin K2 at high yield and application thereof |
| CN113564071A (en) * | 2021-07-16 | 2021-10-29 | 浙江珲达生物科技有限公司 | Bacillus natto for producing menadione-7 and application thereof |
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| CN113564071B (en) * | 2021-07-16 | 2023-02-17 | 湖北美琪健康科技有限公司 | Bacillus natto for producing menadione-7 and application thereof |
| CN113755404A (en) * | 2021-10-13 | 2021-12-07 | 华北制药股份有限公司 | High-yield strain of vitamin K2(MK-7), screening method thereof and method for producing vitamin K2(MK-7) |
| CN114231554A (en) * | 2021-12-27 | 2022-03-25 | 安徽工程大学 | Vitamin k2Biosynthesis method of series products |
| CN114231554B (en) * | 2021-12-27 | 2023-07-28 | 安徽工程大学 | Vitamin k 2 Biosynthesis method of serial products |
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