CN108410775B - One plant height produces bafillus natto and its application of farnoquinone (MK-7) - Google Patents
One plant height produces bafillus natto and its application of farnoquinone (MK-7) Download PDFInfo
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Abstract
The invention discloses bafillus natto and its applications that a plant height produces farnoquinone (MK-7), belong to microorganism field.Bafillus natto (Bacillus subtilis natto) ND-1-A27 of the invention is preserved in China typical culture collection center on March 14th, 2018, 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 bafillus natto and its applications that a plant height produces farnoquinone (MK-7), belong to microorganism neck
Domain.
Background technique
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 significant.
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 lower.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
Studying carefully strain characteristic and fermentation behavior has important scientific value for improving the production performance of fermenting and producing farnoquinone.
It is a kind of at present that new and effective new approaches of physical mutagenesis --- helium atmospheric pressure at room plasma mutation breeding technologies are wide
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 high-intensitive inhereditary material loss to bacterial strain, and then be opened using cell
Dynamic SOS high serious forgiveness repair mechanism, generates the mismatch site of wide variety, it is abundant to ultimately form inheritance stability, type
Mutant strain.Therefore being applied among the mutagenic and breeding of farnoquinone production bacterial strain has important value.
Summary of the invention
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 farnoquinone is produced 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 that a plant height produces farnoquinone (MK-7)
Subtilis natto) ND-1-A27, China typical culture collection center, preservation address are preserved on March 14th, 2018
For Wuhan, China Wuhan University, deposit number is CCTCC NO:M 2018131.
A 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 fermentation 3~6
Farnoquinone (MK-7) is extracted in its obtained fermentation liquid.
Third object of the present invention is to provide the bafillus natto ND-1-A27 fermenting and producing farnoquinone (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 glycerol；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 comprising 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 care product.
Sixth object of the present invention is to provide the bafillus natto ND-1-A27 in food, drug or health care product
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 mention
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.
Biomaterial preservation
Bafillus natto (Bacillus subtilis natto) ND-1-A27, was preserved on March 14th, 2018
State's Type Tissue Collection, preservation address are Wuhan, China Wuhan University, and deposit number is CCTCC NO:M 2018131.
Detailed description of the invention
Fig. 1 is bafillus natto (Bacillus subtilis natto) ND-1 of the invention on nutrient agar
Colonial morphology；
Fig. 2 is bafillus natto (the Bacillus subtilis natto) bat of ND-1 under the microscope of the 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 embodiment
The present invention is further described below with reference to embodiment, the present invention is not limited only to the embodiment.
Embodiment 1: initial strains bafillus natto (Bacillus subtilis natto) ND-1 separation identification 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 sample in 250mL triangular flask, with 10-50mL physiology salt
Then aqueous suspension carries out gradient dilution, take 0.1mL dilution to be coated on nutrient agar panel and cultivated.Under the conditions of 37 DEG C
After cultivating 8h, chooses the preferable single colonie of growing way and carry out fermentation verifying.Picking single colonie 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 fermentation liquid, extract organic film process with 0.22 μm 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 idenfication 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)；Individual morphology observation is carried out to the bacterial strain of culture for 24 hours, finds bacterium
Strain Gram's staining is positive, rod-short, both ends blunt circle (such as Fig. 2).
Physiology and biochemistry identification: having carried out several physiological and biochemical tests to the bacterial strain isolated respectively, including Gram's staining,
Catalase, V-P measurement, V-P culture, pH measurement, gelatin liquefaction, Starch Hydrolysis, D-Glucose fermentation, D- wood-sugar fermentation, D-
The test such as mannose ferment, the growth of NaCl salt tolerant, nitrate reduction.It is compareed in qualification process with bacillus subtilis to increase
The accuracy that qualification result determines.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 idenfication: bafillus natto (Bacillus subtilis is extracted by genomic kit
Natto) the full-length genome of ND-1 bacterial strain transfers the 16s rDNA segment 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 strain one on picking nutrient agar
Ring is seeded in the 250mL conical flask equipped with 30mL seed culture medium, at 37 DEG C, is placed on shaking table and is turned with 120r/min
Speed culture 8h is centrifuged and thallus is suspended with physiological saline to logarithmic phase.Thallus OD value is 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, is put into sterilized glass plate 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, is in slide glass 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 plate, production lethality curve be illustrated in fig. 3 shown below, as can be seen from the figure when mutagenic treatment
Between between bacterial strain lethality there are apparent dose-effect relationship, with the extension of processing time, lethality is gradually risen.
(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 colonie.
Embodiment 3: farnoquinone (MK-7) is produced using the strain fermentation after mutagenesis preliminary screening
(1) the cellular liquid culture of mutagenic strain is prepared
Pick them separately the 31 plants of mutagenic strains and initial strains natto gemma bar on the base of resistance culture containing analogue
Bacterium (Bacillus subtilis natto) ND-1 is seeded in the 250mL conical flask equipped with 30mL seed culture medium, 37
At DEG C, it is placed on shaking table with the revolving speed culture 8h of 120r/min to logarithmic phase, obtains the cellular liquid culture of mutagenic strain.
(2) ingredient and proportion of fermentation medium are as follows:
Glycerol 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 flask of ferment culture medium, at 37 DEG C, fermented and cultured 6 days, obtain fermentation liquid under the conditions of stationary culture.
(4) product detection:
Above-mentioned fermentation liquid is extracted 3 times with extractant (n-hexane: isopropanol=2:1), 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 is prominent then to obtain No. 27 producing strains of number, 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:M
2018131。
Embodiment 4: the fermentation period of bacterial strain ND-1-A27 is probed into
6 are continuously cultivated by shaking flask is carried out through the bacterial strain ND-1-A27 of the authenticated high yield farnoquinone (MK-7) of embodiment 3
It, samples daily, studies the variation of biomass and yield in its fermentation process.Following attached drawing 5, then it can be obtained from the figure that, in fermentation two
It when, thallus 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 verifying of bacterial strain ND-1-A27
5 are continuously cultivated by shaking flask is carried out through the bacterial strain ND-1-A27 of the authenticated high yield farnoquinone (MK-7) of embodiment 3
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 passage has no significant effect fermentation level,
Its produce farnoquinone (MK-7) ability it is basicly stable, maintain same higher level, thus 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 intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (9)
1. a plant height produces bafillus natto (Bacillus subtilis natto) ND-1-A27 of farnoquinone, feature
It is, during bafillus natto (the Bacillus subtilis natto) ND-1-A27 was preserved on March 14th, 2018
State's Type Tissue Collection, preservation address are 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 is in fermenting and producing
Application in farnoquinone.
3. application according to claim 2, which is characterized in that the application is from the bacillus natto to ferment 3~6
Farnoquinone is extracted in its obtained fermentation liquid.
4. bafillus natto (Bacillus subtilis natto) ND-1-A27 fermenting and producing dimension described in claim 1
The method of raw element K2, which is characterized in that the method is the cellular liquid culture by above-mentioned bacterial strains with mass fraction 2~4%
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 glycerol；Ferment
40~60g/L of female powder；180~200g/L of soy peptone；0.5~0.8g/L of dipotassium hydrogen phosphate.
6. one kind includes bafillus natto described in claim 1 (Bacillus subtilis natto) ND-1-A27's
Microbial bacterial 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 into using bafillus natto (Bacillus subtilis natto) ND-1-A27 described in claim 1
Food, drug or the health care product arrived.
9. bafillus natto (Bacillus subtilis natto) ND-1-A27 described in claim 1 prepare food,
Application in drug or health care product.
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| CN110157654B (en) * | 2019-05-21 | 2020-07-28 | 江南大学 | Bacillus natto recombinant strain and construction method and application thereof |
| CN110129234B (en) * | 2019-05-27 | 2021-03-02 | 沈阳农业大学 | Mutagenized bacillus subtilis strain with high natural vitamin K2 yield and application thereof |
| 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 |
| 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 |
| CN113755404B (en) * | 2021-10-13 | 2023-02-28 | 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) |
| 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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