CN104328064A - Bacillus natto and application of Bacillus natto in fermentation production of vitamin K2 - Google Patents
Bacillus natto and application of Bacillus natto in fermentation production of vitamin K2 Download PDFInfo
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/66—Preparation of oxygen-containing organic compounds containing the quinoid structure
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Abstract
The invention discloses bacillus natto which is classified and named bacillus natto and has been preserved in the China Center for Type Culture Collection with the preservation number of CCTCC M 2014405. The strain is extracted from a natto product purchased in the market and is obtained through ultraviolet mutagenesis and screening. By the use of the strain, soybean protein can efficiently be utilized to synthesize vitamin K2. The invention also discloses a method for producing vitamin K2 grease by the utilization of the above bacillus natto. According to the invention, a strain fermentation technology is optimized through a novel oxygen supply strategy, and fermentation cylinder scale-up is carried out. The final output of vitamin K2 reaches 60.54 mg/L. Conversion rate relative to soybean protein is 2.018 mg/g. Production costs are low. By the above method, efficient production of vitamin K2 grease can be realized; the whole process is simple; operation is convenient; production cycle is short; content of vitamin K2 is high; and fermentation cost is reduced. The method is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of bacillus natto and the application in fermentative production multiprenylmenaquinone thereof, belong to biological technical field.
Background technology
Multiprenylmenaquinone is the fat-soluble blood coagulation biostearin of a class, for aphthoquinone series compound, because its content in food is few, have the title of " platinum VITAMIN ", in the prevention and therapy of osteoporosis, there is significant effect, be called as " forth generation osteoporosis product ".The biological activity of multiprenylmenaquinone is mainly reflected in the generation promoting thrombogen and the synthesis increasing Bone Gla protein, play an important role in blood coagulation and bone metabolism, can be used for treating and the disease such as hemorrhage, arteriosteogenesis, osteoarthritis and osteoporosis that prevention vitamin K deficiency causes, in increase human bone mineral density, be better than other vitamin Ks.In recent years, deepen further again the research of its physiological function and understanding, multiprenylmenaquinone also can prevent liver cirrhosis, hepatic fibrosis is converted into liver cancer, promotes liver cell regeneration, also has certain curative effect in prevention and therapy parkinsonism simultaneously.Multiprenylmenaquinone is as human endogenous's property high reactivity VITAMIN, and the authority that its security has obtained U.S. FDA, Chinese food Drug Administration, united states drug study European Parliament and EU Council in one's power assert.
Multiprenylmenaquinone is a compounds---methyl naphthoquinone compounds, 14 kinds of forms are had according to the length difference of C-3 position isoprene side chains on its molecular structure, represent (number that n refers to isoprene unit on side chain) with MK-n, MK-4 (MK-4) and menaquinone-7 (MK-7) the most common, MK-4 is similar to vitamin K1 molecular weight, all there is very short serum half-life, realize blood circulation and need very large dosage, higher than MK-7 recommended dose 1000 times.And MK-7 fast by intestinal absorption, can be transported by large lipid granule, stop in the recycle system for a long time, blood halflife is long, has the ability to play a role in the bone except liver and artery.
Multiprenylmenaquinone was mainly through chemosynthesis in the past, and mostly its isoprene side chains of multiprenylmenaquinone of chemical method synthesis is cis-structure, and activity is lower, and the higher Agua-Mephyton 2 (MK-7) of activity only obtains by microbe fermentation method.For meeting the ever-increasing market requirement of multiprenylmenaquinone, overcoming the shortcoming of chemical method source multiprenylmenaquinone, in recent years, scientific worker has carried out the research utilizing fermentable to produce multiprenylmenaquinone.Mainly comprise and utilize bacillus natto, Flavobacterium etc., bacterial classification of the present invention is bacillus natto, is used for preparing the multiprenylmenaquinone of MK-7 type.Bacillus natto is not the title on taxonomy, is under the jurisdiction of subtilis (Bacillus subtilis).This bacterium is one of 40 kinds of probiotic bacteriums of U.S. FDA announcement, and edible safety, is the excellent species that multiprenylmenaquinone is produced, causes the extensive concern of Chinese scholars.
The domestic patent prepared about multiprenylmenaquinone is mainly concentrated at present: (1) chemical method synthesizes; (2) microbe fermentation method; CN101605883B mainly utilizes milk-acid bacteria to produce, and CN201310521459.1, CN103290077A mainly utilize Flavobacterium to prepare the multiprenylmenaquinone of MK-4.And mainly concentrate on about the patent of bacillus natto: (1) solid state fermentation prepares fodder additives; (2) functional product such as Nattokinase, Synbiotics is prepared; Only two sections relevant with this patent, CN 102827798A disclose a kind of can the bacillus natto of high yield multiprenylmenaquinone, improve growth speed and VK2 output mainly through ultraviolet, chemomorphosis and Protoplast fusion method; CN103898175A also discloses a kind of method of bacillus natto and purification multiprenylmenaquinone, inventive point mainly in method of purification, the fermentation index of not mentioned multiprenylmenaquinone.Above patent does not study the key factor directly affecting multiprenylmenaquinone accumulation in great detail, and cause output not high, industrialization difficulty is larger.Low production efficiency adds production cost, causes microbe-derived multiprenylmenaquinone expensive.Multiprenylmenaquinone is intracellular product, biomass is the key of high yield, the raising of biomass mainly relies on the supply of nutritive element, and the fermentation raw material cost of multiprenylmenaquinone mainly comes from nitrogenous source, the price 70 yuan/Kg of soy peptone, carbon source glycerine is 9 yuan/Kg only, and therefore, increasing the transformation efficiency of nitrogenous source to multiprenylmenaquinone is the key reducing raw materials cost.To sum up, obtain the superior strain of multiprenylmenaquinone, and in conjunction with excellent zymotechnique, obtain higher biomass and multiprenylmenaquinone content, and improve raw material (particularly nitrogenous source) to the transformation efficiency of multiprenylmenaquinone, be the key point of the industrialization promotion of multiprenylmenaquinone.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of autonomous screening, can the bacillus natto of high yield multiprenylmenaquinone.
The technical problem that the present invention also will solve is to provide the application of above-mentioned bacillus natto in fermentative production multiprenylmenaquinone.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of bacillus natto, its Classification And Nomenclature is bacillus natto (Bacillus natto), be preserved in China typical culture collection center (being called for short CCTCC), address: Wuhan, China Wuhan University, postcode: 430072, its deposit number is CCTCC M 2014405, and preservation date is on September 9th, 2014.This bacillus natto obtains from the natto product that market, Nanjing is bought, and by ultraviolet mutagenesis, screening obtains the higher bacterial classification of protease activity.
The multiprenylmenaquinone prepared by this strain fermentation is MK-7 type.
Bacillus natto CCTCC M 2014405 strain characteristic is as follows:
(1) colonial morphology: by bacterial classification dilution spread on solid medium, 30-37 DEG C of cultivation, after 24h, bacterium colony surface irregularity is opaque, dirty white or micro-yellow;
(2) thalli morphology: form and the structure of observing CCTCC M 2014405 under an optical microscope, thalline is shaft-like, the long 0.5-3 micron of individual cells, and gram-positive microorganism is aerobic, containing gemma, is positioned at thalline central authorities or slightly inclined;
(3) liquid culture: quiescent culture surface can produce the microbial film of one deck white, concussion culturing cell fast growth, without white films;
(4) metabolism: main metabolites is the multiprenylmenaquinone of seven alkene methyl naphthoquinone configurations, this bacterial strain all can grow at 25-40 DEG C, and wherein 30-37 DEG C is optimum growth temperature; Thalline is adapted at growing in neutral pH nutrient solution.
(5) extracellular enzyme: can produce proteolytic enzyme, amylase, cellulase etc., the outer nitrogen-containing material of decomposable asymmetric choice net born of the same parents, as soyflour, bacterium slag etc.
Judge the microorganism of this bacterium as gemma Pseudomonas by form and cultural characters, for identifying obtained bacterial strain further, using molecular biology method and its 16sRNA sequence (as shown in SEQ ID No:1) is compared.Find that this bacterium exists the homology of 96% with the 16SRNA sequence of Bacillus subtilis, therefore judge that this bacterial strain belongs to genus bacillus, called after Bacillus natto R127.
The application of above-mentioned bacillus natto in fermentative production multiprenylmenaquinone.
Wherein, described multiprenylmenaquinone is MK-7 type.
Concrete fermentation hair growth method is, CCTCC M 2014405 bacterial strain is starting strain, carries out high density fermentation in liquid medium within, carries out extracting the grease obtaining rich vitamin K2 respectively to the cell collected and filtrate.
Wherein, described high density fermentation, comprises the steps:
(1) carry out actication of culture by the bacterial strain access solid medium being kept at glycerine pipe, cultivate in 37 DEG C of incubators, 20 ~ 28h, continuously two generations of activation;
(2) bacterium on inclined-plane is washed down by the physiological saline after utilizing sterilizing, and access is equipped with in the 250mL shaking flask of 50mL seed culture medium, in the shaking table of 30 ~ 37 DEG C, with the rotating speed of 120 ~ 200rpm, cultivates 24 ~ 48h, obtains primary seed solution;
(3) be equipped with in the 500mL shaking flask of 100mL seed culture medium by first order seed access, inoculum size 2 ~ 10% (v/v), in the shaking table of 30 ~ 37 DEG C, with the rotating speed of 160 ~ 250rpm, is cultivated 18 ~ 24h, is obtained secondary seed solution;
(4) be equipped with in the fermentor tank of fermention medium by secondary seed access, inoculum size 2 ~ 10% (v/v), adds defoamer, air flow 0.2 ~ 2vvm, mixing speed 200 ~ 600rpm, tank temperature 30 ~ 37 DEG C, cultivate 64 ~ 96h, fermentative production multiprenylmenaquinone.
In step (1), (2) or (3), described solid culture based formulas is: 30g/L glucose, 40g/L peptone, 5g/L NaCl, 5g/L extractum carnis, 5g/L yeast extract paste, 30g/L agar, and solvent is water.Seed culture based formulas is 10-50g/L glucose, 20-80g/L peptone, 2-10g/L NaCl, 2-10g/L extractum carnis, 2-10g/L yeast extract paste, and solvent is water.Seed culture medium screening formulation is: 30g/L glucose, 40g/L peptone, 5g/L NaCl, 5g/L extractum carnis, 5g/L yeast extract paste, and solvent is water.Fermentative medium formula is 30-80g/L glycerine, 20-100g/L soy peptone, 0.2-1.5g/L yeast powder, 0.1-1g/LK
2hPO
4, 0.1-1g/LCaCl
2h
2o, 0.1-1g/LMgSO
47H
2o, solvent is water.Fermention medium screening formulation is: 50g/L glycerine, 30g/L soy peptone, 0.6g/L yeast powder, 0.3g/LK
2hPO
4, 0.1g/LCaCl
2h
2o, 0.3g/LMgSO
47H
2o, solvent is water.
In step (4), described defoamer is silicone SE-2, and usage quantity is 0.2-3g/L, preferred 0.3g/L.
In step (4), fermentation cylinder for fermentation is produced multiprenylmenaquinone grease and is adopted Intermittent fermentation or feed supplement formula fermentation process.
In step (4), fermentor tank adopts the gas stone (micropore sparger) of breeding fish as gas distributor.
Wherein, described extraction, comprises the steps:
(I) through microfiltration equipment concentrated broth, obtain concentrated after somatic cells, cell density higher than 100g/L, clarifixator Mechanical Crushing;
(II) filtrate carries out nanofiltration, retains secretion to the multiprenylmenaquinone outside born of the same parents;
(III) step (I) and (II) gained smudge cells and trapped fluid are added organic stirring solvent, extraction;
(IV) by organic solvent reduction vaporization, rich vitamin K2 grease is obtained.
In step (III), described organic solvent is any one or a few the mixing in normal hexane, sherwood oil, ethanol, Virahol and ether, the mixture of preferred Virahol and normal hexane 1:0.5 ~ 2 by volume.
Produce multiprenylmenaquinone by the inventive method, the content of final multiprenylmenaquinone reaches 58.64mg/L, is 1.955mg/g relative to the transformation efficiency of soy peptone.
The processing method that the present invention takes is equally applicable to bacillus natto with other bacterial strains of all belonging to and carry out mutagenic obtained bacterial classification on this basis, as Bacillus subtilis CICC10262, Bacillus subtilis CGMCC NO.8400 etc., the suitableeest bacterial classification is CCTCC M 2014405.
Beneficial effect: the present invention is from the bacterial strain CCTCC M 2014405s of autonomous screening, studying in great detail on growth characteristic basis, novel aeration strategy is adopted to optimize fermenting process, the gas masonry of breeding fish used is utilized to be fermentor tank gas distributor, enhanced oxygen blowing effect, and then a kind of method providing High-efficient Production multiprenylmenaquinone, and be applicable to scale operation, the content of final multiprenylmenaquinone reaches 58.64mg/L, and be 1.955mg/g relative to the transformation efficiency of soy peptone, production cost is lower.Meanwhile, present invention also offers a kind of method extracting multiprenylmenaquinone grease from bacillus natto, extract yield is at about 90-95%.Whole set process is simple, and easy to operate, fermenting process rotating speed is lower than 300rpm, and energy consumption is lower, reduces fermentation costs, is applicable to suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is fermented sample chromatograms.
Fig. 2 is fermented sample multiprenylmenaquinone (MK-7) mass spectrum.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1: bacterial strain screening
Get 1g natto product to be dissolved in 100ml physiological saline, vibration mixes, by the order obtained 10 of sample suspension according to every grade of dilution 10 times
-1-10
-7individual concentration, respectively by 10
-3-10
-7draw 100ul diluent in five concentration to be added in screening culture medium and to be coated with, each concentration is coated with three blocks of plates, 37 DEG C, cultivates 48h, select and can produce hydrolysis circle and larger bacterium colony, continue to rule in screening culture medium, obtain pure bacterium, through the multiple sieve that ferments, high-efficient liquid phase analysis and Mass Spectrometric Identification, acquisition can produce the bacterial classification Bacillus natto R of multiprenylmenaquinone, and multiprenylmenaquinone that this bacterial strain produces is MK-7 configuration (as Fig. 1,2).
For improving the protelytic ability of bacterial classification, mutagenesis screening is carried out to gained Bacillus natto R: by the bacterium liquid brine after activation, bacteria suspension is irradiated under ultraviolet lamp, irradiation distance 30cm, sample every 20s, finally select 180s, lethality rate carries out mutagenesis screening in the condition of about 80%, gained mutagenic bacteria liquid dilution spread is in screening culture medium, the bacterial strain that screening transparent circle is larger, called after Bacillus natto R127, is preserved in Chinese Typical Representative Culture Collection, and its deposit number is CCTCC M 2014405.
Screening culture medium is: 4g casein food grade is dissolved in 100ml water, 115 DEG C of sterilizing 20min, extractum carnis 3g, and peptone 10g, NaCl 5g, agar 20g are dissolved in 900ml water, 121 DEG C, and sterilizing 20min, is cooled to about 50 DEG C, both is mixed, is down flat plate stand-by.
HPLC method: chromatographic column: C18 post, column temperature: 50 DEG C, moving phase: methyl alcohol, flow velocity: 1ml/min, ultraviolet detection wavelength 270nm, sample size: 30ul, detection time: 35min.
Embodiment 2: the basic growth performance of bacterial strain is investigated.
Fundamental property investigation is carried out to the bacterial strain CCTCC M 2014405 that screening obtains, different liqs seed culture medium is adopted to cultivate, investigate cell growth rate, result is as table 1, and used medium is (% represents the quality g containing material in every 100ml substratum):
Substratum 1:100mL bean sprout juice, 5% sucrose
Substratum 2:1% glucose, 1% peptone, 0.5%NaCl
Substratum 3:3% glucose, 4% peptone, 0.5%NaCl, 0.5% extractum carnis, 0.5% yeast extract paste
Substratum 4:0.5% glucose, 1% peptone, 0.5%NaCl, 0.5% extractum carnis, 0.5% yeast extract paste
Table 1 different seed culture medium thalli growth information slip
Drawn by above-mentioned test-results, except No. 2 substratum, other three kinds all can make bacterial classification grow preferably, select No. 3 substratum.
Embodiment 3: strain for accumulating multiprenylmenaquinone ability is investigated.
Four kinds of different fermentations substratum are selected to ferment, the throughput of Primary Study bacterial classification multiprenylmenaquinone.Culture temperature 30 DEG C, rotating speed 120rpm.(% represents the quality g containing material in every 100ml substratum):
Substratum 1:5% glycerine, 3% soy peptone, 0.6g/L yeast powder, 0.3g/L K
2hPO
4, 0.1g/LCaCl
2h
2o, 0.3g/LMgSO
47H
2o
Substratum 2:5% glycerine, 3% analysis for soybean powder, 0.6g/L yeast powder, 0.3mol/LK
2hPO
4, 0.1g/LCaCl
2h2O, 0.3g/LMgSO
47H2O
Substratum 3:15g/L peptone, 25g/L yeast powder, 70g/L glycerine, 0.5g/L K
2hPO
4, pH7.3
Substratum 4:100mL soya-bean milk, 2% glucose, 0.5%NaCl, 2%NaOH adjust pH7.0
Table 2 different fermentations substratum produces multiprenylmenaquinone comparison sheet
Drawn by above-mentioned test-results, No. 3 substratum base consumption speed are lower, and total fat content of No. 2 substratum extractions is up to 6.99g/L, and this is that soyflour is relevant with major nitrogen source in its substratum and raw material, soyflour contains more grease, is proposed when fermentation ends simultaneously.And for the ability of synthesise vitamins K2, the synthesis of all favourable multiprenylmenaquinone of 1-3 substratum, wherein No. 1 substratum reaches 10.5mg/L, is production peak, and follow-up study is further experiment based on this substratum.And No. 2 substratum and No. 1 maximum difference are that soy peptone has changed soyflour into, both are more or less the same at output, and visible cell protein capacity of decomposition is stronger.
Embodiment 4: enhanced oxygen blowing improves multiprenylmenaquinone output.
Multiprenylmenaquinone fermented mainly solid fermentation in the past, and liquid fermenting often adopts low speed conditions, output is lower, according to early-stage Study, find that multiprenylmenaquinone is mainly positioned on cytolemma, and membrane structure is the important symbol of cell division, therefore the present invention adopt Promote cell's growth to divide means to improve the synthesis of multiprenylmenaquinone.The present embodiment selects different speed conditions (120rpm, 150rpm, 180rpm, 210rpm) to inquire into its synthesising law, and substratum is with the substratum 1 in embodiment 3, and specific experiment result is as following table:
Table 3 different rotating speeds condition is on the impact of multiprenylmenaquinone output
Under different rotating speeds condition, VK2 content increases along with rotating speed, and 60h detects, and multiprenylmenaquinone, in the condition of 210rpm, reaches 20.31mg/L.During 84h, VK2 content continues to rise, though 60h glycerol concentration is down to below 5g/L, VK2 still synthesizes rapidly, is up to 25.22mg/L, and in the grease extracted, VK2 content reaches 5.47mg/g.Therefore, need strictly to control fermentation period, put tank at best harvest time.
Embodiment 5: the 5L tank fermentation of multiprenylmenaquinone.
According to embodiment result, find that oxygen supply is conducive to the synthesis of multiprenylmenaquinone, fermentor tank can be ventilated, and can optimize rotating speed, the pH in monitor procedure and dissolved oxygen, and plan selects two kinds of different oxygen supply patterns, ferments.High oxygen supply (600rpm, 1.5vvm), low oxygen supply (200rpm, 1.5vvm), select soy peptone and mushroom powder two kinds of raw materials, ferment, substratum is the same.
Table 4 5L tank different rotating speeds is on the impact of multiprenylmenaquinone output
Utilize 5L to ferment, multiprenylmenaquinone content presents the rear downward trend that first rises, and is conducive to the synthesis of VK2 under high rotating speed 600rpm condition, and wherein 60h output reaches the highest, 35.73mg/L, and multiprenylmenaquinone content accounts for the 5.69mg/g of extracted grease.Contrast substrate glycerol consumption data, learn about 48h, substrate is close to 0, cell enters stationary phase, and VK2 continues synthesis, reaches the maximum of 60h, but continue to extend fermentation period significantly to reduce to 84h, VK2 content, this may to be in decline phase relevant with thalline.Therefore, best harvest time is 60h, and calculate according to 60h fermentation time, the productive rate of multiprenylmenaquinone reaches 595.5ug/L.h.
Embodiment 6: improve the fermentation of 50L tank gas distributor and produce multiprenylmenaquinone.
Consider in large-scale fermentation, rotating speed can not reach 600rpm, therefore the gas stone (micropore sparger) of breeding fish is adopted to improve fermentor tank gas distributor at 50L fermentor tank, rotating speed adjustable reduces energy consumption at below 300rpm, this micropore sparger can by dispersed for bubble one-tenth small bubbles, improve gas transfer efficiency, the results are shown in Table 5, fermentation period foreshortens to 48h, multiprenylmenaquinone output reaches 40.32mg/L, glycerine is reduced to about 5g/L, add glycerine, output reaches 58.64mg/L, 1.955mg/g is reached relative to soy peptone transformation efficiency, for reporting highest level so far, production cost reduces greatly.
Table 5 gas distributor improves
Embodiment 7: multiprenylmenaquinone production technique
By CCTCC M 2014405 bacterial classification according to solid culture based formulas be: 30g/L glucose, 40g/L peptone, 5g/LNaCl, 5g/L extractum carnis, 5g/L yeast extract paste, 30g/L agar, activate, 37 DEG C, cultivate 20h, with physiological saline, inclined-plane thalline is washed down, liquid culture is carried out in substratum 3 in access embodiment 2, 37 DEG C, 120rpm, after cultivating 24h, accessing liquid amount according to 5% inoculum size is carry out two cultures in the 500ml shaking flask of 100ml, then according in the inoculum size access 15L seeding tank of 5%, 1vvm, 200rpm, tank temperature 37 DEG C, cultivate 24h, according to 5% inoculum size, seed liquor is accessed with in the 50L fermentor tank of micropore sparger, liquid amount 35L, substratum is with the substratum 1 in embodiment 3, add 0.3g/L defoamer silicone SE-2, rotating speed 200rpm, ventilation 1vvm, temperature 37 DEG C, detect glycerine Expenditure Levels, glycerine is reduced to about 5g/L, add glycerine, final multiprenylmenaquinone output reaches 60.54mg/L, 2.018mg/g is reached relative to soy peptone transformation efficiency, for reporting highest level so far, production cost reduces greatly.
After fermentation ends, fermented liquid is carried out membrane filtration, collect filtrate and cell respectively, sampling detects two-part multiprenylmenaquinone content.Cell utilizes clarifixator broken, filtrate proceeds nanofiltration again, trapped fluid is mixed into that broken Cell relay is continuous to be extracted, and carries out stirring and evenly mixing, extract three times according to the volume ratio of bacterium liquid, Virahol, normal hexane 1:1:2, collect normal hexane layer, carry out rotary evaporation, obtain the grease of rich vitamin K2, this grease is carried out HPLC analysis, detect the content of multiprenylmenaquinone, extraction yield 95.11%.
Table 6
Note: total amount represents the amount extracting the rear total multiprenylmenaquinone obtained, and detected value is not extracting the multiprenylmenaquinone content in front direct-detection fermented liquid.
Claims (10)
1. a bacillus natto, its Classification And Nomenclature is bacillus natto (Bacillus natto), is preserved in China typical culture collection center, and its deposit number is CCTCC M 2014405, and preservation date is on September 9th, 2014.
2. the application of bacillus natto according to claim 1 in fermentative production multiprenylmenaquinone.
3. application according to claim 2, is characterized in that, described multiprenylmenaquinone is MK-7 type.
4. the application according to Claims 2 or 3, is characterized in that, CCTCC M 2014405 bacterial strain is starting strain, carries out high density fermentation in liquid medium within, carries out extracting the grease obtaining rich vitamin K2 respectively to the cell collected and filtrate.
5. application according to claim 4, is characterized in that, described high density fermentation, comprises the steps:
(1) carry out actication of culture by the bacterial strain access solid medium being kept at glycerine pipe, cultivate in 37 DEG C of incubators, 20 ~ 28h, continuously two generations of activation;
(2) bacterium on inclined-plane is washed down by the physiological saline after utilizing sterilizing, and access is equipped with in the 250mL shaking flask of 50mL seed culture medium, in the shaking table of 30 ~ 37 DEG C, with the rotating speed of 120 ~ 200rpm, cultivates 24 ~ 48h, obtains primary seed solution;
(3) be equipped with in the 500mL shaking flask of 100mL seed culture medium by first order seed access, inoculum size 2 ~ 10% (v/v), in the shaking table of 30 ~ 37 DEG C, with the rotating speed of 160 ~ 250rpm, is cultivated 18 ~ 24h, is obtained secondary seed solution;
(4) be equipped with in the fermentor tank of fermention medium by secondary seed access, inoculum size 2 ~ 10% (v/v), adds defoamer, air flow 0.2 ~ 2vvm, mixing speed 200 ~ 600rpm, tank temperature 30 ~ 37 DEG C, cultivate 64 ~ 96h, fermentative production multiprenylmenaquinone.
6. application according to claim 5, is characterized in that, described solid culture based formulas is 30g/L glucose, 40g/L peptone, 5g/LNaCl, 5g/L extractum carnis, 5g/L yeast extract paste, 30g/L agar, and solvent is water; Seed culture based formulas is 10-50g/L glucose, 20-80g/L peptone, 2-10g/L NaCl, 2-10g/L extractum carnis, 2-10g/L yeast extract paste, and solvent is water; Fermentative medium formula is 30-80g/L glycerine, 20-100g/L soy peptone, 0.2-1.5g/L yeast powder, 0.1-1g/LK
2hPO
4, 0.1-1g/LCaCl
2h2O, 0.1-1g/LMgSO
47H
2o, solvent is water.
7. application according to claim 5, is characterized in that, in step (4), described defoamer is siliconeSE-2, and usage quantity is 0.2-3g/L.
8. application according to claim 5, is characterized in that, in step (4), fermentation cylinder for fermentation is produced multiprenylmenaquinone grease and adopted Intermittent fermentation or feed supplement formula fermentation process; Fermentor tank adopts micropore sparger as the gas distributor of fermentor tank.
9. application according to claim 4, is characterized in that, described extraction, comprises the steps:
(I) through microfiltration equipment concentrated broth, obtain concentrated after somatic cells, cell density higher than 100g/L, clarifixator Mechanical Crushing;
(II) filtrate carries out nanofiltration, retains secretion to the multiprenylmenaquinone outside born of the same parents;
(III) step (I) and (II) gained smudge cells and trapped fluid are added organic stirring solvent, extraction;
(IV) by organic solvent reduction vaporization, rich vitamin K2 grease is obtained.
10. application according to claim 9, is characterized in that, in step (III), described organic solvent is any one or a few the mixing in normal hexane, sherwood oil, ethanol, Virahol and ether.
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| CN104711237A (en) * | 2015-03-20 | 2015-06-17 | 安徽工程大学 | Method for regulating and controlling activity of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase |
| CN106701719A (en) * | 2017-01-16 | 2017-05-24 | 中国科学院合肥物质科学研究院 | Method for simultaneously producing vitamin K2 and nattokinase through Bacillus natto fermentation |
| CN107118991A (en) * | 2017-05-25 | 2017-09-01 | 山东凤凰生物有限公司 | A kind of bafillus natto with the production abilities of MK 7 and its application |
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