WO2023284853A1 - 一种生产甲萘醌-7的纳豆芽孢杆菌及其应用 - Google Patents

一种生产甲萘醌-7的纳豆芽孢杆菌及其应用 Download PDF

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WO2023284853A1
WO2023284853A1 PCT/CN2022/105948 CN2022105948W WO2023284853A1 WO 2023284853 A1 WO2023284853 A1 WO 2023284853A1 CN 2022105948 W CN2022105948 W CN 2022105948W WO 2023284853 A1 WO2023284853 A1 WO 2023284853A1
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fermentation
liquid
bacillus natto
strain
menadione
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French (fr)
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彭湘屏
郑玲辉
孙琼
张敏
陈世敏
高祥
石磊
汪超
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湖北美琪健康科技有限公司
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Priority to EP22841486.8A priority Critical patent/EP4317415A1/en
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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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/111—Aromatic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/174—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/35—Ketones, e.g. benzophenone
    • A61K8/355—Quinones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • 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

Definitions

  • the invention relates to the technical field of microorganisms, in particular to a bacillus natto for producing menadione-7 and an application thereof.
  • Vitamin K2 (2-methyl-3-all-trans-polypentene-1,4-naphthoquinone) or menaquinones is a family of prenyl derivatives.
  • the number of isoprene residues consisting of 5 carbon atoms can be used to distinguish menadione-based compounds.
  • the nomenclature is based on the number of prenyl groups, eg "menadione” or "MK" followed by the number of groups added. Although up to 15 prenyl groups have been found in the side chain of menadione, only menadione containing 2 to 13 prenyl groups have been found in human and animal tissues. Demethylated menadione, ie unbound menadione at carbon 2, has also been found.
  • MK-7 menaquinone-7
  • MK-7 menaquinone-7
  • the method of producing MK-7 in the prior art has: (1) chemical synthesis method, this method needs to use organic solvent, not only unfriendly to environment, operator, and organic solvent residue is arranged in the gained product, and cis-trans form in the product
  • the coexistence of MK-7 of the structure (2) extract from solid fermented natto, the content of MK-7 in natto is extremely low, and has no market competitiveness; (3) obtained by the method of natural Bacillus natto liquid fermentation, the method cycle Long, low yield.
  • the present invention provides a Bacillus natto (Bacillus natto) HDCC00023, which was deposited on February 01, 2021 In the General Microbiology Center of China Committee for Culture Collection of Microorganisms, the preservation number is CGMCC NO.21799.
  • menadione-7-containing medicines health products, food, beverages, cosmetics, additives, and feedstuffs.
  • a fermented liquid containing the bacillus natto A fermented liquid containing the bacillus natto.
  • menadione-7-containing medicines health products, food, beverages, cosmetics, additives, and feedstuffs.
  • a fermentation method for producing menadione-7 by using the bacillus natto is a fermentation method for producing menadione-7 by using the bacillus natto.
  • the fermentation is carried out in a fermentation medium comprising assimilable carbon and/or nitrogen sources.
  • the assimilable carbon source is selected from one or any combination of starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerin, soybean oil, sorbitol, mannitol; preferably glycerol ,starch.
  • the assimilable nitrogen source is selected from yeast extract powder, yeast powder, yeast extract, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone , urea, ammonium salt or any combination of several; preferably soybean peptone.
  • the fermentation medium further includes inorganic salts selected from trisodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, One or any combination of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, manganese sulfate; preferably potassium dihydrogen phosphate, magnesium sulfate, potassium chloride, calcium chloride.
  • the seed liquid is fermented in a fermentation medium, and the dissolved oxygen of the fermentation liquid is controlled to be no less than 5% or 5-15%.
  • the seed liquid is fermented in a fermentation medium, and the total residual sugar concentration of the fermentation liquid is kept not lower than 1.0% or 1.0-1.5%.
  • the seed liquid is fermented in a fermentation medium, and the fermentation temperature is not lower than 10°C, or not lower than 20°C, or 20-60°C, or 20-50°C, or 20-40°C, or 30-50°C, or 30-40°C, or 35-40°C, or 37°C.
  • the fermentation time is not less than 12 hours, or not less than 1 day, or not less than 2 days, or not less than 3 days, or not less than 4 days, or not less than 5 days, or not less than Within 6 days, more preferably 7 days.
  • the seed liquid is primary seed liquid or secondary seed liquid.
  • the preparation method of the first-level seeds is as follows: take an activated single colony, scrape the colonies, and then inoculate them into a Erlenmeyer flask, shake them on a shaking table, and control the OD value ⁇ 4.0; the preparation method of the second-level seeds For, take first-class seeds, inoculate them into seed tanks, control dissolved oxygen ⁇ 40%, and control OD value ⁇ 6.0.
  • the preparation method of the primary seed is as follows: take an activated single colony, scrape the colony, and inoculate it into a conical flask, shake it on a shaker, and control the OD value to ⁇ 4.0, and the culture temperature is not lower than 10°C.
  • the incubation time is not less than 0.5h, or not less than 1h, or not less than 2h, or not less than 3h, or not less than 4h, or not less than 5h, or not less than 6h, or not less than Less than 7h, or 8h;
  • the preparation method of the secondary seeds is as follows: take the primary seeds, inoculate them into the seed tank, control the dissolved oxygen ⁇ 40%, control the OD value ⁇ 6.0, and the cultivation temperature is not lower than 10°C, or not lower than 20°C.
  • the incubation time is not less than 0.5h, or not less than 1h, or not less than 2h, or not less than 3h, or not less than 4h, or not less than 5h, or not less than 6h, or 7h, or 8h.
  • the preparation method of the secondary seeds is as follows: take the primary seeds and inoculate them into a seed tank, the pressure of the tank is 0.05Mpa, the dissolved oxygen is controlled to be ⁇ 40%, and the OD value is controlled to be ⁇ 6.0.
  • the preparation method of the secondary seeds is as follows: take the primary seeds and inoculate them into a seed tank, the tank pressure is 0.05Mpa, the air flow rate is 1vvm, the dissolved oxygen is controlled to be ⁇ 40%, and the OD value is controlled to be ⁇ 6.0.
  • the preparation method of the secondary seeds is as follows: take the primary seeds and inoculate them into the seed tank at a ratio of 0.02-0.2% (V/W). A more preferable ratio is 0.05% (V/W).
  • the fermentation medium includes 2% glycerol, 5% soluble starch and 2% soybean peptone according to mass percentage.
  • the fermentation method further comprises adding a feed medium.
  • the feed medium includes 30% maltodextrin and 10% soybean peptone in terms of mass percentage.
  • feeding is initiated when the total sugars drop below 1.5%.
  • the seed liquid is inoculated into the fermenter of the liquid fermentation medium at a ratio of 4% (V/W), the set temperature is 37°C, the tank pressure is 0.05Mpa, the air flow rate is 0.7vvm, and the dissolved oxygen drops below 15%. , control the stirring so that the dissolved oxygen is 5-15%.
  • V/W the set temperature
  • the tank pressure is 0.05Mpa
  • the air flow rate is 0.7vvm
  • the dissolved oxygen drops below 15%.
  • control the stirring so that the dissolved oxygen is 5-15%.
  • the bacterial strain of the present invention is obtained by mutagenesis, and the effective transformation rate is significantly improved, and more MK-7 is produced under the condition of consuming very little nutrient;
  • the strain (HDCC00023) consumes nutrients more peacefully, and the control of the fermentation process is easier.
  • the strain (HDCC00023) has stronger vitality and can realize continuous fermentation production.
  • Figure 1 is a comparison of fermentation yield, substrate consumption, and by-product production (viscosity characterization) in 50L tanks of different strains.
  • Figure 2 is a comparison of parameters in the fermentation process of 50L tanks of different strains.
  • Embodiment 1 strain mutagenesis screening
  • the pure MK-7-producing strain (HDCC00001) was isolated from the feces of adults who ate natto all year round.
  • the strain was identified as Bacillus natto by 16S RNA sequencing (see SEQ ID NO: 1 for the sequence). Using adult feces as the source of isolation, the strains obtained have higher safety and are more suitable for the production of health care products.
  • HDCC00003 bacterial strain glycerol tube thawing solution Take 0.1ml of HDCC00003 bacterial strain glycerol tube thawing solution, inoculate it into 50ml LB liquid medium, cultivate overnight at 37°C, collect the bacteria after centrifugation of the seed solution, resuspend with normal saline, and mutate the bacteria suspension under 15W ultraviolet light for 10s , and then continued mutagenesis for 10 s with a final concentration of 0.2% lithium chloride.
  • the bacterial strain was obtained.
  • the highest fermentation level of the shake flask of this bacterial strain was 584 ⁇ g/ml, and the distribution range of the fermentation level of 40 single colony shake flasks was 564 ⁇ 20 ⁇ g/ml.
  • the strain has been deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on February 1, 2021, and the preservation number is CGMCC NO.21799.
  • Primary screening fermentation medium 1% glycerol, 0.5% soybean peptone, pH 7.0, sterilized at 121°C for 15 minutes, 3ml/15ml.
  • Re-screened fermentation medium glycerin 4%, soybean peptone 1.5%, pH 7.0, sterilized at 121°C for 20 minutes, 30ml/250ml.
  • Embodiment 2 original strain (HDCC00001) 50L tank fermentation test
  • the pH of LB liquid medium was adjusted to 7.0 before disinfection, and the disinfection conditions were 121-123°C for 30 minutes.
  • the pH of LB liquid medium was adjusted to 7.0 before disinfection, and the disinfection conditions were 121-123°C and holding pressure for 30 minutes.
  • the formula of the liquid fermentation medium is as follows: 2% glycerin, 5% soluble starch, 2% soybean peptone, the pH is adjusted to 8.0 before disinfection, and the disinfection conditions are 121-123° C., holding pressure for 30 minutes.
  • the highest titer of the strain (HDCC00001) was 20.3 ⁇ g/ml when fermented for 3 days, and then the dissolved oxygen rose rapidly, and CER (refers to the carbon dioxide of the culture in the biological fermentation process) release amount) completely dropped to zero, microscopic examination found that a large number of spores were formed, and the potency no longer increased. Through a large amount of feeding, CER rebounded and dissolved oxygen decreased, indicating that the vitality of the bacteria was restored, but the potency still could not be effectively increased, and even fell down. At the same time, the by-products continue to accumulate, resulting in a continuous increase in the viscosity of the feed liquid with the prolongation of the fermentation culture period, resulting in a worse mass transfer effect of the fermentation liquid.
  • Embodiment 3 50L tank fermentation test of mutagenic strain (HDCC00003)
  • the fermentation cycle of the strain (HDCC00003) can be extended to 4 days, and the highest titer is 124 ⁇ g/ml at this time.
  • the carbon and nitrogen sources are consumed rapidly, the dissolved oxygen drops to zero rapidly, the agitator runs at the maximum speed, and the feeding amount increases linearly.
  • the results of microscopic examination showed that the increase of feed amount effectively avoided the formation of spores, but the rate of titer growth was still unable to be maintained.
  • the effective fermentation period of the strain (HDCC00023) can last for 7 days, and the fermentation titer can maintain continuous growth, reaching a maximum of 526 ⁇ g/ml, successfully expressing the strain Productivity demonstrated in shake flasks.
  • strain (HDCC00003) and strain (HDCC00001) strain (HDCC00023) consumed the least nutrient substance and produced the most MK-7 by comparison between titer and cumulative feed.
  • the viscosity of the feed liquid is maintained at a level of about 100 cps, which indicates that the formation of by-products is effectively controlled. That is to say, the effective transformation rate of the strain (HDCC00023) was significantly increased and more MK-7 was produced under the condition of consuming very little nutrient.
  • the decrease of feed liquid viscosity reduces the difficulty of oxygen entering the fermentation liquid, making it easier to control dissolved oxygen through stirring. It is not necessary to frequently increase the stirring speed to maintain dissolved oxygen.
  • the required dissolved oxygen can be achieved by maintaining around 400rpm, and the entire fermentation process dissolves Oxygen is controllable. And because it does not need to run at high speed frequently, the machine loss and energy consumption are also reduced accordingly. Further, since the viscosity of the fermentation broth is reduced, the difficulty of subsequent purification is correspondingly reduced.
  • the fluctuation range of pH can reflect the stability of the strain metabolizing nutrients, and the small fluctuation range of pH reflects that the strain (HDCC00023) consumes nutrients more peacefully. Therefore, compared with other strains, the fermentation process of the strain (HDCC00023) Control is easier.
  • Embodiment 5 mutagenic strain (HDCC00023) 30 tons (T) tank fermentation test
  • the pH of LB liquid medium was adjusted to 7.0 before disinfection, and the disinfection conditions were 121-123°C for 30 minutes.
  • the pH of LB liquid medium was adjusted to 7.0 before disinfection, and the disinfection conditions were 121-123°C and holding pressure for 30 minutes.
  • the formula of the liquid fermentation medium is as follows: 2% glycerin, 5% soluble starch, 2% soybean peptone, the pH is adjusted to 8.0 before disinfection, and the disinfection conditions are 121-123° C., holding pressure for 30 minutes.
  • the results of fermentation culture show that after the strain (HDCC00023) is scaled up to 30T scale, the process is stable and controllable, with good reproducibility, the effective fermentation period can last for more than 7 days, and the fermentation titer reaches 384 ⁇ g/ml.
  • the titer of the strain (HDCC00023) is much higher than that of the strain (HDCC00003) and the strain (HDCC00001), the fermentation titer can maintain continuous growth, the formation of by-products is effectively controlled, the growth and metabolism are mild, and the whole fermentation
  • the dissolved oxygen in the process is controllable, the feeding control of nutrients is easier, the process stability is good, and it is suitable for industrial scale-up. It fundamentally solves the key problems of difficult amplification (poor repeatability of process control), low effective substrate conversion rate and high production cost in the process of MK-7 industrialization, and reduces the difficulty of purification of fermentation broth.

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Abstract

本发明提供了一种生产甲萘醌-7的纳豆芽孢杆菌，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.21799。该菌种营养物质需求少、产量高、代谢温和、易于调控、适合产业化放大。

Description

一种生产甲萘醌-7的纳豆芽孢杆菌及其应用 技术领域
本发明涉及微生物技术领域，具体涉及一种生产甲萘醌-7的纳豆芽孢杆菌及其应用。
背景技术
维生素K2(2-甲基-3-全反式-聚戊烯-1,4-萘醌)或甲萘醌类是一族异戊烯基衍生物。由5个碳原子组成的异戊二烯残基的数量可用于区分甲萘醌系化合物。其命名规则是基于异戊二烯基的数量，例如“甲萘醌”或“MK”之后加上基团的数量。尽管甲萘醌侧链上已经被发现最多达到15个异戊二烯基，但在人类和动物组织中出现的只有含有2到13个异戊二烯基的甲萘醌。脱甲基化的甲萘醌，即在2号碳原子位点上未结合的甲萘醌也已经被发现。
维生素K2的一般性名称或常用名为甲萘醌(Menaquinone)，其基本成分为甲萘醌-4(MK-4)和甲萘醌-7(MK-7)。已知MK-7是活性最强的维生素K2中的一种。
现有技术中生产MK-7的方法有：(1)化学合成法，该方法需要用到有机溶剂，不仅对环境、操作人员不够友好，而且所得产品中有有机溶剂残留，产品中顺反式结构的MK-7共存；(2)从固体发酵纳豆中提取，纳豆中MK-7含量极低，没有市场竞争力；(3)用天然纳豆菌液体发酵的方法获得，该方法周期长，产量低。
在利用纳豆芽孢杆菌发酵生产MK-7过程中，原始菌种生长代谢非常旺盛，非常容易形成大量芽孢导致产物合成停止，为了解决这一问题，必须提供大量的营养物质消耗，这使得对于生长和代谢的调控非常困难。并且这些被大量消耗的营养物质被大量转化为副产物导致发酵液粘稠，一方面不利于氧气进入发酵液，使得溶氧不易控制，另一面后续分离提纯困难。另外，发酵设备及培养条件(譬如溶氧)差异导致工艺重复性非常差。上述这些原因使得规模化生产难以实现。
发明内容
为了获得产量高、营养物质需求少、代谢温和、易于调控、适合产业化放大的纳豆芽孢杆菌，本发明提供了一种纳豆芽孢杆菌(Bacillus natto)HDCC00023，于2021年02月01日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.21799。
一种所述纳豆芽孢杆菌的用途，用于制备甲萘醌-7；
或者，用于制备含甲萘醌-7的药品、保健品、食品、饮料、化妆品、添加剂、饲料中的一个或多个。
一种含有所述纳豆芽孢杆菌的发酵液。
一种所述发酵液的用途，用于制备甲萘醌-7；
或者，用于制备含甲萘醌-7的药品、保健品、食品、饮料、化妆品、添加剂、饲料中的一个或多个。
一种用所述纳豆芽孢杆菌生产甲萘醌-7的发酵方法。
优选地，包括在含有可同化的碳源和/或氮源的发酵培养基里，进行发酵。
优选地，所述可同化的碳源选自淀粉、麦芽糊精、葡萄糖、蔗糖、乳糖、麦芽糖、工业糖蜜、甘油、豆油、山梨醇、甘露醇之一或者任意几种的组合；优选为甘油、淀粉。
优选地，所述可同化的氮源选自酵母抽提粉、酵母粉、酵母膏、大豆卵磷脂、黄豆饼粉、棉籽饼粉、花生饼粉、麸质粉、玉米浆干粉、豆粕、蛋白胨、尿素、铵盐之一或者任意几种的组合；优选为大豆蛋白胨。
优选地，所述发酵培养基还包括无机盐，所述无机盐选自柠檬酸三钠、磷酸二氢钾、磷酸氢二钾、硫酸铵、碳酸钙、硫酸亚铁、硫酸锌、硫酸铜、氯化钠、氯化钾、氯化钙、硫酸镁、氯化铁、硫酸锰之一或任意几种的组合；优选为磷酸二氢钾、硫酸镁、氯化钾、氯化钙。
优选地，取种子液在发酵培养基中发酵，控制发酵液的溶氧为不低于5％或者5～15％。
优选地，取种子液在发酵培养基中发酵，维持发酵液的总残糖浓度不低于1.0％或者1.0～1.5％。
优选地，取种子液在发酵培养基中发酵，发酵温度不低于10℃，或者不低于20℃，或者为20～60℃，或者为20～50℃，或者为20～40℃，或者为30～50℃，或者为30～40℃，或者为35～40℃，或者为37℃。
优选地，所述发酵时间不低于12h、或者不低于1天、或者不低于2天、或者不低于3天、或者不低于4天、或者不低于5天、或者不低于6天，更优选地为7天。
优选地，所述种子液为一级种子液或二级种子液。
优选地，所述一级种子的制备方法为，取活化好的单菌落，刮取菌落，然后接种到三角瓶中，摇床上振荡培养，控制OD值≥4.0；所述二级种子的制备方法为，取一级种子，接种到种子罐中，控制溶氧≥40％，控制OD值≥6.0。
优选地，所述一级种子的制备方法为，取活化好的单菌落，刮取菌落，然后接种到三角瓶中，摇床上振荡培养，控制OD值≥4.0，培养温度不低于10℃，或者不低于20℃，或者为20～60℃，或者为20～50℃，或者为20～40℃，或者为30～50℃，或者为30～40℃，或者为35～40℃，或者为37℃，培养时间不低于0.5h、或不低于1h、或不低于2h、或不低于3h、或不低于4h、或不低于5h、或不低于6h、或不低于7h、或为8h；
优选地，所述二级种子的制备方法为，取一级种子，接种到种子罐中，控制溶氧≥40％，控制OD值≥6.0，培养温度不低于10℃、或者不低于20℃、或者为20～60℃、或者为20～50℃、或者为20～40℃、或者为30～50℃、或者为25～45℃、或者为30～45℃、或者为30～40℃、或者为35～45℃、或者为35～40℃、或者为37℃，培养时间不低于0.5h、或不低于1h、或不低于2h、或不低于3h、或不低于4h、或不低于5h、或不低于6h、或为7h、或为8h。
优选地，所述二级种子的制备方法为，取一级种子，接种到种子罐中，罐压0.05Mpa，控制溶氧≥40％，控制OD值≥6.0。
优选地，所述二级种子的制备方法为，取一级种子，接种到种子罐中，罐压0.05Mpa，空气流量1vvm，控制溶氧≥40％，控制OD值≥6.0。
优选地，所述二级种子的制备方法为，取一级种子，以0.02～0.2％(V/W)的比例接种到种子罐中。更优选的比例为0.05％(V/W)。
优选地，所述发酵培养基，按照质量百分比，包括甘油2％，可溶性淀粉5％，大豆蛋白胨2％。
优选地，所述发酵方法还包括加入补料培养基。
优选地，所述补料培养基，按照质量百分比，包括麦芽糊精30％，大豆蛋白胨10％。
优选地，当总糖下降到1.5％以下时，开始补料。
优选地，种子液以4％(V/W)的比例接种到液体发酵培养基的发酵罐中，设定温度37℃，罐压0.05Mpa，空气流量0.7vvm，待溶氧下降到15％以下，控制搅拌，使溶氧为5～15％。当总糖下降到1.5％以下时，开始补料，控制总糖浓度为1.0～1.5％。
有益效果：
1、通过诱变得到本发明的菌种，在消耗营养量极少的情况下，有效转化率显著提高，产出了更多的MK-7；
2、由于料液粘度降低，整个发酵过程溶氧可控，并且由于不需要经常高转速运行，使得机器损耗以及能耗也相应降低；
3、进一步地，由于发酵液的粘度降低，使得后续提纯的难度也相应降低。
4、菌种(HDCC00023)对营养物质消耗更加平和，发酵过程的控制更容易。
5、菌种(HDCC00023)的活力更强，可以实现持续发酵生产。
附图说明
图1为不同菌种50L罐发酵产量、底物消耗、副产物生成量(粘度表征)对比。
图2为不同菌种50L罐发酵过程参数对比。
具体实施方式
下述实施例中所用的实验方法如无特殊说明，均为常规方法。
下述实施例中采用的材料、试剂等如无特殊说明，皆为普通市售品，皆可于市场购得。
下面将通过实施例对本发明作进一步的描述，这些描述并不是对本发明内容作进一步的限定。本领域的技术人员应理解，对本发明内容所作的等同替换，或相应的改进，仍属于本发明的保护范围之内。
以下实施例所使用的培养基中，物料配比所用的百分数(％)计量单位，除特殊说明外均为质量百分比。
实施例1菌种诱变筛选
从常年食用纳豆的成人粪便中分离获得产MK-7的纯菌种(HDCC00001)。该菌种经16SRNA测序鉴定为纳豆芽孢杆菌(序列详见SEQ ID NO:1)。以成人粪便为分离源，获得的菌种具有更高的安全性，更适合用于保健品的生产。
取分离获得的原始菌种(HDCC00001)甘油管解冻液0.1ml，接种到50ml LB液体培养基中，37℃培养过夜，种子液用终浓度为0.2％的氯化锂诱变30s，用无菌生理盐水梯度稀释至10 -2～10 -5后分离于LB固体平板培养基，于37℃恒温避光培养24小时，长出菌落后，用牙签转种到新鲜LB固体培养基上，37℃培养过夜，用接种针接种于装有3ml液体的初筛发酵培养基的试管(15ml)中，37℃，250rpm振摇96小时，加入等体积分析纯异丙醇浸泡，离心后取上清液测定效价，最终获得MK19283菌株，该菌株试管初筛发酵水平为112μg/ml。
取MK19283菌株甘油管，用无菌生理盐水梯度稀释至10 -3～10 -6后分离于LB固体平板培养基，于37℃恒温培养24小时，长出菌落后，随机挑选40颗菌落，用接种针分别接种一环于装有20ml液体LB培养基的种瓶，37℃、250rpm培养过夜，然后以4％的比例(v/v)接种到装有30ml液体复筛发酵培养基的250ml三角瓶中，37℃，250rpm振摇96小时。取发酵液1ml，加入等体积分析纯异丙醇浸泡，离心后取上清液测定效价。选取高产的菌株，采用上述方法，连续进行自然分离纯化，直到各个菌落发酵水平稳定。连续三轮纯化后，获得菌种(HDCC00003)，该菌株摇瓶最高发酵水平为228μg/ml，40颗单菌落摇瓶发酵水平分布范围为200±10μg/ml。
取HDCC00003菌种甘油管解冻液0.1ml，接种到50ml LB液体培养基中，37℃培养过夜，种子液离心后收集菌体，用生理盐水重悬，菌悬液于15W紫外灯下诱变10s，然后用终浓度为0.2％的氯化锂继续诱变10s。参照上述得到菌种(HDCC00003)的分离纯化方法，获得菌株(HDCC00023)，该菌株摇瓶最高发酵水平为584μg/ml，40颗单菌落摇瓶发酵水平分布范围为564±20μg/ml。该菌种已于2021年02月01日保藏于中国微生物菌种保藏管理委员会普通 微生物中心，保藏编号为CGMCC NO.21799。
初筛发酵培养基：甘油1％，大豆蛋白胨0.5％，pH7.0，121℃消毒15min，3ml/15ml。
复筛发酵培养基：甘油4％，大豆蛋白胨1.5％，pH7.0，121℃消毒20min，30ml/250ml。
实施例2原始菌种(HDCC00001)50L罐发酵试验
1、菌种活化
取原始菌种(HDCC00001)甘油管，在室温下解冻，吸取0.1ml菌悬液接种到LB固体平板，涂布均匀，置于37℃培养箱内培养16h，得到活化好的单菌落。
2、一级种子制备
取活化好的单菌落，用接种环刮取一环菌落，然后接种到盛有100ml LB液体培养基的500ml三角瓶中，包扎好置于37℃、250rpm的摇床上振荡培养8h，控制种子液OD值≥4.0。
LB液体培养基消毒前调pH至7.0，消毒条件为121～123℃、30min。
3、二级种子制备
取培养合格的一级种子，以0.05％(V/W)的比例接种到装有10L的LB液体培养基的种子罐中，设定温度37℃，罐压0.05Mpa，空气流量1vvm，初始搅拌100rpm，搅拌联动控制溶氧≥40％，培养周期7h，控制种子液OD值≥6.0。
LB液体培养基消毒前调pH至7.0，消毒条件为121～123℃、保压30min。
4、发酵培养
取培养合格的二级种子，以4％(V/W)的比例接种到装有30L液体发酵培养基的发酵罐中，设定温度37℃，罐压0.05Mpa，空气流量0.7vvm，初始搅拌150rpm，待溶氧下降到15％以下，开启搅拌联动，控制溶氧5～15％。维持总残糖浓度1.0～1.5％，发酵周期为7天。
液体发酵培养基配方组成为：甘油2％，可溶性淀粉5％，大豆蛋白胨2％，消毒前调pH至8.0，消毒条件为121～123℃、保压30min。
5、发酵过程指标监测
自发酵开始，每12h取样测定总糖、氨基氮、效价，并完成补料量统计记录。
根据菌种的发酵过程和结果，以及参考图1和图2，菌种(HDCC00001)发酵3天时最高效价20.3μg/ml，之后溶氧快速回升，CER(指生物发酵过程中培养物的二氧化碳释放量)完全掉零，镜检发现形成大量芽孢，效价不再增长。通过大量补料，CER出现回升，溶氧下降，说明菌体活力得到恢复，但是效价仍无法有效增长，甚至出现倒跌。与此同时，副产物在持续积累，导致料液粘度随着发酵培养周期的延长持续增加，导致发酵液传质效果更差。
实施例3：诱变菌种(HDCC00003)50L罐发酵试验
除了菌种为诱变菌种(HDCC00003)外，其它同实施例2。
根据菌种的发酵过程和结果，以及参考图1和图2，菌种(HDCC00003)发酵周期可以延长至4天，此时最高效价124μg/ml。发酵中前期，碳氮源被迅速消耗，溶氧快速掉零，搅拌最大转速运行，补料量呈直线上升。镜检结果显示，补料量的增加有效避免了芽孢的形成，但是仍然无法维持效价增长的速度。此外，发酵过程搅拌、溶氧、CER曲线显示，84h后发酵参数出现反复波动，说明氯化锂诱变后得到的菌种虽然效价提高，但是菌种发酵过程稳定性差，难以进行等比例放大控制。
实施例4诱变菌种(HDCC00023)50L罐发酵试验
除了菌种为诱变菌种(HDCC00023)外，其它同同实施例2。
根据菌种的发酵过程和结果，以及参考图1和图2，菌种(HDCC00023)有效发酵周期可以持续7天，发酵效价可以维持持续增长，最高达到526μg/ml，成功表达出了该菌种在摇瓶上表现出的生产能力。通过效价与累积补料之间的对比发现，与菌种(HDCC00003)和菌种(HDCC00001)相比，菌种(HDCC00023)消耗最少的营养物质而产出最多的MK-7。而料液粘度维持在100cps左右水平，则表明副产物的形成得到有效控制。即菌种(HDCC00023)在消耗营养量极少的情况下，有效转化率显著提高，产出了更多的MK-7。
料液粘度降低导致氧气进入发酵液难度降低，使得通过搅拌来控制溶氧更加容易，不需要经常提高搅拌速度来维持溶氧，维持在400rpm左右就可以达到所需溶氧量，整个发酵过程溶氧可控。并且由于不需要经常高转速运行，使得机器损耗以及能耗也相应降低。进一步地，由于发酵液的粘度降低，使得后续提纯的难度也相应降低。
由于CER相对稳定，镜检整个过程也未形成大量芽孢，表明菌种(HDCC00023)的活力更强，可以实现持续发酵产。另外pH的波动幅度可以反映菌种代谢营养物质的稳定性，pH波动幅度小反映出菌种(HDCC00023)对营养物质消耗更加平和，因此与其它菌种相比，菌种(HDCC00023)发酵过程的控制更容易。
实施例5诱变菌种(HDCC00023)30吨(T)罐发酵试验
1、菌种活化
取生产菌种(HDCC00023)工作甘油管，在室温下解冻，吸取0.1ml菌悬液接种到LB固体平板，涂布均匀，置于37℃培养箱内培养16h，得到活化好的单菌落。
2、一级种子制备
取活化好的单菌落，用接种环刮取一环菌落，然后接种到盛有500ml LB液体培养基的2800ml三角瓶中，包扎好置于37℃、220rpm的摇床上振荡培养8h，控制种子液OD值≥4.0。
LB液体培养基消毒前调pH至7.0，消毒条件为121～123℃、30min。
3、二级种子制备
取培养合格的一级种子，以0.05％(V/W)的比例接种到装有2吨LB液体培养基的种子罐中，设定温度37℃，罐压0.05Mpa，空气流量1vvm，初始搅拌100rpm，搅拌联动控制溶氧≥40％，培养周期7h，控制种子液OD值≥6.0。
LB液体培养基消毒前调pH至7.0，消毒条件为121～123℃、保压30min。
4、发酵培养
取培养合格的二级种子，以4％(V/W)的比例接种到装有20吨液体发酵培养基的发酵罐中，设定温度37℃，罐压0.05Mpa，空气流量0.7vvm，初始搅拌80rpm，待溶氧下降到15％以下，开启搅拌联动，控制溶氧5～15％。控制总糖浓度为1.0～1.5％，发酵周期为7天。
液体发酵培养基配方组成为：甘油2％，可溶性淀粉5％，大豆蛋白胨2％，消毒前调pH至8.0，消毒条件为121～123℃、保压30min。
发酵培养结果显示，菌种(HDCC00023)在按照比例放大到30T规模后，工艺稳定可控，重现性好，有效发酵周期可以持续7天以上，发酵效价达到384μg/ml。
综上所述，菌种(HDCC00023)的效价远高于菌种(HDCC00003)和菌种(HDCC00001)，发酵效价可以维持持续增长，副产物的形成得到有效控制，生长代谢温和，整个发酵过程溶氧可控，对营养物质的投料控制更容易，工艺稳定性好，适合产业化放大。从根本上解决了MK-7产业化过程中放大困难(工艺调控重复性差)、底物有效转化率低、生产成本高的关键性难题，以及降低了发酵液的提纯难度。
现有技术中存在多种利用纳豆芽孢杆菌生产MK-7的发酵方法，以上的实施例仅用于示例性说明本发明提供的纳豆芽孢杆菌所采用的发酵方法。可以理解的是，获知本发明提供的纳豆芽孢杆菌后，本领域技术人员可以利用本发明提供的纳豆芽孢杆菌发酵生产MK-7和其它产物(如种子液、发酵液、发酵产物、菌渣)，以及利用本发明提供的纳豆芽孢杆菌或其生产的MK-7或其它产物生产其它产品，如制造药品、保健品、食品、饮料、化妆品、添加剂、饲料。

Claims (12)

  1. 一种纳豆芽孢杆菌(Bacillus natto)HDCC00023，于2021年02月01日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.21799。
  2. 一种如权利要求1所述纳豆芽孢杆菌的用途，其特征在于：
    用于制备甲萘醌-7；
    或者，用于制备含甲萘醌-7的药品、保健品、食品、饮料、化妆品、添加剂、饲料中的一个或多个。
  3. 一种含有如权利要求1所述纳豆芽孢杆菌的发酵液。
  4. 一种如权利要求3所述发酵液的用途，其特征在于：
    用于制备甲萘醌-7；
    或者，用于制备含甲萘醌-7的药品、保健品、食品、饮料、化妆品、添加剂、饲料中的一个或多个。
  5. 一种用如权利要求1所述纳豆芽孢杆菌生产甲萘醌-7的发酵方法。
  6. 如权利要求5所述的发酵方法，其特征在于：
    包括在含有可同化的碳源和/或氮源的发酵培养基里，进行发酵。
  7. 如权利要求6所述的发酵方法，其特征在于：
    所述可同化的碳源选自淀粉、麦芽糊精、葡萄糖、蔗糖、乳糖、麦芽糖、工业糖蜜、甘油、豆油、山梨醇、甘露醇之一或者任意几种的组合；优选为甘油、淀粉。
  8. 如权利要求6所述的发酵方法，其特征在于：
    所述可同化的氮源选自酵母抽提粉、酵母粉、酵母膏、大豆卵磷脂、黄豆饼粉、棉籽饼粉、花生饼粉、麸质粉、玉米浆干粉、豆粕、蛋白胨、尿素、铵盐之一或者任意几种的组合；优选为大豆蛋白胨。
  9. 如权利要求6所述的发酵方法，其特征在于：
    所述发酵培养基还包括无机盐，所述无机盐选自柠檬酸三钠、磷酸二氢钾、磷酸氢二钾、硫酸铵、碳酸钙、硫酸亚铁、硫酸锌、硫酸铜、氯化钠、氯化钾、氯化钙、硫酸镁、氯化铁、硫酸锰之一或任意几种的组合；优选为磷酸二氢钾、硫酸镁、氯化钾、氯化钙。
  10. 如权利要求5～9所述的发酵方法，其特征在于：
    取种子液在发酵培养基中发酵，控制发酵液的溶氧为不低于5％或者5～15％。
  11. 如权利要求5～9所述的发酵方法，其特征在于：
    取种子液在发酵培养基中发酵，维持发酵液的总残糖浓度不低于1.0％或者1.0～1.5％。
  12. 如权利要求5～9所述的发酵方法，其特征在于：
    取种子液在发酵培养基中发酵，发酵温度不低于10℃，或者不低于20℃，或者为20～60℃，或者为20～50℃，或者为20～40℃，或者为30～50℃，或者为30～40℃，或者为35～40℃，或者为37℃。
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