CN104694589A - Method for synthesizing vitamin K2 based on using flavobacterium in-situ fermentation, separation and coupling - Google Patents

Method for synthesizing vitamin K2 based on using flavobacterium in-situ fermentation, separation and coupling Download PDF

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Publication number
CN104694589A
CN104694589A CN201510080787.1A CN201510080787A CN104694589A CN 104694589 A CN104694589 A CN 104694589A CN 201510080787 A CN201510080787 A CN 201510080787A CN 104694589 A CN104694589 A CN 104694589A
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culture
fermentation
oil
flavobacterium
situ
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郑之明
王鹏
王丽
赵根海
刘会
王晗
吴荷芳
孙孝娟
刘红霞
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Hefei Institutes of Physical Science of CAS
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C46/00—Preparation of quinones
    • C07C46/10—Separation; Purification; Stabilisation; Use of additives

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a method for synthesizing vitamin K2 based on using flavobacterium in-situ fermentation, separation and coupling. The method comprises the following steps of performing seed culture, inoculated culture and fermented culture, standing, layering and extracting vitamin K2, wherein active ingredients are added to a fermentation medium after inoculated culture, and the active ingredients comprise 10-60g/L surface active agent, 5-40g/L glycerol and 100-250g/L vegetable oil. Compared with the prior art, the method for synthesizing vitamin K2 based on using the flavobacterium in-situ fermentation, separation and coupling has the advantages that through the surface active agent, glycerol and the vegetable oil, matched with high mixing and ventilation volume, an oil-in-water emulsion is formed in a fermentation tank, and on the basis that dissolved oxygen and mass transfer are improved, extracellular fat-soluble vitamin K2 is extracted by the vegetable oil, so that the in-situ fermentation, separation and coupling are realized.

Description

Based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method
Technical field
What the present invention relates to is a kind of based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method.
Background technology
Multiprenylmenaquinone (menaquinone, is abbreviated as MK, aphthoquinone) is a kind of derivative with the bioactive naphthoquinones group of phylloquinone, liposoluble vitamin.According to the difference of isoprene side chains length on C-3, MK has 14 kinds, usually represents with MK-n, and wherein n refers to the number of isoprene unit on side chain.MK-4, MK-7, MK-9 biological activity is all significantly higher than vitamin K1.When human homergy and functions of intestines and stomach standard state, vitamin K1 and vitamin K3, methylnaphthohydroquinone just can be utilized by intestinal absorption after being converted into multiprenylmenaquinone active body under normal intestinal flora participates in.Multiprenylmenaquinone has good promotion blood coagulation, the osteoporotic effect of prevention and therapy.
The bacterial classification that can carry out fermentative production multiprenylmenaquinone has the Flavobacterium (Flavobacterium.sp) belonging to Gram-negative bacteria, main product MK-4 and MK-6; Belong to the subtilis (Bacillus subtilis natto, Japanese document is referred to as Bacillus natto) of gram-positive microorganism, mainly produce MK-7.It is reported that some lactobacilluss can produce the MK-8,9,10 of 29-123 μ g/L in skim-milk and soymilk in addition, but because output is lower, generally can not use as fermentative production as foodstuff additive.
According to patent and reported in literature, during the fermentation, suitable tensio-active agent can improve the output of MK-4 and MK-6 to Flavobacterium.Its reason adds tensio-active agent MK-4 and MK-6 can be made to escape to outside born of the same parents, can remove Product inhibiton to a certain extent, thus improve output.But along with the outer multiprenylmenaquinone concentration of born of the same parents improves gradually, can be declined to the speed outside born of the same parents by born of the same parents' internal diffusion, and then affect productive rate.And how multiprenylmenaquinone effectively extracts also is problem in the urgent need to address.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, providing a kind of based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method.
The present invention is achieved by the following technical solutions: a kind of based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, comprise the steps: seed culture, inoculation culture, fermentation culture and stratification extract vitamin K 2, in fermentation culture process, add activeconstituents by the fermention medium after inoculation culture, described activeconstituents is composed of the following components: 10g/L-60g/L tensio-active agent, 5g/L-40g/L glycerine and 100g/L-250g/L vegetables oil.
Optimize, a kind of based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, specifically comprise the steps:
(1) seed culture, gets in the 500mL triangular flask that a ring Flavobacterium is inoculated in containing 50-150mL seed culture medium, under temperature is 30-37 DEG C of condition, with rotating speed 100-200rpm shaking culture 24-48h from preserving inclined-plane;
Described seed culture medium is composed of the following components: 1-5g/L glucose, 2-10g/L glycerine, 10-20g/L peptone, 1.5-4g/L yeast extract, 1-4g/L K 2hPO 4, 1-3g/L NaCl and 0.1-1g/L MgSO 47H 2o;
(2) inoculation culture, accesses in the fermention medium in fermentor tank by inoculum size 5%-10%, culture temperature 30-37 DEG C, under condition, with rotating speed 100-200rpm, air flow 0.5-1vvm stir culture 24-36 hour;
Described fermention medium is composed of the following components: 5-30g/L analysis for soybean powder, 0-10g/L glucose, 10-20g/L peptone, 3-10g/L yeast extract, 1-3g/L K 2hPO 4, 1-4g/L NaCl, 0.1-1g/L MgSO 47H 2o;
(3) fermentation culture, 10g/L-60g/L tensio-active agent, 5g/L-40g/L glycerine and 100g/L-250g/L vegetables oil will be added in fermention medium after inoculation culture, and stir speed (S.S.) 500-800rpm, air flow 1.5-3vvm stirs, form oil-in-water microemulsion, fermentation culture 72-96 hour;
(4) stratification extracts, and will stop stirring through the mixture of step (3) fermentation culture, and after leaving standstill the little layered of 3-5, extract upper strata vegetables oil, containing total multiprenylmenaquinone in oil phase.
As the further optimization of such scheme, in above-mentioned steps (3), tensio-active agent is selected from one or more in sucrose fatty ester (SE), Polyoxyethylene Sorbitol Fatty Acid Esters-80 (Tween80), poly-Labraso (Labrasol).
As the further optimization of such scheme, vegetables oil is selected from soybean oil, Semen Maydis oil, peanut oil in above-mentioned steps (3), one or more in sweet oil, camellia wet goods.
The present invention has the following advantages compared to existing technology: one of the present invention is based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, by tensio-active agent, glycerine, vegetables oil, coordinate high stirring, high air flow, forms O/w emulsion in fermentor tank, on the basis of improving dissolved oxygen and mass transfer, make the outer fat-soluble multiprenylmenaquinone of born of the same parents by vegetable oil extraction, realize the fermentation separation coupling of original position.After having fermented, the multiprenylmenaquinone containing the overwhelming majority in vegetables oil.The present invention is while raising multiprenylmenaquinone output, and multiprenylmenaquinone is extracted into vegetables oil by original position, greatly reduces later stage separation costs, for realizing vitamin K 2heavy industrialization in-situ fermentation separation coupling provide possibility.
Accompanying drawing explanation
Fig. 1 is that one of the present invention is based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2the process flow sheet of method.
Embodiment
Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
A kind of based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, with Flavobacterium (Flavobacterium.sp) for fermentation strain, see Fig. 1, operate according to the following step successively:
1. seed culture, get in the 500mL triangular flask that a ring Flavobacterium is inoculated in containing 100mL seed culture medium from preserving inclined-plane, described seed culture medium is composed of the following components: glucose 3g/L, glycerine 5g/L, peptone 10g/L, yeast extract 3g/L, K 2hPO 43g/L, NaCl 3g/L, MgSO 47H 2o 0.3g/L, pH nature.37 DEG C, with rotating speed 200rpm shaking culture 24h.
2. inoculation culture, accesses in the fermention medium in fermentor tank by inoculum size 5%.Fermention medium is composed of the following components: analysis for soybean powder 20g/L, glucose 10g/L, peptone 10g/L, yeast extract 10g/L, K 2hPO 43g/L, NaCl 2g/L, MgSO 47H 2o 1g/L.With culture temperature 37 DEG C, rotating speed 150rpm, air flow 0.8vvm, incubation time 24 hours.
3. fermentation culture, 50g/L sucrose fatty ester (SE) is added in fermentor tank, 10g/L glycerine, 150g/L soybean oil, stir speed (S.S.) is increased to 600rpm, and air flow is increased to 2vvm, forms oil-in-water (O/W) micro emulsion, fermentation culture 96 hours, multiprenylmenaquinone output is about 85mg/L.
4. stratification extracts, and stops stirring and ventilation, leaves standstill 5 hours, after layering, extracts upper strata soybean oil, multiprenylmenaquinone collection rate about 80% in oil phase.
Embodiment 2
A kind of based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, with Flavobacterium (Flavobacterium.sp) for fermentation strain, see Fig. 1, operate according to the following step successively:
1. seed culture, gets in the 500mL triangular flask that a ring Flavobacterium is inoculated in containing 80mL seed culture medium from preserving inclined-plane, seed culture medium: glucose 5g/L, glycerine 10g/L, peptone 20g/L, yeast extract 3g/L, K 2hPO 42g/L, NaCl 2g/L, MgSO 47H 2o 0.4g/L, pH nature.At 37 DEG C, shaking table 180rpm shaking culture 24h.
2. inoculation culture, accesses fermentor tank by inoculum size 5%.Fermention medium: analysis for soybean powder 30g/L, glucose 5g/L, peptone 15g/L, yeast extract 5g/L, K 2hPO 41g/L, NaCl 1g/L, MgSO 47H 2o 0.5g/L, culture temperature 35 DEG C, rotating speed 200rpm, air flow 1vvm, incubation time 36 hours.
3. fermentation culture, incubation time is after 36 hours, 40g/L Polyoxyethylene Sorbitol Fatty Acid Esters-80 (Tween80) is added in fermentor tank, 20g/L glycerine, 200g/L Semen Maydis oil, stir speed (S.S.) is increased to 700rpm, air flow is by being increased to 1.8vvm, form oil-in-water (O/W) micro emulsion, fermentation culture 80 hours, multiprenylmenaquinone output is about 72mg/L.
4. stratification extracts, and stops stirring and ventilation, leaves standstill 3 hours, after layering, extracts upper strata Semen Maydis oil, multiprenylmenaquinone collection rate about 70% in oil phase.
Embodiment 3
A kind of based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, with Flavobacterium (Flavobacterium.sp) for fermentation strain, see Fig. 1, operate according to the following step successively:
1. seed culture, gets in the 500mL triangular flask that a ring Flavobacterium is inoculated in containing 150mL seed culture medium from preserving inclined-plane, seed culture medium: glucose 5g/L, glycerine 5g/L, peptone 15g/L, yeast extract 10g/L, K 2hPO 41g/L, NaCl 1g/L, MgSO 47H 2o1g/L, pH nature.At 37 DEG C, shaking table 200rpm shaking culture 36h.
2. inoculation culture, accesses fermentor tank by inoculum size 10%.Fermention medium: analysis for soybean powder 25g/L, glucose 8g/L, peptone 10g/L, yeast extract 10g/L, K 2hPO 42g/L, NaCl 2g/L, MgSO 47H 2o 0.5g/L, culture temperature 37 DEG C, rotating speed 150rpm, air flow 1vvm, incubation time 24 hours.
3. fermentation culture, incubation time is after 24 hours, in fermentor tank, add 50g/L gather Labraso (Labrasol), 10g/L glycerine, 15g/L sweet oil, stir speed (S.S.) is increased to 650rpm, air flow is by being increased to 2vvm, form oil-in-water (O/W) micro emulsion, fermentation culture 96 hours, multiprenylmenaquinone output is about 80mg/L.
4. stratification extracts, and stops stirring and ventilation, leaves standstill 4 hours, after layering, extracts upper strata sweet oil, multiprenylmenaquinone collection rate about 82% in oil phase.
Embodiment 4
A kind of based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, with Flavobacterium (Flavobacterium.sp) for fermentation strain, see Fig. 1, operate according to the following step successively:
1. get in the 500mL triangular flask that a ring Flavobacterium is inoculated in containing 150mL seed culture medium from preserving inclined-plane, seed culture medium: glucose 5g/L, glycerine 10g/L, peptone 15g/L, yeast extract 10g/L, K 2hPO 41.5g/L, NaCl 1.5g/L, MgSO 47H 2o0.5g/L, pH nature.At 37 DEG C, shaking table 200rpm shaking culture 36h.
2. inoculation culture, accesses fermentor tank by inoculum size 10%.Fermention medium: analysis for soybean powder 15g/L, glucose 4g/L, peptone 10g/L, yeast extract 5g/L, K 2hPO 41.5g/L, NaCl 1.5g/L, MgSO 47H 2o 0.8g/L, culture temperature 37 DEG C, rotating speed 200rpm, air flow 1vvm, incubation time 36 hours.
3. fermentation culture, incubation time is after 36 hours, 1% sucrose fatty ester (SE), 1% Polyoxyethylene Sorbitol Fatty Acid Esters-80 (Tween80) is added in fermentor tank, 20g/L glycerine, 100g/L sweet oil, 10g/L soybean oil stir speed (S.S.) is increased to 800rpm, air flow is by being increased to 3vvm, form oil-in-water (O/W) micro emulsion, fermentation culture 96 hours, multiprenylmenaquinone output is about 88mg/L.
4. stratification extracts, and stops stirring and ventilation, leaves standstill 5 hours, after layering, extracts upper strata sweet oil and soybean oil, multiprenylmenaquinone collection rate about 75% in oil phase.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

Claims (5)

1. based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, comprise the steps: seed culture, inoculation culture, fermentation culture and stratification extract vitamin K 2, it is characterized in that: in fermentation culture process, add activeconstituents in the fermention medium after inoculation culture, described activeconstituents is composed of the following components: 10g/L-60g/L tensio-active agent, 5g/L-40g/L glycerine and 100g/L-250g/L vegetables oil.
2. one according to claim 1 is based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, it is characterized in that: comprise the steps:
(1) seed culture, gets in the 500mL triangular flask that a ring Flavobacterium is inoculated in containing 50-150mL seed culture medium, under temperature is 30-37 DEG C of condition, with rotating speed 100-200rpm shaking culture 24-48h from preserving inclined-plane;
Described seed culture medium is composed of the following components: 1-5g/L glucose, 2-10g/L glycerine, 10-20g/L peptone, 1.5-4g/L yeast extract, 1-4g/L K 2hPO 4, 1-3g/L NaCl and 0.1-1g/L MgSO 47H 2o;
(2) inoculation culture, accesses in the fermention medium in fermentor tank by inoculum size 5%-10%, culture temperature 30-37 DEG C, under condition, with rotating speed 100-200rpm, air flow 0.5-1vvm stir culture 24-36 hour;
Described fermention medium is composed of the following components: 5-30g/L analysis for soybean powder, 0-10g/L glucose, 10-20g/L peptone, 3-10g/L yeast extract, 1-3g/L K 2hPO 4, 1-4g/L NaCl, 0.1-1g/L MgSO 47H 2o;
(3) fermentation culture, activeconstituents is added by the fermention medium after inoculation culture, described activeconstituents is composed of the following components: 10g/L-60g/L tensio-active agent, 5g/L-40g/L glycerine, 100g/L-250g/L vegetables oil, and stir speed (S.S.) 500-800rpm, air flow 1.5-3vvm stirs, form oil-in-water microemulsion, fermentation culture 72-96 hour;
(4) stratification extracts, and will stop stirring through the mixture of step (3) fermentation culture, and after leaving standstill the little layered of 3-5, extract upper strata vegetables oil, containing total multiprenylmenaquinone in oil phase.
3. according to claim 2 based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, it is characterized in that: tensio-active agent is selected from one or more in sucrose fatty ester, Polyoxyethylene Sorbitol Fatty Acid Esters-80, poly-Labraso in above-mentioned steps (3).
4. according to Claims 2 or 3 based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, it is characterized in that: vegetables oil is selected from soybean oil, Semen Maydis oil, peanut oil in above-mentioned steps (3), one or more in sweet oil, camellia wet goods.
5. according to claim 2 based on utilizing Flavobacterium in-situ fermentation separation coupling synthesise vitamins K 2method, it is characterized in that: in step (3) fermentation culture process, in fermentor tank, add 50g/L sucrose fatty ester, 10g/L glycerine, 150g/L soybean oil, stir speed (S.S.) 600rpm, air flow 2vvm, forms oil-in-water microemulsion, fermentation culture 96 hours.
CN201510080787.1A 2015-02-12 2015-02-12 Method for synthesizing vitamin K2 based on using flavobacterium in-situ fermentation, separation and coupling Pending CN104694589A (en)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
CN105541585A (en) * 2016-02-03 2016-05-04 中国科学院合肥物质科学研究院 Method for extracting microbial wet cell intracellular vitamin K2 crude product
CN113004134A (en) * 2021-03-04 2021-06-22 中国科学院合肥物质科学研究院 Method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract
CN114574419A (en) * 2021-06-30 2022-06-03 中国科学院合肥物质科学研究院 Preparation of vitamin K by fermenting bacillus natto2Method (2)

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CN103865835A (en) * 2013-12-17 2014-06-18 江南大学 Menadione-7(MK-7) high-yielding strain and application thereof
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105541585A (en) * 2016-02-03 2016-05-04 中国科学院合肥物质科学研究院 Method for extracting microbial wet cell intracellular vitamin K2 crude product
CN113004134A (en) * 2021-03-04 2021-06-22 中国科学院合肥物质科学研究院 Method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract
CN113004134B (en) * 2021-03-04 2023-04-07 中国科学院合肥物质科学研究院 Method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract
CN114574419A (en) * 2021-06-30 2022-06-03 中国科学院合肥物质科学研究院 Preparation of vitamin K by fermenting bacillus natto2Method (2)

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Application publication date: 20150610