WO2021253546A1

WO2021253546A1 - Method for preparing vitamin k2 by microbial fermentation

Info

Publication number: WO2021253546A1
Application number: PCT/CN2020/101767
Authority: WO
Inventors: 陈必钦; 钟锦潮; 严必能; 李丹; 吴轶; 詹光煌
Original assignee: 内蒙古金达威药业有限公司; 厦门金达威集团股份有限公司
Priority date: 2020-06-18
Filing date: 2020-07-14
Publication date: 2021-12-23
Also published as: CN111549079B; CN111549079A

Abstract

A method for preparing vitamin K2 by microbial fermentation, comprising: controlling, in stages, at least one among oxygen uptake rate, oxidation-reduction potential in the fermentation liquid, and lactic acid concentration within a predetermined range; and/or, after 5 to 20 hours of fermentation, adding auxiliary materials into the fermentation liquid, said auxiliary materials being selected from at least one among nicotinamide, vitamin B12 and methionine. Preparation of vitamin K2 by means of the described method satisfies different requirements for the culturing conditions for vitamin K2 fermentation in the stages of growth, synthesis etc., promoting bacterial growth and product synthesis, efficiently improving the fermentation level of vitamin K2, and achieving high vitamin K2 content in the fermentation product.

Description

A method for preparing vitamin K2 by adopting microbial fermentation method

Technical field

The invention belongs to the field of vitamin K2 preparation, and relates to a method for preparing vitamin K2 by adopting a microbial fermentation method.

Background technique

Vitamin K2 is an essential fat-soluble vitamin, also known as menadione. It can regulate the synthesis of various coagulation factors by promoting the synthesis of prothrombin by the liver. It is also an essential cofactor for the carboxylation of osteocalcin. It plays an important role in bone development and helps to transfer calcium ions into the bones. It can play a key role in blood clotting and prevention of osteoporosis. In addition, as an electron transfer carrier in respiratory metabolism, vitamin K2 also participates in many important metabolic reactions in the organism. Recent studies have found that vitamin K2 has many different functions in clinical applications. For example, it can be used to prevent and treat cardiovascular and cerebrovascular diseases, Parkinson's disease and type II diabetes, and promote the recovery of liver function. It has broad application prospects.

Vitamin K2 has multiple isomers. According to the number of isoprene units at the C3 position on its molecular structure, it is represented by MK-n, of which MK-7 has the highest biological activity and the longest half-life. The current research and development methods of vitamin K2 mainly include natural extraction methods, chemical synthesis methods and microbial fermentation methods. Among them, the natural extraction method has low yield; the chemical synthesis method has complicated steps and also has the problem of low yield, which also leads to the high production cost of vitamin K2. In addition, chemical synthesis methods are prone to produce different cis-isomers with low activity and a large number of by-products, and cause environmental pollution, making it difficult to form large-scale production. The microbial fermentation method has the characteristics of low pollution, low cost, high product activity, and easy to scale up production scale, and has gradually become a hot area of research. Through screening, mutagenesis and modification, domestic and foreign research institutions have constructed many potential strains for the production of vitamin K2, including Bacillus subtilis natto, Flavobacterium, Bacillus subtilis, Bacillus amyloliquefaciens, lactic acid bacteria, etc. However, the fermentation level is still generally low. For example, CN104328064B mutagenesis selected a vitamin K2 producing strain that efficiently utilizes soy protein, with a vitamin K2 output of 60.54 mg/L; CN108410775B discloses a vitamin K2 producing Bacillus natto with a vitamin output of about 70 mg/L CN107475312A discloses a method for improving the permeability of cell membranes and increasing vitamin K2. After the method is improved, the level of vitamin K2 produced by Flavobacterium is 48.8mg/L; Tang Guixiang et al. optimized the fermentation conditions of a strain of Bacillus amyloliquefaciens The post-vitamin K2 output is about 70mg/L (Tang Guixiang, Chen You, etc.; Selection of high-yield vitamin K2 strains and optimization of fermentation conditions[J]., Science and Technology of Food Industry, 2019, 24(40): 68-73.). The low fermentation level limits the process of industrial production of vitamin K2.

Summary of the invention

The purpose of the present invention is to overcome the disadvantages that the fermentation level of vitamin K2 prepared by the existing microbial fermentation method is generally low, and the content of vitamin K2 in the obtained fermentation product is relatively low, and to provide a method that can effectively improve the fermentation level and the obtained fermentation product The method with higher vitamin K2 content.

After in-depth research, the inventor of the present invention found that the oxygen consumption rate (Oxygen Uptake Rate, hereinafter abbreviated as OUR), oxidation reduction potential (Oxidation Reduction Potential, hereinafter abbreviated as ORP) and lactic acid concentration in the fermentation process can be very good. Ground feedback fermentation process, by controlling the range of OUR, ORP and lactic acid concentration in the fermentation broth in stages, it can provide the most suitable fermentation and culture conditions for each stage of the bacterial fermentation process, and meet the needs of vitamin K2 fermentation in each stage of growth and synthesis. The different requirements of culture conditions promote the growth of bacteria and the synthesis of vitamin K2, shorten the fermentation cycle and reduce the energy consumption of the equipment. In addition, the inventors of the present invention have also found after in-depth research that after 5-20 hours of fermentation and culture, supplementation of at least one of nicotinamide, vitamin B12 and methionine can effectively promote the synthesis of vitamin K2. Based on this, the present invention has been completed.

Specifically, the present invention provides a method for preparing vitamin K2 by using a microbial fermentation method, wherein the method includes stepwise controlling at least one of OUR, ORP in the fermentation broth, and lactic acid concentration within a predetermined range during the fermentation culture stage And/or, after 5-20 hours of fermentation and cultivation, add auxiliary materials to the fermentation broth, the auxiliary materials are selected from at least one of nicotinamide, vitamin B12 and methionine;

The way to control OUR in stages is as follows: fermentation 0～4h OUR is controlled at 20～80mmol/L·h, 4～12h OUR is controlled at 80～160mmol/L·h, 12～24h OUR is controlled at 60～140mmol/L·h ，24～60h OUR is controlled at 40～100mmol/L·h, 60～140h OUR is controlled at 20～90mmol/L·h;

The way to control the ORP in the fermentation broth in stages is as follows: the ORP in the fermentation broth for 0～4h is controlled at 60～150mV, the ORP in the fermentation broth at 4～12h is controlled at -80～100mV, and the ORP in the fermentation broth at 12～24h is controlled at -50 ～120mV, the ORP in the fermentation broth for 24～60h is controlled at -20～160mV, and the ORP in the fermentation broth at 60～140h is controlled at 20～220mV;

The way to control the concentration of lactic acid in the fermentation broth in stages is as follows: the concentration of lactic acid in the fermentation broth is controlled at 1-20mg/L for 0～4h, the concentration of lactic acid in the fermentation broth at 4～12h is controlled at 5～50mg/L, and the fermentation broth is 12～24h. The concentration of medium lactic acid is controlled at 10-100mg/L, the concentration of lactic acid in the fermentation broth for 24-60h is controlled at 30-150mg/L, and the concentration of lactic acid in the fermentation broth at 60-140h is controlled at 50-100mg/L.

Preferably, in the fermentation culture stage, at least one of OUR, ORP in the fermentation broth, and lactic acid concentration is monitored online, and at least one of the air flow, rotation speed, and tank pressure is adjusted in different stages of the fermentation to realize the control of OUR, At least one of ORP and lactic acid concentration in the fermentation broth is regulated in stages.

Preferably, the addition ratio of the nicotinamide is 0.01-0.1 wt%.

Preferably, the addition ratio of the vitamin B12 is 0.03 to 0.15 wt%.

Preferably, the addition ratio of the methionine is 0.03 to 0.1 wt%.

Preferably, the bacteria used in the fermentation are selected from Bacillus subtilis natto, Flavobacterium, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis (Bacillus licheniformis) at least one.

Preferably, the temperature of the fermentation culture is 28-40° C., the pH value is 6.0-7.5, and the seed solution inoculum is 1-5%.

Preferably, the volume of the fermentor used in the fermentation culture is 0.5L to 500m ³ .

Preferably, the method for preparing vitamin K2 by microbial fermentation provided by the present invention further includes supplementing glycerol during the fermentation process to control the glycerol concentration in the fermentation system at 1-10 g/L.

The preparation of vitamin K2 by the method provided by the present invention can well meet the different requirements for the culture conditions of vitamin K2 in various stages of growth and synthesis, promote the growth of bacteria and product synthesis, and can very effectively improve the fermentation level of vitamin K2. The obtained fermentation product has a relatively high content of vitamin K2. Furthermore, according to the fluctuation of OUR, ORP, and lactic acid concentration during the fermentation process, the fine-tuning of the process can also reduce the fermentation difference caused by the fluctuation of seed quality, which is beneficial to the fermentation level between batches. stability.

detailed description

The present invention uses online monitoring of at least one of the fermentation process OUR, the ORP in the fermentation broth and the concentration of lactic acid to feedback and regulate the fermentation process process parameters, and adjusts at least one of the air flow rate, the rotation speed and the tank pressure in different stages of the fermentation. In the stage, at least one of the OUR, ORP in the fermentation broth and the concentration of lactic acid is controlled within a predetermined range, and supplementary materials are added in conjunction with the fermentation process to promote the growth of vitamin K2 producing strains and product synthesis, thereby effectively improving the fermentation level of vitamin K2. Among them, OUR can be detected by a tail gas mass spectrometer; ORP in the fermentation broth can be detected by a redox electrode; the concentration of lactic acid in the fermentation broth can be detected by a lactic acid meter.

In some embodiments of the present invention, the concentration of OUR, ORP, and lactic acid is adjusted in five stages (ie, 0-4h, 4-12h, 12-24h, 24-60h, 60-140h). It should be noted that 0～4h includes 1h, 2h, 3h, 4h in total 4h, 4-12h includes 5h, 6h...12h in total 8h, 12-24h includes 13h, 14h... The 24h is 12h, the 24-60h includes the 25h, the 26h...the 60th is 26h, and the 60-140h includes the 61h, the 62h...the 140h is 80h.

In some embodiments of the present invention, for the control of OUR during the fermentation process, increasing the air flow rate can increase the OUR level, and reducing the air flow rate can decrease the OUR level; increasing the fermentation tank speed can increase the OUR level, and reducing the fermentation tank speed can reduce the OUR level. OUR level; increasing the fermenter pressure can increase the OUR level, and reducing the fermentor pressure can reduce the OUR level. The fermentation of vitamin K2 can be ensured by adjusting at least one of air flow, rotation speed and tank pressure to ensure that the fermentation 0～4h OUR value range is controlled within 20～80mmol/L·h, such as 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80mmol/L·h, etc.; 4～12h OUR value range is controlled within 80～160mmol/L·h, such as 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160mmol/L·h, etc.; 12～24h OUR value range is controlled within 60～140mmol/L·h, such as 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140mmol/L·h etc.; 24～60h OUR value range is controlled within 40～100mmol/L·h, such as 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mmol/L·h, etc.; 60～140h OUR value range is controlled within 20～90mmol/L·h, such as 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90mmol/L·h, etc.

In some embodiments of the present invention, for the control of ORP in the fermentation process, increasing the air flow can increase the ORP level in the fermentation broth, and reducing the air flow can reduce the ORP level in the fermentation broth; increasing the speed of the fermentation tank can increase the ORP level in the fermentation broth. ORP level, reducing the speed of the fermentation tank can reduce the ORP level in the fermentation broth; increasing the pressure of the fermentation tank can increase the ORP level in the fermentation broth, and reducing the pressure in the fermentation tank can reduce the ORP level in the fermentation broth. The fermentation of vitamin K2 can be ensured by adjusting at least one of the aeration rate, rotation speed and tank pressure to ensure that the ORP value range of the fermentation broth in the 0～4h fermentation is controlled within 60～150mV, such as 60, 65, 70, 75, 80, 85, 90 , 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150mV, etc.; the ORP value range of 4-12h fermentation broth is controlled within -80～100mV, such as -80, -75,- 70, -65, -60, -55, -50, -45, -40, -35, -30, -25, -20, -15, -10, -5, 0, 5, 10, 15, 20 , 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mV, etc.; the ORP value range in the 12-24h fermentation broth is controlled within -50～120mV, Such as -50, -45, -40, -35, -30, -25, -20, -15, -10, -5, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120mV, etc.; the ORP range of 24-60h fermentation broth is controlled within -20～160mV, such as- 20, -15, -10, -5, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 , 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160mV, etc.; the ORP value range of 60～140h fermentation broth is controlled within 20～220mV, such as 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220mV, etc.

In some embodiments of the present invention, for the control of lactic acid concentration in the fermentation process, increasing the air flow can reduce the concentration of lactic acid in the fermentation broth, and reducing the air flow can increase the concentration of lactic acid in the fermentation broth; increasing the speed of the fermentation tank can reduce the concentration of lactic acid in the fermentation broth. The concentration of lactic acid in the fermentation broth, reducing the speed of the fermentation tank can increase the concentration of lactic acid in the fermentation broth; increasing the pressure of the fermentation tank can reduce the concentration of lactic acid in the fermentation broth, and reducing the pressure of the fermentation tank can increase the concentration of lactic acid in the fermentation broth. The fermentation of vitamin K2 can be ensured by adjusting at least one of the aeration rate, rotation speed and tank pressure to ensure that the lactic acid concentration value range in the fermentation broth of fermentation 0～4h is controlled within 1～20mg/L, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mg/L, etc.; the concentration of lactic acid in the fermentation broth for 4 to 12 hours is controlled within 5 to 50 mg/L , Such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg/L, etc.; the concentration of lactic acid in the fermentation broth for 12 to 24 hours is controlled within 10 to 100 mg/L, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mg/L, etc.; the concentration of lactic acid in the fermentation broth for 24 to 60 hours is controlled within 30 to 150 mg /L, such as 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150mg/L, etc.; the range of lactic acid concentration in the fermentation broth for 60～140h is controlled within 50～100mg/L, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mg/L, etc. .

In some embodiments of the present invention, the adjuvant includes at least one of nicotinamide, vitamin B12 and methionine, and specifically may be a mixture of nicotinamide, vitamin B12, methionine, nicotinamide and vitamin B12 , A mixture of nicotinamide and methionine, a mixture of vitamin B12 and methionine, a mixture of nicotinamide, vitamin B12 and methionine, particularly preferably a mixture of nicotinamide, vitamin B12 and methionine. In addition, based on the total weight of the culture medium, the addition ratios of nicotinamide, vitamin B12, and methionine are preferably 0.01 to 0.1 wt%, 0.03 to 0.15 wt%, and 0.03 to 0.1 wt%, respectively. When the adjuvant is a mixture of nicotinamide, vitamin B12 and methionine, and/or the amount of the adjuvant is controlled within the above-mentioned preferred range, the fermentation level of vitamin K2 can be further improved.

In some embodiments of the present invention, the bacteria used are conventional strains that can produce vitamin K2, specifically including Bacillus subtilis natto, Flavobacterium, Bacillus subtilis, and Bacillus subtilis. Bacillus amyloliquefaciens, Bacillus licheniformis, etc., preferably Bacillus subtilis natto. In addition, there are no specific restrictions on the seeds and fermentation medium used in the present invention, and they can be various conventional culture media, and the medium suitable for the growth of bacteria can be selected according to different bacterial species. If you know, I won't repeat it here.

The present invention does not specifically limit the conditions of fermentation culture, for example, the temperature may be 28-40° C., the pH value may be 6.5-7.5, and the seed liquid inoculation amount may be 1-5%. Among them, the reagents for adjusting the pH value include but are not limited to: sodium hydroxide solution, potassium hydroxide solution, ammonia water and the like. In addition, the volume of the fermentor used for the fermentation culture may be 0.5L to 500m ³ .

In addition, in some embodiments of the present invention, the provided method for preparing vitamin K2 by microbial fermentation further includes adding glycerin during the fermentation process to control the concentration of glycerin in the fermentation system at 1-10 g/L, in order to ensure that the bacteria In the process of body culture, appropriate amount of carbon source is taken in for growth and product synthesis.

According to a specific embodiment of the present invention, the method for preparing vitamin K2 by using a microbial fermentation method includes the following steps:

(1) Strain activation: under aseptic conditions, insert the frozen stock of strain seeds into beef extract peptone agar medium according to the gradient dilution method, and cultivate it at 28-40℃ for 15-25h;

(2) Seed culture: Pick a single colony from the cultivated plate seeds, insert it into a shaker flask of beef extract peptone medium containing 0.5-2% glucose, and place it in a shaker at 28-40℃, 200 Cultivate for 10-20h at ～240rpm;

(3) Fermentation culture: The cultured seed liquid is connected to the fermentor at 1～5% inoculum, the culture temperature is 28～40℃, the culture pH is 6.5～7.5, and the OUR and ORP in the fermentation broth are monitored throughout the fermentation process. At least one of and lactic acid concentration, the air flow rate, rotation speed and tank pressure of the fermentation tank are adjusted according to OUR, ORP in the fermentation broth and lactic acid concentration changes; 40-60% glycerol is added to the fermentation process to control the glycerol concentration at 1-10g/ L. When the fermentation is cultured for 5-20 hours, add at least one of nicotinamide, vitamin B12 and methionine to the fermentation broth, and continue the culture to 140 hours and then put it in the tank.

The following describes the embodiments of the present invention in detail. The examples of the embodiments are intended to explain the present invention, but should not be understood as a limitation to the present invention. For those who do not indicate specific technologies or conditions in the examples, it shall be carried out in accordance with the technologies or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

In the following examples and comparative examples:

OUR is detected by exhaust gas mass spectrometer; ORP in fermentation broth is detected by redox electrode; lactic acid concentration in fermentation broth is detected by online lactic acid meter.

The content of vitamin K2 in the fermentation broth is detected by high performance liquid chromatography, the specific method is as follows:

Sample processing: Measure a proper volume of fermentation broth, add 4 times the volume of the extract (the volume ratio of n-hexane and isopropanol is 2:1), shake for 40 minutes in the dark, stand for layering, absorb the supernatant and pass 0.22μm organic After the filter membrane is filtered, it is tested on the machine. Among them, the detection conditions: the chromatographic column uses a C18 column, the column temperature is 30°C, the mobile phase methanol, the flow rate is 1.0 mL/min, the detection wavelength is 254 nm (248 nm is used in Example 13, and the others are detected at this wavelength), and the injection volume is 20 uL. The detection time is 35 minutes. The test results were calculated by the external standard method to obtain the vitamin K2 content in the fermentation broth.

Example 1: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(1) Strain activation: Under aseptic conditions, the frozen seed stock of Bacillus subtilis natto (strain number: ATCC 15245) was inserted into the beef extract peptone agar medium according to the gradient dilution method, and cultured at 30°C 20h.

(2) Seed culture: Pick a single colony from the cultured plate seeds, insert it into a 1L shaker flask of beef extract peptone medium containing 1% glucose, and then place it in a shaker at 30°C and 220 rpm for 14 hours.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 4% inoculum, and the culture temperature is 30℃. During the fermentation process, use sodium hydroxide solution or ammonia to control the pH at 7.0, and monitor the whole fermentation process. OUR value, rotation speed, aeration volume and tank pressure are adjusted according to the change of OUR value, fermentation 0～4h control OUR level at 30～60mmol/L·h, fermentation 4～12h control OUR level at 90～140mmol/L· h, fermentation for 12-24h, control OUR level at 70-110mmol/L·h, fermentation for 24-60h control OUR level at 50-80mmol/L·h, fermentation 60-140h, control OUR level at 35-75mmol/L·h, During the fermentation process, the rotation speed is controlled at 200-400rpm, the aeration ratio is controlled at 0.3-1.0vvm, and the tank pressure is controlled at 0.03-0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation is cultured to 16h, 0.02wt% nicotinamide, 0.03wt% vitamin B12 and 0.03wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

Each liter of fermentation medium contains the following components: soybean meal powder 40g, corn steep liquor 10g, sodium chloride 0.4g, magnesium sulfate 1.2g, disodium hydrogen phosphate 0.5g, zinc chloride 0.2g, ferrous sulfate 0.15g, glycerin 20g, 0.5g foam enemy, use sodium hydroxide solution to adjust the pH to 7.0 before elimination.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 18.4 g/L, and the content of vitamin K2 in the fermentation broth was 117 mg/L by high performance liquid chromatography.

Example 2: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 4% inoculum, and the culture temperature is 30℃. During the fermentation process, use sodium hydroxide solution or ammonia to control the pH at 7.0, and monitor the whole fermentation process. The ORP value in the fermentation broth, rotation speed, aeration volume and tank pressure are adjusted according to the changes in the ORP value in the fermentation broth. The ORP level in the fermentation broth is controlled at 90-120mV for 0～4h, and the ORP in the fermentation broth is controlled at 4～12h. The level is -50～60mV, the ORP level in the fermentation broth is controlled at -30～70mV for 12～24h, the ORP level in the fermentation broth is controlled at -10～100mV at 24～60h, the ORP level in the fermentation broth is controlled at 60～140h 20～160mV, during the fermentation process, the rotation speed is controlled at 200～400rpm, the aeration ratio is controlled at 0.3～1.0vvm, and the tank pressure is controlled at 0.03～0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation is cultured to 16h, 0.02wt% nicotinamide, 0.03wt% vitamin B12 and 0.03wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 19.9g/L, and the content of vitamin K2 in the fermentation broth was 113mg/L by high performance liquid chromatography.

Example 3: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 4% inoculum, and the culture temperature is 30℃. During the fermentation process, use sodium hydroxide solution or ammonia to control the pH at 7.0, and monitor the whole fermentation process. The value of the lactic acid concentration in the fermentation broth, the speed, the aeration rate and the tank pressure are adjusted according to the numerical change of the lactic acid concentration in the fermentation broth. The lactic acid concentration in the fermentation broth is controlled at 2-10mg/L for 0～4h, and the lactic acid concentration in the fermentation broth is controlled at 2～10mg/L, and the fermentation is controlled for 4～12h. The concentration of lactic acid in the fermentation broth is 5-30mg/L, the concentration of lactic acid in the fermentation broth is controlled at 15-60mg/L for 12-24h, the concentration of lactic acid in the fermentation broth is controlled at 35-100mg/L, and the fermentation is 60-140h for 24-60h Control the concentration of lactic acid in the fermentation broth at 50-80mg/L, the speed during the fermentation process at 200-400rpm, the aeration ratio at 0.3-1.0vvm, and the tank pressure at 0.03-0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation is cultured to 16h, 0.02wt% nicotinamide, 0.03wt% vitamin B12 and 0.03wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 19.9 g/L, and the content of vitamin K2 in the fermentation broth was 119 mg/L by high performance liquid chromatography.

Example 4: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 4% inoculum, and the culture temperature is 30℃. During the fermentation process, use sodium hydroxide solution or ammonia to control the pH at 7.0, and monitor the whole fermentation process. OUR value, rotation speed, aeration volume and tank pressure are adjusted according to the change of OUR value, fermentation 0～4h control OUR level at 30～60mmol/L·h, fermentation 4～12h control OUR level at 90～140mmol/L· h, fermentation for 12-24h, control OUR level at 70-110mmol/L·h, fermentation for 24-60h control OUR level at 50-80mmol/L·h, fermentation 60-140h, control OUR level at 35-75mmol/L·h, During the fermentation process, the rotation speed is controlled at 200-400rpm, the aeration ratio is controlled at 0.3-1.0vvm, and the tank pressure is controlled at 0.03-0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 18.7 g/L, and the content of vitamin K2 in the fermentation broth was 136 mg/L by high performance liquid chromatography.

Example 5: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 4% inoculum, and the culture temperature is 30℃. During the fermentation process, use sodium hydroxide solution or ammonia to control the pH at 7.0, and monitor the whole fermentation process. The values of OUR and ORP, speed, aeration, and tank pressure are adjusted according to the changes of OUR and ORP values. Fermentation 0～4h control OUR and ORP levels at 30～60mmol/L·h and 80～120mV, respectively, fermentation 4～12h Control the levels of OUR and ORP at 90～140mmol/L·h and -80～70mV respectively, and control the levels of OUR and ORP at 70～110mmol/L·h and -30～60mV respectively after 12～24h fermentation, and control OUR at 24～60h after fermentation. And ORP levels are 50～80mmol/L·h and -20～110mV, respectively, fermentation 60～140h, OUR and ORP levels are controlled at 35～75mmol/L·h and 60～150mV, respectively, and the rotation speed is controlled at 200～400rpm during fermentation. , The ventilation ratio is controlled at 0.3～1.0vvm, and the tank pressure is controlled at 0.03～0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation is cultured to 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 19.1 g/L, and the content of vitamin K2 in the fermentation broth was 139 mg/L by high performance liquid chromatography.

Example 6: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 4% inoculum, and the culture temperature is 30℃. During the fermentation process, use sodium hydroxide solution or ammonia to control the pH at 7.0, and monitor the whole fermentation process. The values of OUR and lactic acid, speed, ventilation and tank pressure are adjusted according to the changes of OUR and lactic acid values. Fermentation is 0～4h to control OUR and lactic acid levels at 30～60mmol/L·h and 1～10mg/L, respectively. Fermentation 4 ～12h control OUR and lactic acid levels at 90～140mmol/L·h and 5～20mg/L, respectively, fermentation 12～24h control OUR and lactic acid levels at 70～110mmol/L·h and 20～50mg/L, respectively, fermentation 24 ～60h control OUR and lactic acid levels at 50～80mmol/L·h and 35～100mg/L respectively, fermentation 60～140h control OUR and lactic acid levels at 35～75mmol/L·h and 50～70mg/L, respectively, fermentation process The middle speed is controlled at 200-400rpm, the ventilation ratio is controlled at 0.3-1.0vvm, and the tank pressure is controlled at 0.03-0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 18.6 g/L, and the content of vitamin K2 in the fermentation broth was 134 mg/L by high performance liquid chromatography.

Example 7: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 4% inoculum, and the culture temperature is 30℃. During the fermentation process, use sodium hydroxide solution or ammonia to control the pH at 7.0, and monitor the whole fermentation process. The values of ORP and lactic acid, speed, ventilation, and tank pressure are adjusted according to the changes of ORP and lactic acid values. The levels of ORP and lactic acid are controlled at 80～120mV and 1～10mg/L respectively for 0～4h, and ORP is controlled at 4～12h after fermentation. And lactic acid levels were at -80～70mV and 5～20mg/L, fermentation for 12～24h to control ORP and lactic acid levels at -30～60mV and 20～50mg/L, fermentation for 24～60h to control ORP and lactic acid levels at- 20～110mV and 35～100mg/L, 60～140h of fermentation, control ORP and lactic acid levels at 60～150mV and 50～70mg/L, respectively, during the fermentation process, the rotation speed is controlled at 200～400rpm, the aeration ratio is controlled at 0.3～1.0vvm, The tank pressure is controlled at 0.03～0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation is cultured to 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then put in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 19.0 g/L, and the content of vitamin K2 in the fermentation broth was 135 mg/L by high performance liquid chromatography.

Example 8: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 4% inoculum, and the culture temperature is 30℃. During the fermentation process, use sodium hydroxide solution or ammonia to control the pH at 7.0, and monitor the whole fermentation process. The values of OUR, ORP and lactic acid, speed, ventilation and tank pressure are adjusted according to the changes of OUR, ORP and lactic acid values. Fermentation 0～4h control OUR, ORP and lactic acid levels at 30～60mmol/L·h, 80～ 120mV and 1～10mg/L, fermentation for 4～12h to control OUR, ORP and lactic acid levels at 90～140mmol/L·h, -80～70mV and 5～20mg/L, fermentation for 12～24h to control OUR, ORP and lactic acid The levels are 70～110mmol/L·h, -30～60mV and 20～50mg/L respectively, and the levels of OUR, ORP and lactic acid are controlled at 50～80mmol/L·h, -20～110mV and 35～ after 24～60h fermentation. 100mg/L, fermentation 60～140h, control the OUR, ORP and lactic acid levels at 35～75mmol/L·h, 60～150mV and 50～70mg/L, respectively. During the fermentation process, the rotation speed is controlled at 200～400rpm, and the aeration ratio is controlled at 0.3 ~1.0vvm, the tank pressure is controlled at 0.03～0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 19.4 g/L, and the content of vitamin K2 in the fermentation broth was 138 mg/L by high performance liquid chromatography.

Example 9: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed liquid with 4% inoculum to a 100L fermentor, the culture temperature is 30℃, and the fermentation process uses sodium hydroxide solution or ammonia to control the pH value at 7.0, and the whole fermentation process is wrong. OUR, ORP and lactic acid concentration in the fermentation broth are detected and adjusted, the speed is controlled at 300rpm, the ventilation ratio is controlled at 0.6vvm, and the tank pressure is controlled at 0.05MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 17.4 g/L, and the content of vitamin K2 in the fermentation broth was 91 mg/L by high performance liquid chromatography.

Example 10: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 4% inoculum, and the culture temperature is 30℃. During the fermentation process, use sodium hydroxide solution or ammonia to control the pH at 7.0, and monitor the whole fermentation process. OUR value, rotation speed, aeration volume and tank pressure are adjusted according to the change of OUR value, fermentation 0～4h control OUR level at 30～60mmol/L·h, fermentation 4～12h control OUR level at 90～140mmol/L· h, fermentation for 12-24h, control OUR level at 70-110mmol/L·h, fermentation for 24-60h control OUR level at 50-80mmol/L·h, fermentation 60-140h, control OUR level at 35-75mmol/L·h, During the fermentation process, the rotation speed is controlled at 200-400rpm, the aeration ratio is controlled at 0.3-1.0vvm, and the tank pressure is controlled at 0.03-0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. At least one of nicotinamide, vitamin B12 and methionine (see Table 1) was added to the fermentation broth when the fermentation was cultured to 16 hours, and the culture was continued for 140 hours and then put in the tank.

The fermentation broth was collected to obtain the bacteria, and the results of the dry weight of the bacteria and the content of vitamin K2 are shown in Table 1 below.

Table 1 The effect of adding different excipients on the fermentation of vitamin K2

所补加辅料Supplementary materials	菌体干重Dry weight	维生素K2含量Vitamin K2 content
0.04％烟酰胺+0.04％维生素B12+0.03％甲硫氨酸0.04% Niacinamide + 0.04% Vitamin B12 + 0.03% Methionine	21.9g/L21.9g/L	128mg/L128mg/L
0.04％烟酰胺+0.07％维生素B120.04% Niacinamide + 0.07% Vitamin B12	20.3g/L20.3g/L	120mg/L120mg/L
0.11％烟酰胺0.11% Niacinamide	18.5g/L18.5g/L	100mg/L100mg/L
0.11％维生素B120.11% Vitamin B12	21.7g/L21.7g/L	90mg/L90mg/L
0.11％甲硫氨酸0.11% methionine	18.8g/L18.8g/L	94mg/L94mg/L
0.04％烟酰胺+0.06％甲硫氨酸0.04% Niacinamide + 0.06% Methionine	20.5g/L20.5g/L	125mg/L125mg/L
0.07％维生素B12+0.06％甲硫氨酸0.07% Vitamin B12+0.06% Methionine	21.6g/L21.6g/L	114mg/L114mg/L
0.04％烟酰胺0.04% Niacinamide	21.0g/L21.0g/L	109mg/L109mg/L
0.07％维生素B120.07% Vitamin B12	21.7g/L21.7g/L	96mg/L96mg/L
0.06％甲硫氨酸0.06% methionine	21.0g/L21.0g/L	104mg/L104mg/L

Example 11: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(1) Strain activation: Under aseptic conditions, the flavobacterium (strain number: CICC10651) seed freezing solution was inserted into the LB solid medium according to the gradient dilution method, and cultured at 37°C for 24h.

(2) Seed culture: Pick a single colony from the cultivated plate seeds, insert it into a 1L shake flask containing 1% glucose LB medium, and then place it in a shaker at 37°C and 200 rpm for 12 hours.

(3) Fermentation culture: Connect the cultured seed tank seed liquid to a 100L fermentor with 2% inoculum, the culture temperature is 37℃, the fermentation process uses sodium hydroxide solution or ammonia water to control the pH value at 7.4, and the whole fermentation is monitored. The values of OUR, ORP and lactic acid, speed, ventilation and tank pressure are adjusted according to the changes of OUR, ORP and lactic acid values. Fermentation 0～4h control OUR, ORP and lactic acid levels at 30～50mmol/L·h, 80～ respectively 120mV and 1～10mg/L, fermentation for 4～12h to control OUR, ORP and lactic acid levels at 80～100mmol/L·h, -80～70mV and 15～40mg/L, fermentation for 12～24h to control OUR, ORP and lactic acid The levels are 60～90mmol/L·h, -30～60mV, and 30～60mg/L, respectively, and the levels of OUR, ORP and lactic acid are controlled at 40～60mmol/L·h, -20～110mV and 45～24～60h after fermentation. 90mg/L, fermentation 60～140h, control the OUR, ORP and lactic acid levels at 30～50mmol/L·h, 60～150mV and 60～80mg/L, respectively, the rotation speed during the fermentation process is controlled at 200～400rpm, the aeration ratio is controlled at 0.3 ~1.0vvm, the tank pressure is controlled at 0.03～0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

Each liter of fermentation medium contains the following components: 30g of yeast powder, 10g of fish meal, 2g of sodium chloride, 0.6g of magnesium sulfate, 3.5g of disodium hydrogen phosphate, 20g of glycerol, 0.5g of foam, and sodium hydroxide solution before elimination. Adjust the pH to 7.4.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 9.4 g/L, and the content of vitamin K2 in the fermentation broth was 74 mg/L by high performance liquid chromatography.

Example 12: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 2% inoculum. The culture temperature is 37℃. Sodium hydroxide solution or ammonia water is used to control the pH value at 7.4 during the fermentation process. The whole fermentation process is wrong. OUR, ORP and lactic acid concentration in the fermentation broth are detected and adjusted, the speed is controlled at 300rpm, the ventilation ratio is controlled at 0.6vvm, and the tank pressure is controlled at 0.05MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 8.7g/L, and the content of vitamin K2 in the fermentation broth was 32 mg/L by high performance liquid chromatography.

Example 13: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(1) Strain activation: Under aseptic conditions, the Bacillus subtilis (strain number: CGMCC 1.8886) seed freezing solution was inserted into the beef extract peptone agar medium according to the gradient dilution method, and cultured at 37°C for 20 hours.

(2) Seed culture: Pick a single colony from the cultured plate seeds, insert it into a 1L shaker flask of beef extract peptone medium containing 1% glucose, and then place it in a shaker at 37°C and 220 rpm for 12 hours.

(3) Fermentation culture: Connect the cultured seed tank seed solution to a 100L fermentor with 2% inoculum, the culture temperature is 37℃, the fermentation process uses sodium hydroxide solution or ammonia to control the pH value at 7.0, and the whole fermentation is monitored. The values of OUR, ORP and lactic acid, speed, ventilation and tank pressure are adjusted according to the changes of OUR, ORP and lactic acid values. Fermentation 0～4h control OUR, ORP and lactic acid levels at 20～70mmol/L·h, 100～ 140mV and 1～5mg/L, fermentation for 4～12h to control OUR, ORP and lactic acid levels at 80～120mmol/L·h, -20～100mV and 5～10mg/L, fermentation for 12～24h to control OUR, ORP and lactic acid The levels are 80～120mmol/L·h, 10～80mV and 15～30mg/L respectively, and the levels of OUR, ORP and lactic acid are controlled at 60～80mmol/L·h, 20～140mV and 30～50mg/L after 24～60h fermentation. L, fermentation 60～140h, control the OUR, ORP and lactic acid levels at 30～80mmol/L·h, 70～200mV and 50～70mg/L, respectively. During the fermentation process, the rotation speed is controlled at 200～400rpm, and the aeration ratio is controlled at 0.3～1.0 vvm, the tank pressure is controlled at 0.03～0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

Each liter of fermentation medium contains the following components: 30g soybean meal powder, 10g yeast powder, 0.5g calcium chloride, 0.5g magnesium sulfate, 0.5g disodium hydrogen phosphate, 20g glycerol, 0.5g foam enemy, and use hydrogen before elimination The sodium solution adjusts the pH to 7.0.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 15.7 g/L, and the content of vitamin K2 in the fermentation broth was 116 mg/L by high performance liquid chromatography.

Example 14: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(1) Strain activation: Under aseptic conditions, the frozen seed solution of Bacillus amyloliquefaciens (strain number CGMCC 1.921) was inserted into the beef extract peptone agar medium according to the gradient dilution method, and cultured at 32°C for 24 hours.

(2) Seed culture: Pick a single colony from the cultured plate seeds, insert it into a 1L shaker flask of beef extract peptone medium containing 1% glucose, and then place it in a shaker at 32°C and 200rpm for 16h.

(3) Fermentation culture: Connect the cultured seed tank seed liquid with 1% inoculum to a 100L fermentor, the culture temperature is 32℃, the fermentation process uses sodium hydroxide solution or ammonia water to control the pH value at 7.2, and the whole fermentation is monitored. The values of OUR, ORP and lactic acid, speed, ventilation and tank pressure are adjusted according to the changes of OUR, ORP and lactic acid values. Fermentation 0～4h control OUR, ORP and lactic acid levels at 20～60mmol/L·h, 90～ 150mV and 1～10mg/L, fermentation for 4～12h to control OUR, ORP and lactic acid levels at 80～100mmol/L·h, -60～80mV and 10～25mg/L, fermentation for 12～24h to control OUR, ORP and lactic acid The levels were 70～100mmol/L·h, -40～60mV and 25～60mg/L, and the levels of OUR, ORP and lactic acid were controlled at 55～75mmol/L·h, -10～120mV and 30～ 110mg/L, fermentation 60～140h, control the OUR, ORP and lactic acid levels at 25～40mmol/L·h, 40～150mV and 60～80mg/L, respectively, the rotation speed during the fermentation process is controlled at 200～400rpm, the aeration ratio is controlled at 0.3 ~1.0vvm, the tank pressure is controlled at 0.03～0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

Each liter of fermentation medium contains the following components: soybean meal powder 30g, starch 10g, yeast powder 15g, calcium chloride 0.3g, magnesium sulfate 1.2g, dipotassium hydrogen phosphate 0.5g, glycerol 10g, foam enemy 0.5g, before eliminating Use sodium hydroxide solution to adjust the pH to 7.2.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 23.3 g/L, and the content of vitamin K2 in the fermentation broth was 104 mg/L by high performance liquid chromatography.

Example 15: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(1) Strain activation: Under aseptic conditions, the frozen seed solution of Bacillus licheniformis (strain number CGMCC1.15832) was inserted into the beef extract peptone agar medium according to the gradient dilution method, and cultured at 37°C for 24 hours.

(2) Seed culture: pick a single colony from the cultured plate seeds, insert it into a 1L shake flask of beef extract peptone medium containing 1% glucose, and then place it in a shaker at 37°C and 220 rpm for 16 hours.

(3) Fermentation culture: Connect the cultured seed tank seed liquid with 4% inoculum to a 100L fermentor, the culture temperature is 37℃, the fermentation process uses sodium hydroxide solution or ammonia water to control the pH value at 7.0, and the whole fermentation is monitored. The values of OUR, ORP and lactic acid, speed, ventilation and tank pressure are adjusted according to the changes of OUR, ORP and lactic acid values. Fermentation 0～4h control OUR, ORP and lactic acid levels at 25～65mmol/L·h, 60～ 100mV and 1～10mg/L, fermentation for 4～12h to control OUR, ORP and lactic acid levels at 90～130mmol/L·h, -50～70mV and 5～30mg/L, fermentation for 12～24h to control OUR, ORP and lactic acid The levels are 60～100mmol/L·h, -40～90mV and 15～50mg/L, respectively. The levels of OUR, ORP and lactic acid are controlled at 55～80mmol/L·h, -10～140mV and 30～ after 24～60h fermentation. 65mg/L, fermentation 60～140h, control the OUR, ORP and lactic acid levels at 30～70mmol/L·h, 40～100mV and 55～80mg/L, respectively, the rotation speed during the fermentation process is controlled at 200～400rpm, the aeration ratio is controlled at 0.3 ～1.0vvm, the tank pressure is controlled at 0.03～0.07MPa. During the fermentation process, 50% glycerol is added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

Each liter of fermentation medium contains the following components: 20g soybean meal powder, 5g corn steep liquor, 0.4g sodium chloride, 0.8g magnesium sulfate, 0.5g disodium hydrogen phosphate, 0.2g zinc chloride, 0.1g ferrous sulfate, glycerin 20g, 0.5g foam enemy, use sodium hydroxide solution to adjust the pH to 7.0 before elimination.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 16.4g/L, and the content of vitamin K2 in the fermentation broth was 96mg/L by high performance liquid chromatography.

Example 16: A method for preparing vitamin K2 by fermentation, using a 100L fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) fermentation culture.

(3) Fermentation culture: Connect the cultured seed tank seed liquid to a 100L fermentor with 4% inoculum, the culture temperature is 37℃, and the fermentation process uses sodium hydroxide solution or ammonia to control the pH value at 7.0. The whole fermentation process is wrong. OUR, ORP and lactic acid concentration in the fermentation broth are detected and adjusted, the speed is controlled at 300rpm, the ventilation ratio is controlled at 0.6vvm, and the tank pressure is controlled at 0.05MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 17.1 g/L, and the content of vitamin K2 in the fermentation broth was 57 mg/L by high performance liquid chromatography.

Example 17: A method for preparing vitamin K2 by fermentation, using a 160m ³ fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) seed expansion; (4) fermentation culture .

(2) Seed culture: Pick a single colony from the cultured plate seeds, insert it into a 2L shake flask of beef extract peptone medium containing 1% glucose, and then place it in a shaker at 30°C and 220 rpm for 14 hours.

(3) Seed expansion: the cultured seed liquid is connected to a 100L seed tank with 1% inoculum for culture, the culture temperature is 30°C, the aeration ratio is 0.3vvm, the rotation speed is 250rpm, the tank pressure is 0.03MPa, and the culture time is 10h. After the cultivation is completed, a 5m ³ seed tank is connected with 1% inoculum, the cultivation temperature is 30°C, the aeration ratio is 0.3vvm, the rotation speed is 150rpm, the tank pressure is 0.03MPa, and the cultivation time is 8h.

Each liter of seed expansion medium contains the following components: 20g soybean meal, 10g corn syrup, 0.4g disodium hydrogen phosphate, 0.3g magnesium sulfate, 10g glycerol, 0.5g soaked enemies, use sodium hydroxide solution to adjust pH before elimination To 7.0.

(4) Fermentation culture: The seed liquid of the cultivated seed tank is connected to a 160m ³ fermentor with 4% inoculum, and the culture temperature is 30°C. During the fermentation process, sodium hydroxide solution or ammonia water is used to control the pH value at 7.0, and the fermentation process is complete. Monitor the value of OUR, and adjust the speed, aeration volume and tank pressure according to the value of OUR. Fermentation 0～4h control OUR level at 30～70mmol/L·h, fermentation 4～12h control OUR level at 100～140mmol/L ·H, fermentation 12-24h, control OUR level at 75-120mmol/L·h, fermentation 24-60h control OUR level at 50-90mmol/L·h, fermentation 60-140h control OUR level at 30-70mmol/L·h During the fermentation process, the rotation speed is controlled at 70-120rpm, the aeration ratio is controlled at 0.3-1.0vvm, and the tank pressure is controlled at 0.03-0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 19.3 g/L, and the content of vitamin K2 in the fermentation broth was 141 mg/L by high performance liquid chromatography.

Example 18: A method for preparing vitamin K2 by fermentation, using a 160m ³ fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) seed expansion; (4) fermentation culture .

(4) Fermentation culture: The seed liquid of the cultivated seed tank is connected to a 160m ³ fermentor with 4% inoculum, and the culture temperature is 30°C. During the fermentation process, sodium hydroxide solution or ammonia water is used to control the pH value at 7.0, and the fermentation process is complete. Monitor the ORP value in the fermentation broth. The speed, aeration volume and tank pressure are adjusted according to the changes in the ORP value in the fermentation broth. The ORP level in the fermentation broth is controlled at 80-130mV for 0～4h, and the fermentation broth is controlled at 4～12h. ORP level in -60～50mV, fermentation for 12～24h, control the ORP level in fermentation broth at -40～80mV, fermentation for 24～60h control the ORP level in fermentation broth at -20～100mV, fermentation 60～140h to control the ORP level in fermentation broth At 20-150mV, during the fermentation process, the speed is controlled at 70-120rpm, the aeration ratio is controlled at 0.3-1.0vvm, and the tank pressure is controlled at 0.03-0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 18.4 g/L, and the content of vitamin K2 in the fermentation broth was 135 mg/L by high performance liquid chromatography.

Example 19: A method for preparing vitamin K2 by fermentation, using a 160m ³ fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) seed expansion; (4) fermentation culture .

(4) Fermentation culture: The seed liquid of the cultivated seed tank is connected to a 160m ³ fermentor with 4% inoculum, and the culture temperature is 30°C. During the fermentation process, sodium hydroxide solution or ammonia water is used to control the pH value at 7.0, and the fermentation process is complete. Monitor the value of the lactic acid concentration in the fermentation broth, and adjust the speed, aeration volume and tank pressure according to the value of the lactic acid concentration in the fermentation broth. Fermentation 0～4h control the lactic acid concentration in the fermentation broth at 2～10mg/L, fermentation 4～12h Control the lactic acid concentration in the fermentation broth at 5-20mg/L, fermentation for 12-24h, control the lactic acid concentration in the fermentation broth at 15-50mg/L, fermentation for 24-60h, control the lactic acid concentration in the fermentation broth at 35-90mg/L, and fermentation at 60～ The concentration of lactic acid in the fermentation broth is controlled at 50-80mg/L for 140h, the speed is controlled at 70-120rpm during the fermentation process, the aeration ratio is controlled at 0.3-1.0vvm, and the tank pressure is controlled at 0.03-0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 17.9 g/L, and the content of vitamin K2 in the fermentation broth was 133 mg/L by high performance liquid chromatography.

Example 20: A method for preparing vitamin K2 by fermentation, using a 120m ³ fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) seed expansion; (4) fermentation culture .

(4) Fermentation culture: The seed liquid of the cultured seed tank is connected to a 120m ³ fermentor with 2% inoculum, and the culture temperature is 30℃. Sodium hydroxide solution or ammonia water is used to control the pH value at 7.0 during the fermentation process, and the whole fermentation process Monitor the value of OUR, and adjust the speed, aeration volume and tank pressure according to the value of OUR. Fermentation 0～4h control OUR level at 20～60mmol/L·h, fermentation 4～12h control OUR level at 90～130mmol/L ·H, fermentation 12～24h control OUR level at 70～100mmol/L·h, fermentation 24～60h control OUR level at 50～80mmol/L·h, fermentation 60～140h control OUR level at 40～70mmol/L·h During the fermentation process, the speed is controlled at 70-130rpm, the aeration ratio is controlled at 0.3-1.0vvm, and the tank pressure is controlled at 0.03-0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 20.5g/L, and the content of vitamin K2 in the fermentation broth was 137mg/L by high performance liquid chromatography.

Example 21: A method for preparing vitamin K2 by fermentation, using a 120m ³ fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) seed expansion; (4) fermentation culture .

(4) Fermentation culture: The seed liquid of the cultured seed tank is connected to a 120m ³ fermentor with 2% inoculum, and the culture temperature is 30℃. Sodium hydroxide solution or ammonia water is used to control the pH value at 7.0 during the fermentation process, and the whole fermentation process Monitor the ORP value in the fermentation broth. The speed, aeration volume and tank pressure are adjusted according to the change in the ORP value in the fermentation broth. The ORP level in the fermentation broth is controlled at 80-120mV for 0～4h, and the fermentation broth is controlled at 4～12h. ORP level in -60～60mV, fermentation for 12～24h, control the ORP level in the fermentation broth at -40～100mV, fermentation for 24～60h, control the ORP level in the fermentation broth at -10～120mV, fermentation for 60～140h, control the ORP level in the fermentation broth At 20～150mV, during the fermentation process, the speed is controlled at 70～130rpm, the aeration ratio is controlled at 0.3～1.0vvm, and the tank pressure is controlled at 0.03～0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 21.3 g/L, and the content of vitamin K2 in the fermentation broth was 139 mg/L by high performance liquid chromatography.

Example 22: A method for preparing vitamin K2 by fermentation, using a 120m ³ fermentor for fermentation culture, including the following steps: (1) strain activation; (2) seed culture; (3) seed expansion; (4) fermentation culture .

(4) Fermentation culture: The seed liquid of the cultured seed tank is connected to a 120m ³ fermentor with 2% inoculum, and the culture temperature is 30℃. Sodium hydroxide solution or ammonia water is used to control the pH value at 7.0 during the fermentation process, and the whole fermentation process Monitor the value of lactic acid concentration in the fermentation broth, and adjust the speed, aeration volume and tank pressure according to the value of the lactic acid concentration in the fermentation broth. Fermentation 0～4h control the lactic acid concentration in the fermentation broth at 1～10mg/L, fermentation 4～12h Control the concentration of lactic acid in the fermentation broth at 5-15mg/L, fermentation for 12-24h, control the concentration of lactic acid in the fermentation broth at 15-40mg/L, fermentation for 24-60h, control the concentration of lactic acid in the fermentation broth at 30-80mg/L, and fermentation at 60～ The concentration of lactic acid in the fermentation broth is controlled at 50-90mg/L for 140h, the rotation speed is controlled at 70-130rpm during the fermentation process, the aeration ratio is controlled at 0.3-1.0vvm, and the tank pressure is controlled at 0.03-0.07MPa. During the fermentation process, 50% glycerol was added to control the glycerol concentration at 1-10g/L. When the fermentation culture reaches 16h, 0.04wt% nicotinamide, 0.07wt% vitamin B12 and 0.06wt% methionine are added to the fermentation broth, and the culture is continued to 140h and then placed in the tank.

Each liter of fermentation medium contains the following components: 40g soybean meal powder, 10g corn steep liquor, 0.4g sodium chloride, 1.2g magnesium sulfate, 0.5g disodium hydrogen phosphate, 0.2g zinc chloride, 0.15g ferrous sulfate, glycerin 20g, 0.5g foam enemy, use sodium hydroxide solution to adjust the pH to 7.0 before elimination.

The fermentation broth was collected to obtain the bacteria, the dry weight of the bacteria was 20.6 g/L, and the content of vitamin K2 in the fermentation broth was 143 mg/L by high performance liquid chromatography.

Examples 23-29 and Comparative Example 1: A method for preparing vitamin K2 by fermentation. The difference from Examples 1 to 3, 5 to 8 and Example 12 is that the fermentation is cultured to 16 hours, and the fermentation broth is not Add nicotinamide, vitamin B12 and methionine, and other experimental conditions are the same as in Examples 1 to 3, 5 to 8 and Example 12. After putting the tank, the fermentation broth is collected to obtain the bacteria, and the dry weight of the bacteria and vitamins are measured. The K2 content is shown in Table 2 below.

Table 2 The effect of different oxygen supply regulation methods on the fermentation of vitamin K2

实施例Example	供氧调控方式Oxygen supply regulation method	菌体干重Dry weight	维生素K2含量Vitamin K2 content
实施例23Example 23	同实施例1Same as Example 1	20.7g/L20.7g/L	102mg/L102mg/L
实施例24Example 24	同实施例2Same as Example 2	21.2g/L21.2g/L	105mg/L105mg/L
实施例25Example 25	同实施例3Same as Example 3	20.4g/L20.4g/L	97mg/L97mg/L
实施例26Example 26	同实施例5Same as Example 5	19.7g/L19.7g/L	104mg/L104mg/L
实施例27Example 27	同实施例6Same as Example 6	20.1g/L20.1g/L	99mg/L99mg/L
实施例28Example 28	同实施例7Same as Example 7	21.4g/L21.4g/L	102mg/L102mg/L
实施例29Example 29	同实施例8Same as Example 8	20.6g/L20.6g/L	105mg/L105mg/L
对比例1Comparative example 1	同实施例12Same as Example 12	7.9g/L7.9g/L	24mg/L24mg/L

Comparative Example 2: A method for preparing vitamin K2 by fermentation. The difference from Example 23 is that during the fermentation process, the value of OUR is monitored throughout the fermentation process, and the speed, ventilation, and tank pressure are performed according to the value of OUR. Adjust, control the OUR level at 30-60mmol/L·h for 0～4h fermentation, control the OUR level at 40～70mmol/L·h for 4～12h fermentation, and control the OUR level at 40～55mmol/L·h for 12～24h fermentation. The OUR level was controlled at 30-50 mmol/L·h during the fermentation for 24 to 60 hours, and the OUR level was controlled at 20-40 mmol/L·h during the fermentation for 60 to 140 hours. The dry weight of the bacteria was 15.0 g/L, and the content of vitamin K2 in the fermentation broth was 59 mg/L by high performance liquid chromatography.

Comparative Example 3: A method for preparing vitamin K2 by fermentation. The difference from Example 24 is that during the fermentation process, the ORP value in the fermentation broth is monitored throughout the fermentation process. The speed, aeration, and tank pressure are based on the fermentation broth. Adjust the value of ORP, fermentation 0～4h control the ORP level in the fermentation broth at 90～120mV, fermentation 4～12h control the ORP level in the fermentation broth at -120～-90mV, fermentation 12～24h control the ORP level in the fermentation broth Control the ORP level in the fermentation broth at -100～-70mV, fermentation for 24～60h, control the ORP level in the fermentation broth at -50～10mV, and control the ORP level in the fermentation broth at 0～50mV after fermentation for 60～140h. Other experimental conditions are the same as in Example 24. The fermentation broth obtains the bacteria. The dry weight of the bacteria was 15.5g/L, and the content of vitamin K2 in the fermentation broth was 64mg/L by high performance liquid chromatography.

Comparative Example 4: A method for preparing vitamin K2 by fermentation. The difference from Example 25 is that during the fermentation process, the value of the lactic acid concentration in the fermentation broth is monitored throughout the fermentation process. The speed, aeration, and tank pressure are based on the fermentation broth. The lactic acid concentration in the fermentation broth is controlled at 2-20mg/L for 0～4h, the lactic acid concentration in the fermentation broth is controlled at 55～75mg/L for 4～12h, and the fermentation is controlled at 12～24h for fermentation. The concentration of lactic acid in the broth is 100～120mg/L, the concentration of lactic acid in the fermentation broth is controlled at 150～170mg/L for 24～60h, the concentration of lactic acid in the fermentation broth is controlled at 120～150mg/L after 60～140h, and the other experimental conditions are the same. Example 25, collect the fermentation broth after putting the tank to obtain the bacteria. The dry weight of the bacteria was 12.1g/L, and the content of vitamin K2 in the fermentation broth was 42mg/L by high performance liquid chromatography.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art will not depart from the principle and purpose of the present invention. Under the circumstances, changes, modifications, substitutions and modifications can be made to the above-mentioned embodiments within the scope of the present invention.

Claims

A method for preparing vitamin K2 by using a microbial fermentation method, characterized in that the method includes controlling at least one of OUR, ORP in the fermentation broth, and lactic acid concentration within a predetermined range in stages during the fermentation culture stage; and, After 5-20 hours of fermentation and cultivation, add auxiliary materials to the fermentation broth, and the auxiliary materials are selected from at least one of nicotinamide, vitamin B12 and methionine;

The way to control OUR in stages is as follows: fermentation 0～4h OUR is controlled at 20～80mmol/L·h, 4～12h OUR is controlled at 80～160mmol/L·h, 12～24h OUR is controlled at 60～140mmol/L·h ，24～60h OUR is controlled at 40～100mmol/L·h, 60～140h OUR is controlled at 20～90mmol/L·h;

The way to control the ORP in the fermentation broth in stages is as follows: the ORP in the fermentation broth for 0～4h is controlled at 60～150mV, the ORP in the fermentation broth at 4～12h is controlled at -80～100mV, and the ORP in the fermentation broth at 12～24h is controlled at -50 ～120mV, the ORP in the fermentation broth for 24～60h is controlled at -20～160mV, and the ORP in the fermentation broth at 60～140h is controlled at 20～220mV;

The way to control the concentration of lactic acid in the fermentation broth in stages is as follows: the concentration of lactic acid in the fermentation broth is controlled at 1-20mg/L for 0～4h, the concentration of lactic acid in the fermentation broth at 4～12h is controlled at 5～50mg/L, and the fermentation broth is 12～24h. The concentration of medium lactic acid is controlled at 10-100mg/L, the concentration of lactic acid in the fermentation broth for 24-60h is controlled at 30-150mg/L, and the concentration of lactic acid in the fermentation broth at 60-140h is controlled at 50-100mg/L.
The method for preparing vitamin K2 by microbial fermentation according to claim 1, characterized in that at least one of OUR, ORP in the fermentation broth and lactic acid concentration is monitored online during the fermentation and culture stage, and passed at different stages of the fermentation Adjusting at least one of air flow, rotation speed, and tank pressure to achieve stepwise control of at least one of OUR, ORP in the fermentation broth, and lactic acid concentration.
The method for preparing vitamin K2 by microbial fermentation according to claim 1 or 2, wherein the addition ratio of nicotinamide is 0.01-0.1 wt%.
The method for preparing vitamin K2 by microbial fermentation according to claim 1 or 2, wherein the addition ratio of vitamin B12 is 0.03 to 0.15 wt%.
The method for preparing vitamin K2 by microbial fermentation according to claim 1 or 2, wherein the addition ratio of methionine is 0.03 to 0.1 wt%.
The method for preparing vitamin K2 by using a microbial fermentation method according to claim 1 or 2, wherein the bacteria used in the fermentation are selected from Bacillus subtilis natto, Flavobacterium, At least one of Bacillus subtilis (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and Bacillus licheniformis (Bacillus licheniformis).
The method for preparing vitamin K2 by microbial fermentation according to claim 1 or 2, characterized in that the temperature of the fermentation culture is 28-40°C, the pH value is 6.0-7.5, and the seed liquid inoculum is 1-5 %.
The method for preparing vitamin K2 by microbial fermentation according to claim 1 or 2, characterized in that the volume of the fermentation tank used in the fermentation culture is 0.5L to 500m 3 .
The method for preparing vitamin K2 by microbial fermentation according to claim 1 or 2, characterized in that the method further comprises adding glycerol during the fermentation process to control the glycerol concentration in the fermentation system at 1-10 g/L.