Extraction method of vitamin K2

Abstract

The invention relates to the technical field of vitamin K2 extraction, in particular to an extraction method of vitamin K2. According to the method, the vitamin K2 fermentation liquor is heated, the lysozyme is added at a proper temperature, ultrasonic treatment is combined, wall breaking is carried out on the thallus cells, in the process, on one hand, wall breaking is carried out on the thallus cells through ultrasonic treatment, on the other hand, under the low-intensity ultrasonic condition, the activity of the lysozyme is improved, and the yield of the lysozyme is increased. The adding amount of the lysozyme is reduced, and meanwhile, the breakage rate of thallus cells is effectively guaranteed; besides, in the method, the fermentation liquor is heated in a manner of directly connecting a gas heat source, a microporous aeration manner is adopted in the process, bubbles introduced into the fermentation liquor show an enhancement effect on the breakage rate of thallus cells under an ultrasonic condition, and particularly, when low-temperature steam is adopted, the effect is more obvious, so that the fermentation liquor can be recycled. After being treated by the method disclosed by the invention, the breakage rate of thallus cells can reach 99% or above, and the yield of the vitamin K2 in the fermentation liquor is effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of vitamin K2 extraction, and in particular to a method for extracting vitamin K2. Background Art

[0002] Vitamin K2 is an essential endogenous vitamin with high activity in the human body. It has many important physiological functions such as preventing and treating osteoporosis, arterial calcification, cardiovascular disease, tumors and Parkinson's disease. Vitamin K2 is a series of menadione compounds, light yellow crystals, mainly synthesized by intestinal bacteria. There are 14 forms according to the length of the isoprene side chain at the C-3 position in its molecular structure, represented by MK-n (n refers to the number of isoprene units on the side chain), among which MK-7 has the most significant biological activity.

[0003] Among the methods for preparing vitamin K2, microbial fermentation is favored because of its readily available raw materials, mild conditions, low environmental pressure, high biological activity and high biocompatibility, and easy absorption and utilization by the human body. Currently, the microorganisms that can synthesize vitamin K2 include Flavobacterium, Mycobacterium, Nocardia, Lactobacillus, Streptomyces, and Bacillus natto. Among them, Bacillus natto has become the most important microorganism for fermentation production of vitamin K2 due to its advantages such as fast growth rate, easy cultivation, and high vitamin K2 content. It is one of the ideal strains for industrial production.

[0004] After the microbial fermentation is completed, the vitamin K2 in the fermentation broth needs to be extracted, separated and purified. The existing operation methods mostly use organic solvent extraction and gel chromatography separation. Since about half of the vitamin K2 produced during the fermentation process is located in the cells of the microorganisms, the microbial cells need to be crushed. In the industrial extraction process, mechanical crushing is mostly used. However, considering the actual production cost, the actual cell crushing rate is only about 80%, which leads to the loss of some vitamin K2, resulting in a reduction in product yield and also affecting production costs. Therefore, an extraction method that is easy to industrialize and can efficiently extract vitamin K2 produced by microbial fermentation is a problem that needs to be solved. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting vitamin K2, so as to solve the problem of low yield of vitamin K2 due to incomplete extraction when extracting vitamin K2 from fermentation broth.

[0006] To achieve the above object, the present invention adopts the following technical scheme: a method for extracting vitamin K2, comprising the following steps:

[0007] S1, taking vitamin K2 fermentation liquid with Bacillus natto as the strain, and passing a gas heat source directly into the fermentation liquid to heat the fermentation liquid;

[0008] S2. After the temperature is raised, the temperature is kept stable, lysozyme is added to the fermentation liquid, and the cell wall of the microorganisms in the fermentation liquid is broken under the condition of combined ultrasound;

[0009] S3. The fermentation liquid treated by S2 is extracted with an organic solvent, and then impurities are removed and refined to obtain a vitamin K2 product.

[0010] Furthermore, in the above-mentioned S1, the temperature of the gas heat source is 50-60°C; the gas heat source includes but is not limited to low-temperature steam, nitrogen and rare gas.

[0011] Furthermore, in the S1-S2, the gas heat source is directly introduced into the fermentation liquid by microporous aeration, and the ventilation volume in the temperature rising stage is 0.5-1V / (V·min), raising the temperature of the fermentation liquid to 45-55°C; the ventilation volume in the temperature stabilization stage is 0.1-0.5V / (V·min), stabilizing the temperature of the fermentation liquid at 50±2°C.

[0012] Furthermore, in the S1-S2, the stirring intensity is 60-120 rpm.

[0013] Furthermore, in the S2, the added amount of lysozyme is 0.5-2% of the mass of the fermentation liquid.

[0014] Furthermore, the frequency of the ultrasound is 20kHz, the power is 50-100W, and the duration of the ultrasound action is 30-120min.

[0015] Furthermore, in the S3, the volume ratio of the organic solvent to the fermentation liquid is 3 to 10:1, and the extraction time after adding the organic solvent is 2 to 4 hours.

[0016] Furthermore, the organic solvent is a mixed solution of isopropanol and n-hexane, and the volume ratio of isopropanol to n-hexane is 1:1-3.

[0017] Furthermore, in the S3, the organic phase after the organic solvent extraction is taken, impurities are removed by gel chromatography, and the eluent is eluted and then distilled under reduced pressure to obtain the vitamin K2 product.

[0018] Furthermore, the gel chromatography adopts one or more stages, the chromatography column is filled with molecular sieve filler, and the eluent includes but is not limited to one or more of ethanol, acetone and dichloromethane.

[0019] Beneficial effects of the present invention:

[0020] 1. The method of the present invention adopts a method of breaking bacterial cells and then extracting. Compared with directly using an organic solvent for extraction, on the one hand, the extraction is more thorough, which helps to improve the yield of vitamin K2. On the other hand, the extraction process only needs to be performed once. Compared with the previous multiple extraction method, the process is simpler, which is beneficial to reducing production costs and improving production efficiency;

[0021] 2. The method of the present invention uses a combination of lysozyme + ultrasound + aeration to break the bacterial cells, and the process has a good breaking effect. In actual production, when low-temperature steam is used, the bacterial cell breaking rate can reach more than 95%, which effectively improves the yield of vitamin K2. Compared with the traditional mechanical breaking, the single breaking rate is low and the energy consumption of multiple breaking is high. The method of the present invention is more convenient for use in industrial production. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0023] Example 1

[0024] A batch of vitamin K2 fermentation broth was taken and the vitamin K2 content therein was tested. During the process, a high-pressure homogenizer was used to crush the cells three times, and the cell crushing rate was found to be >99%. High-performance liquid chromatography was used for testing, and the vitamin K2 content in this batch of fermentation broth was 73.7 mg / L.

[0025] Take 100L of the fermentation liquid from this batch and add it to the reactor, and start stirring at a stirring intensity of 80rmp.

[0026] A gas heat source is introduced into the fermentation liquid through a microporous aeration disk. In this embodiment, the gas heat source is nitrogen, which is heated to 50-60°C and then introduced into the reactor through the microporous aeration disk. The temperature of the fermentation liquid is monitored during the process. When the temperature reaches above 45°C, the fermentation liquid is slowly ventilated and the temperature of the fermentation liquid is finally controlled to be stable at 50±2°C.

[0027] Take 1.5 kg of lysozyme, dissolve it and gradually add it to the reactor. After the addition is completed, turn on the ultrasound, the frequency of the ultrasound is 20 kHz, the intensity is 60 W, and the action is continued for 60 minutes; after the action is completed, take samples for testing, and the bacterial cell disruption rate is 91.28%.

[0028] An organic solvent is added to the fermentation broth. In this embodiment, the organic solvent is a mixed solution of isopropanol and n-hexane, wherein the volume ratio of isopropanol to n-hexane is 1:2, and the amount of organic solvent added is 600 L. After the addition of the organic solvent, the stirring conditions are maintained unchanged, and stirring and extraction are continued for 3 hours.

[0029] After the extraction is completed, the organic solvent in the upper layer is separated and impurities are removed by gel chromatography. In this embodiment, two-stage gel chromatography is used for treatment, the molecular sieve filler in the chromatography column is selected as BIO-Beads S-X3, and the eluent is n-hexane.

[0030] The refined solution was distilled under reduced pressure at 55°C, and finally 7.81 g of vitamin K2 was obtained. After testing, the purity was 89.82%, and the yield of vitamin K2 was 95.18%.

[0031] Example 2

[0032] In this embodiment, the same batch of fermentation liquid as in Embodiment 1 is selected, and in this embodiment, the gas heat source is selected to be low-temperature steam, and the rest is the same as in Embodiment 1.

[0033] After the final ultrasonic treatment was completed, the bacterial cell disruption rate was 96.31%, and a total of 8.03 g of vitamin K2 was obtained with a purity of 90.53%. The yield of vitamin K2 was 98.64%.

[0034] Example 3

[0035] In this embodiment, the same batch of fermentation liquid as in Embodiment 1 is selected. The difference from Embodiment 1 is that in this embodiment, the gas heat source is selected as argon gas, and the rest is the same as in Embodiment 1.

[0036] After the final ultrasonic treatment, the cell disruption rate was 90.75%, and a total of 7.69 g of vitamin K2 was obtained with a purity of 90.88%. The yield of vitamin K2 was 94.83%.

[0037] By comparing Examples 1-3, it can be found that nitrogen, low-temperature steam and argon can all achieve good yields, but in comparison, similar effects are obtained by using nitrogen and argon, and the yield of vitamin K2 is about 95%. Although argon can also achieve a good yield in the method of the present invention, it is not suitable for use in actual production due to its high cost. Even if it can be recycled, it will still incur higher costs. In comparison, nitrogen is more economical and reasonable.

[0038] However, it can be found from Example 2 of the present invention that when low-temperature steam is used, the yield of vitamin K2 is significantly improved, even reaching more than 98%. By comparing the breakage rate of bacterial cells during the treatment process, it can be found that when low-temperature steam is used, the breakage rate of bacterial cells is significantly improved. One possible reason is that the low-temperature steam is in a negative pressure state. After entering the reactor, the volume of the bubbles is further compressed under the external pressure. In this process, part of the bacterial cells will be broken, and more tiny bubbles will be formed. Then, under the action of ultrasound, the breakage rate of the bacterial cells is further improved.

[0039] Although in the above embodiment, the use of low-temperature steam enables a good yield of vitamin K2, low-temperature steam requires special equipment to be prepared in actual applications, and its cost is relatively high. In industrial production, a reasonable choice can be made based on actual production practices.

[0040] Example 4

[0041] In this embodiment, the impurity removal method after organic solvent extraction is changed. In this embodiment, the two-stage gel chromatography treatment method is still used. The difference from Example 2 is that in this embodiment, the molecular sieve filler recorded in the Chinese invention patent previously applied by the applicant: "CN115304464A A method for extracting vitamin K2 (35)" is used, which is different from the existing molecular sieve filler BIO-Beads S-X3 in Example 1.

[0042] Finally, the mass of vitamin K2 obtained was 7.26 g, the purity was 99.35%, and the yield was 97.87%.

[0043] It can be seen that although the purity of the vitamin K2 obtained in the methods described in Examples 1-3 of the present invention is relatively low, at about 90%, by selecting a reasonable molecular sieve filler to optimize the molecular sieve filler described in Examples 1-3 of the present invention, the purity of vitamin K2 can be effectively improved while ensuring the yield, making it more convenient for use in industrial production.

[0044] Comparative Example 1

[0045] This comparative example adopts the same batch of vitamin K2 fermentation broth as in Example 1. In this comparative example, the fermentation broth is heated by electric heating outside the reactor, which is different from the direct heating by the gas heat source in Example 1. At the same time, during the reaction process, only lysozyme is added to break the bacterial cells, and ultrasound is not applied during the breaking process. Others are the same as in Example 1.

[0046] Finally, after the lysozyme action was completed, the bacterial cell disruption rate was 58.94%. After the extraction was completed, a total of 6.35 g of vitamin K2 was obtained with a purity of 91.33%. The yield of vitamin K2 was 78.69%.

[0047] It can be seen that when ultrasound is not applied but lysozyme is used, there is a certain disruption effect on bacterial cells, but the effect is obviously inferior to the combination of lysozyme and ultrasound, and the yield of vitamin K2 is only about 80%.

[0048] Comparative Example 2

[0049] On the basis of comparative example 1, further experimental exploration was carried out. In this process, the dosage of lysozyme was increased to 2kg, 3kg, 4kg and 5kg respectively. The other conditions were the same as comparative example 1. The final results are shown in the following table:

[0050] Table 1 Effect of lysozyme addition on cell disruption rate and vitamin K2 yield

[0051]

[0052] It can be seen that with the increase in the amount of lysozyme, the bacterial cell breakage rate and the yield of vitamin K2 are promoted, but the final yield of vitamin K2 is only about 82%, indicating that with the increase in the amount of lysozyme, the improvement in the cell breakage rate is limited. When the amount of lysozyme continues to increase, the bacterial cell breakage rate and the yield of vitamin K2 cannot be significantly and effectively improved. At the same time, in actual production applications, considering factors such as cost, it is not allowed to continue to increase the amount of lysozyme added.

[0053] Comparative Example 3

[0054] Further research was conducted on the basis of Comparative Examples 1 and 2. In this process, only ultrasound was used, and the intensity of ultrasound was increased to 100 W. Lysozyme was not added, and the other conditions were the same as those of Comparative Example 1.

[0055] The cell breakage rate after ultrasonic treatment was detected, and the cell breakage rate was only 32.55%. The mass of the obtained vitamin K2 was 5.13 g, and the purity was 92.10%. The yield of vitamin K2 was 62.72%.

[0056] It can be seen from Comparative Examples 1-3 that the use of lysozyme alone, even if the amount of lysozyme is increased, cannot achieve a good disruption effect on bacterial cells. Since the power of ultrasound in the method of the present invention is low, the use of ultrasound alone has a poor disruption effect on bacterial cells.

[0057] Comparative Example 4

[0058] In this comparative example, the fermentation liquid is still heated by electric heating outside the reactor, and the bacterial cell disruption method combining lysozyme and ultrasound is used in this comparative example. The parameter conditions are the same as those in Example 1.

[0059] Finally, after the ultrasonic treatment, the bacterial cell disruption rate was 81.31%. After the extraction, the mass of vitamin K2 obtained was 7.19 g, the purity was 90.94%, and the yield was 88.72%.

[0060] It can be seen that lysozyme and ultrasound play a combined role. The combination of the two can, on the one hand, improve the fragmentation effect of bacterial cells, and on the other hand, also play a positive role in improving the yield of vitamin K2. However, the effect is still not as good as the direct gas heat source treatment method in the method of the present invention.

[0061] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims

1. A method for extracting vitamin K2, characterized in that: The following steps are involved: S1, taking vitamin K2 fermentation liquid with Bacillus natto as the strain, and passing a gas heat source directly into the fermentation liquid to heat the fermentation liquid; S2. After the temperature is raised, the temperature is kept stable, lysozyme is added to the fermentation liquid, and the cell wall of the microorganisms in the fermentation liquid is broken under the condition of combined ultrasound; S3. The fermentation liquid treated by S2 is extracted with an organic solvent, and then impurities are removed and refined to obtain a vitamin K2 product.

2. The method for extracting vitamin K2 according to claim 1, characterized in that: In the above-mentioned S1, the temperature of the gas heat source is 50-60° C.; the gas heat source includes but is not limited to low-temperature steam, nitrogen and rare gas.

3. The method for extracting vitamin K2 according to claim 2, characterized in that: In the S1-S2, the gas heat source is directly introduced into the fermentation liquid by microporous aeration. The ventilation volume in the temperature rising stage is 0.5-1V / (V·min), and the temperature of the fermentation liquid is increased to 45-55°C; the ventilation volume in the temperature stabilization stage is 0.1-0.5V / (V·min), and the temperature of the fermentation liquid is stabilized at 50±2°C.

4. The method for extracting vitamin K2 according to claim 1, characterized in that: In the above-mentioned S1-S2, the stirring intensity is 60-120 rpm.

5. The method for extracting vitamin K2 according to claim 1, characterized in that: In the S2, the added amount of lysozyme is 0.5-2% of the mass of the fermentation liquid.

6. The method for extracting vitamin K2 according to claim 6, characterized in that: The frequency of the ultrasound is 20kHz, the power is 50-100W, and the ultrasound action time is 30-120min.

7. The method for extracting vitamin K2 according to claim 1, characterized in that: In the S3, the volume ratio of the organic solvent to the fermentation liquid is 3 to 10:1, and the extraction time after adding the organic solvent is 2 to 4 hours.

8. The method for extracting vitamin K2 according to claim 7, characterized in that: The organic solvent is a mixed solution of isopropanol and n-hexane, and the volume ratio of isopropanol to n-hexane is 1:1-3.

9. The method for extracting vitamin K2 according to claim 1, characterized in that: In the above-mentioned S3, the organic phase after the organic solvent extraction is taken, and impurities are removed by gel chromatography. After elution with an eluent, the organic phase is distilled under reduced pressure to obtain the vitamin K2 product.

10. The method for extracting vitamin K2 according to claim 9, characterized in that: The gel chromatography adopts one or more stages, the chromatography column is filled with molecular sieve filler, and the eluent includes but is not limited to one or more of ethanol, acetone, n-hexane and dichloromethane.

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