A method for extracting vitamin K2

Abstract

The present invention relates to the technical field of vitamin K2 extraction, and in particular to a method for extracting vitamin K2. The method of the present invention heats a vitamin K2 fermentation broth, adds lysozyme at a suitable temperature, and combines ultrasound to break the bacterial cell walls. During the process, on the one hand, the bacterial cell walls are broken by ultrasound, and on the other hand, under low-intensity ultrasound conditions, the activity of lysozyme is improved, the amount of lysozyme added is reduced, and the cell breakage rate is effectively guaranteed. In addition, in the method of the present invention, the fermentation broth is heated by a direct gas heat source. During the process, microporous aeration is adopted. The bubbles introduced into the fermentation broth show an enhanced effect on the cell breakage rate under ultrasound conditions. In particular, when low-temperature steam is used, the effect is more obvious. After treatment by the method of the present invention, the cell breakage rate can reach more than 99%, effectively improving the yield of vitamin K2 in the fermentation broth.

Description

Technical Field

[0001] The present 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, highly active endogenous vitamin for the human body, with 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, pale yellow crystals, mainly synthesized by intestinal bacteria. There are 14 forms based on 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 highly favored due to its readily available raw materials, mild conditions, low environmental stress, high bioactivity and biocompatibility, and ease of absorption and utilization by the human body. Currently, microorganisms capable of synthesizing vitamin K2 include Flavobacterium, Mycobacterium, Nocardia, Lactobacillus, Streptomyces, and Bacillus natto. Bacillus natto, due to its rapid growth, ease of cultivation, and high vitamin K2 content, has become the primary microorganism for fermentation-based vitamin K2 production and is currently one of the ideal strains for industrial production.

[0004] After microbial fermentation is completed, vitamin K2 in the fermentation broth needs to be extracted, separated and purified. 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 decrease 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 to solve the problem of low vitamin K2 yield due to incomplete extraction when extracting vitamin K2 from fermentation broth.

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

[0007] S1. Taking a vitamin K2 fermentation broth produced by Bacillus natto as the bacterial strain, passing a gas heat source directly into the fermentation broth to increase the temperature of the fermentation broth;

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

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

[0010] Furthermore, in said 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), which increases 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), which stabilizes the temperature of the fermentation liquid at 50±2°C.

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

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

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

[0015] Furthermore, in the S3, the volume ratio of the organic solvent to the fermentation broth 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 isopropyl alcohol and n-hexane, and the volume ratio of isopropyl alcohol to n-hexane is 1:1-3.

[0017] Furthermore, in the above-mentioned 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 uses a method of disrupting 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 increase the yield of vitamin K2. On the other hand, the extraction process only needs to be performed once. Compared with the previous method of multiple extractions, 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 crush bacterial cells, and the process has a good crushing effect. In actual production, when low-temperature steam is used, the bacterial cell crushing rate can reach more than 95%, which effectively improves the yield of vitamin K2. Compared with the traditional mechanical crushing, the single crushing rate is low and the energy consumption of multiple crushing 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 disrupt the cells three times, and the cell disruption 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] 100 L of the fermentation broth of this batch was taken and added to the reactor, and stirring was started at an intensity of 80 rpm.

[0026] A gas heat source is introduced into the fermentation liquid through a microporous aeration disk. In this embodiment, nitrogen is selected as the gas heat source. The nitrogen 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, slow ventilation is performed 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 with a frequency of 20 kHz and an intensity of 60 W, and continue the action 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 was added to the fermentation broth. In this embodiment, the organic solvent was a mixed solution of isopropanol and n-hexane, wherein the volume ratio of isopropanol to n-hexane was 1:2, and the amount of organic solvent added was 600 L. After the addition of the organic solvent, the stirring conditions were maintained unchanged and the stirring extraction was continued for 3 h.

[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 purified solution was subjected to reduced pressure distillation at 55° C. to obtain 7.81 g of vitamin K2. The purity was found to be 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 Example 1 was selected, and in this embodiment, the gas heat source was selected as low-temperature steam, and the rest was the same as in Example 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 Example 1 was selected. The difference from Example 1 was that in this embodiment, argon was selected as the gas heat source. The rest was the same as in Example 1.

[0036] After the final ultrasonic treatment, the bacterial cell disruption rate was 90.75%, and a total of 7.69g 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 achieved 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, due to the high cost of argon, it is not suitable for use in actual production. 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, some bacterial cells will be broken, and even smaller bubbles will be formed. Then, under the action of ultrasound, the breakage rate of bacterial cells is further improved.

[0039] Although the use of low-temperature steam in the above embodiment 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 for 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 fillers 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 uses 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 method of the gas heat source in Example 1. At the same time, during the reaction process, only lysozyme is added to disrupt the bacterial cells, and ultrasound is not applied during the disruption process. Other aspects 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 significantly 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 amount of lysozyme was increased to 2kg, 3kg, 4kg and 5kg respectively. 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 of the cell breakage rate is limited. When the amount of lysozyme is further increased, 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 exploration was conducted based on Comparative Examples 1 and 2. During 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 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 also has a poor effect on the disruption of bacterial cells.

[0057] Comparative Example 4

[0058] In this comparative example, the fermentation liquid was still heated by electric heating outside the reactor, and bacterial cell disruption was performed by combining lysozyme with ultrasound. The parameters were the same as those in Example 1.

[0059] Finally, after the ultrasonic treatment was completed, the bacterial cell disruption rate was 81.31%. After the extraction was completed, 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 effect of bacterial cell disruption, 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 forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that is the same or similar to that of the present application falls within the scope of protection of the present invention.

Claims

1. A method for extracting vitamin K2, characterized in that: The following steps are involved: S1. Taking a vitamin K2 fermentation broth produced by Bacillus natto as the bacterial strain, passing a gas heat source directly into the fermentation broth to increase the temperature of the fermentation broth; The temperature of the gas heat source is 50-60°C, and the gas heat source includes low-temperature steam. The gas heat source is directly introduced into the fermentation liquid by microporous aeration. During the heating phase, the temperature of the fermentation liquid is raised to 45-55°C. During the temperature stabilization phase, the temperature of the fermentation liquid is stabilized at 50±2°C. S2. After the temperature is raised, the temperature is maintained stable, lysozyme is added to the fermentation broth, and the cell wall of the microorganisms in the fermentation broth is broken under the condition of combined ultrasound; The power of the ultrasound is 50-100W; S3. The fermentation liquid treated in S2 is extracted with an organic solvent, and then purified by removing impurities to obtain a vitamin K2 product.

2. The method for extracting vitamin K2 according to claim 1, wherein: In the above-mentioned S1, the gas heat source also includes nitrogen and rare gas.

3. The method for extracting vitamin K2 according to claim 2, wherein: In the above-mentioned S1-S2, the ventilation volume in the temperature rising stage is 0.5~1V / (V·min); the ventilation volume in the temperature maintaining stable stage is 0.1~0.5V / (V·min).

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

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

6. The method for extracting vitamin K2 according to claim 1, wherein: The frequency of the ultrasound is 20 kHz, and the duration of the ultrasound action is 30 to 120 minutes.

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

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

9. The method for extracting vitamin K2 according to claim 1, wherein: In the above-mentioned 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.

10. The method for extracting vitamin K2 according to claim 9, wherein: The gel chromatography adopts one or more stages, the chromatography column is filled with molecular sieve filler, and the eluent is one or more of ethanol, acetone, n-hexane and dichloromethane.

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