Method for preparing vitamin B12 for removing cyanide by adopting fermentation method

Abstract

The invention belongs to the technical field of chemical medicines, and relates to a method for preparing vitamin B12 for removing cyanide by adopting a fermentation method. Comprising the following steps: (1) preparing vitamin B12 fermentation liquor, and performing flocculation and filtration to obtain filtrate 1; (2) vitamin B12 conversion: adding a ferricyanide complex and ammonia water into the filtrate 1 for conversion to obtain a conversion solution; (3) crude extraction of vitamin B12: performing flocculation and ultrafiltration on the conversion liquid to obtain filtrate 2; and (4) refining the vitamin B12: crystallizing the filtrate 2, and drying to obtain the vitamin B12. The method not only ensures higher yield of vitamin B12, but also improves the conversion efficiency, and meanwhile, the product cyanide is less in residue, higher in purity and more economical and practical.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical medicine and relates to a method for preparing vitamin B12 with cyanide removed by fermentation. Background Art

[0002] Vitamin B12, abbreviated as VB12, also known as cyanocobalamin, has the largest and most complex chemical formula of all vitamins. It contains the metal ion cobalt, so the scientific name of vitamin B12 is "cobalamin". It is an important element for maintaining the body's liver function and the operation of the brain and nervous system.

[0003] Since vitamin B12 is easier to extract and more stable after combining with cyanide, cyanide is usually used in the industry to prepare vitamin B12, that is, cyanocobalamin vitamin B12. Chinese invention patent application CN101736061A discloses a method for preparing vitamin B12, which uses ferrocyanide complex to achieve conversion and then produce vitamin B12, but requires the addition of a defoaming agent, which is not easy to remove and has the problem of low conversion rate; Chinese invention patent application CN116284191A discloses a method for converting vitamin B12 using sodium nitrite and potassium ferrocyanide, wherein the addition of sodium nitrite is still not ideal for improving the conversion rate, and the conversion needs to be carried out in a sealed system, which increases the difficulty of preparation.

[0004] In addition, ferrocyanide complexes such as potassium ferrocyanide have lower toxicity than other cyanides such as sodium cyanide and potassium cyanide, but they also have certain toxicity and are chemically active. They will decompose when exposed to acid or heat, especially when exposed to heat, they will produce nitrogen, potassium cyanide and iron-carbon compounds. When using ferrocyanide complexes to prepare vitamin B12, if the conversion temperature is too high, potassium cyanide will easily be produced. If it cannot be removed, its use will be affected.

[0005] Conventional treatment methods for cyanide-containing wastewater include acidification, chlorination, ozone oxidation, electrolytic oxidation and biological treatment. The most widely used and mature method in China is chlorination, which uses chlorine gas or liquid chlorine and bleaching powder to oxidize cyanide in wastewater into non-toxic substances such as carbon dioxide and ammonia. However, the alkaline chlorination method consumes a large amount of reagents and is costly; the acid chlorination method requires fully enclosed operation and is difficult.

[0006] The current technology for preparing vitamin B12 using ferrocyanide complexes has not yet introduced cyanide removal technology, resulting in the presence of cyanide residues in the prepared vitamin B12, which has certain limitations in use. Therefore, it is necessary to perform a series of pre-treatment or post-treatment operations when converting ferrocyanide complexes to vitamin B12 to avoid the generation of cyanide or remove cyanide residues. Summary of the invention

[0007] The present invention aims at the problems existing in the prior art and provides a method for removing cyanide and a method for preparing vitamin B12 (VB12). The method combines a ferrocyanide complex with ammonia water to improve the yield of VB12 as a whole. Meanwhile, a special treatment process is introduced before and after the conversion, so that the obtained VB12 has less cyanide residue and high purity.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] The present invention provides a method for preparing vitamin B12 with cyanide removed by fermentation, comprising the following steps:

[0010] (1) preparing vitamin B12 fermentation broth, and performing flocculation and filtration to obtain filtrate 1;

[0011] (2) Vitamin B12 conversion: adding ferrocyanide complex and ammonia water to the filtrate 1 for conversion to obtain a conversion solution;

[0012] (3) Crude extraction of vitamin B12: Flocculation and ultrafiltration of the conversion solution were performed to obtain filtrate 2;

[0013] (4) Purification of vitamin B12: The filtrate 2 is crystallized and dried to obtain vitamin B12.

[0014] Furthermore, the fermentation broth is obtained by fermenting, hydrolyzing and filtering one or more of Bacillus megaterium, Salmonella typhimurium and Propionibacterium freudenreichii.

[0015] As a supplementary explanation, the fermentation broth is prepared by anaerobic fermentation using the above-mentioned strains according to conventional means in the art.

[0016] Furthermore, in step (1), the flocculation adopts flocculants including perlite, diatomaceous earth and polyaniline;

[0017] The filtration is carried out by ceramic membrane filtration.

[0018] Furthermore, the flocculant is used in an amount of 1-5% (w / v).

[0019] Furthermore, the mass ratio of the perlite, diatomaceous earth and polyaniline is 1:2-4:0.1-0.5.

[0020] Furthermore, the ceramic membrane is selected from titanium oxide membrane or zirconium oxide membrane, and the pore size is 0.5-1 μm.

[0021] Furthermore, in step (2), the ferrocyanide complex is potassium ferrocyanide; wherein the amount of potassium ferrocyanide accounts for 0.1-2% (w / v) of the vitamin B12 fermentation broth.

[0022] Furthermore, in step (2), the amount of ammonia water used is based on controlling the pH value of the system to be 6.8-8.

[0023] Furthermore, the concentration of the aqueous ammonia is 25-28%.

[0024] In the process of preparing VB12 fermentation liquid, the present invention adopts anaerobic fermentation, glucose is degraded to produce acetic acid, propionic acid and CO2, and VB12 is synthesized intracellularly; however, the presence of acetic acid and propionic acid will inhibit the conversion of VB12, so in the present invention, ammonia water is used to regulate the pH value of the fermentation liquid, which can neutralize the acetic acid and propionic acid produced in the fermentation process and promote the conversion of VB12, and on the other hand, the weak reducing property of ammonia water itself can be used to protect VB12 from being destroyed, thereby further ensuring the yield of VB12.

[0025] Furthermore, the vitamin B12 conversion temperature is 50-80°C, and the conversion time is 30-60min.

[0026] Furthermore, in step (3), the flocculant includes polyaluminium ferric chloride and polyaniline.

[0027] Furthermore, the mass ratio of the polyaluminium ferric chloride to polyaniline is 0.05-1:1-10.

[0028] Furthermore, in step (3), the ultrafiltration is performed in a cross-flow mode for circulation filtration.

[0029] The cross-flow mode conditions are: cross-flow flow rate 7-9 L / s, pressure 0.1-0.2 MPa, filtrate temperature 30-50°C; the ultrafiltration membrane material is modified acrylic acid polymer, and the retention molecular weight is greater than 6 kDa.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) In the VB12 conversion process, the present invention uses a combination of ferrocyanide complex and ammonia water. While the ammonia water regulates the pH value of the conversion process system, it also acts as a reducing agent to protect VB12 from being oxidized and destroyed, thereby improving the yield of VB12 as a whole;

[0032] (2) This method can effectively remove colloidal impurities in the fermentation broth by flocculating the fermentation broth before conversion and flocculating and ultrafiltration the fermentation mixed liquor after conversion, providing a basis for subsequent separation and purification. The obtained VB12 cyanide residue is small and has a high purity;

[0033] (3) The VB12 preparation process of the present invention is simple and easy to operate, has a short reaction time, high conversion efficiency, and is more economical and practical. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited.

[0035] Example 1

[0036] The preparation method of vitamin B12 of the present invention is as follows:

[0037] (1) Preparation of vitamin B12 fermentation broth: Bacillus megaterium, Salmonella typhimurium and Propionibacterium freudenreichii were inoculated into a culture medium (composition of the culture medium: glucose 80 g / L, yeast extract 20 g / L, calcium carbonate 2 g / L, magnesium sulfate 2 g / L, potassium dihydrogen phosphate 2.5 g / L, potassium hydrogen phosphate 2.5 g / L, agar 18 g / L, vitamin B1 5 mg / L) at a volume ratio of 1:1:1 and cultured to a density of 3 × 10 8 cfu / mL of bacterial liquid; inoculating the bacterial liquid at an inoculum amount of 5% into a fermentation tank containing a fermentation medium (2% peptone, 0.5% yeast extract, 0.5% sodium chloride, 5% beef extract, 0.04% cysteine, 0.5% hemin chloride beef extract, pH adjusted to 7.4), stirring and mixing, and performing anaerobic fermentation at a fermentation temperature of 33° C., a tank pressure of 0.05 MPa, and a fermentation time of 72 hours to obtain a vitamin B12 fermentation liquid (titer 427 μg / mL);

[0038] 20.00L of vitamin B12 fermentation liquid was pumped into a flocculation tank, and a flocculant (perlite, diatomaceous earth and polyaniline in a mass ratio of 1:2:0.5; the flocculant accounted for 3% (w / v) of the vitamin B12 fermentation liquid) was added. After stirring for 30 minutes, it was filtered with a zirconia ceramic membrane with a pore size of 0.5 μm to obtain 18.50L of filtrate 1 (titer 454 μg / mL), with a yield of 98.4%;

[0039] (2) Vitamin B12 conversion: add potassium ferrocyanide (0.5% (w / v) of filtrate 1) to filtrate 1, adjust the pH value of the solution to 6.8-8 with aqueous ammonia, heat to 60°C and stir for 30 minutes to obtain a conversion solution;

[0040] (3) Crude extraction of vitamin B12: The conversion liquid was pumped into a flocculation tank, and a flocculant (polyaluminium chloride iron and polyaniline in a mass ratio of 1:1; the amount was 3% (w / v) of the conversion liquid) was added for flocculation and stirring for 30 min, and a cross-flow mode was used for circulation filtration, wherein the cross-flow flow rate was 8 L / s, the pressure was 0.1 MPa, and the filtrate temperature was 50°C; the ultrafiltration membrane material was a modified acrylic acid polymer with a molecular weight cutoff of 8 kDa; 16.85 L of filtrate 2 (titer 476 μg / mL) was obtained, and the yield was 95.6%;

[0041] (4) Purification of vitamin B12: Acetone was added to the filtrate 2 for recrystallization, and then filtered and dried to obtain 7.52 mg of crude vitamin B12 (detected by HPLC, the vitamin B12 content was 95.24%, CN - Content 0.9μg / mg), yield 89.3%. Total yield 84.0%.

[0042] In the vitamin B12 preparation process of the present invention, the titer of vitamin B12 in the vitamin B12 fermentation broth and the filtrate 2, and the VB12 and cyanide (CN) in the vitamin B12 crude product are determined. - ) content was obtained by high performance liquid chromatography.

[0043] 1. Investigation of preparation process

[0044] Vitamin B12 was prepared according to the preparation method in Table 1, and the total conversion rate of vitamin B12, the purity of crude vitamin B12 and the cyanide (CN - ) content was tested, and the results are shown in Table 2.

[0045] Table 1

[0046]

[0047]

[0048] Table 2

[0049]

[0050] Conclusion: According to the above table, the filtrates 1 and 2 in Examples 2 and 5 were obtained by adsorption, desorption and filtration of macroporous resin. Although the VB12 content in the crude product was high (higher than 90%), CN -The content is low (less than 1.5 μg / mg), but the total yield decreases significantly, which may be due to the partial loss of VB12 caused by incomplete desorption; Example 3 uses cyanamide for conversion, and the yield is significantly lower than that of Example 1, and the effect is not as good as that of ferrocyanide complex; and Example 4 uses simple membrane filtration technology for post-flocculation treatment of the conversion liquid, and the total yield is acceptable, but the VB12 content in the crude product is low, CN - The content is high, the impurity content is high, and the post-processing effect is not ideal.

[0051] 2. Determination of process parameters

[0052] Example 6: Determination of the dosage of potassium ferrocyanide

[0053] According to the preparation method of Example 1, the amount of potassium ferrocyanide used was the amount shown in Table 3, and the total yield of vitamin B12, the purity of crude vitamin B12 and the cyanide (CN - ) content was tested, and the results are shown in Table 4.

[0054] Table 3

[0055]

[0056] Table 4

[0057]

[0058] Conclusion: According to the above table, when the dosage of potassium ferroxanthate is 0.05% (w / v), the yield is low; when the dosage is 0.05% (w / v), the VB12 content in the crude product is low, and CN - The content is relatively high; when the dosage is between 0.1-3% (w / v), the total yield is above 80%, and the VB12 content in the crude product is higher than 94%, CN - The content is less than 0.12 μg / mg. Therefore, the dosage of potassium ferroxanthate in the preparation method of the present invention is preferably 0.1-3% (w / v).

[0059] Example 7: Selection of transformation substances

[0060] According to the preparation method of Example 1, the conversion of vitamin B12 in step (2) was carried out using the conversion products shown in Table 5, and the total yield of vitamin B12, the purity of crude vitamin B12 and the cyanide (CN - ) content was tested, and the results are shown in Table 6.

[0061] Table 5

[0062]

[0063] Table 6

[0064]

[0065] Conclusion: In step (2), the pH adjusting substance has a great influence on the total yield during the conversion of vitamin B12, among which ammonia water is the best, followed by sodium monohydrogen phosphate / sodium dihydrogen phosphate system, and sodium nitrite is the worst. By comparison, it is found that ammonia water has weak reducing properties, sodium monohydrogen phosphate / sodium dihydrogen phosphate do not have reducing properties, and sodium nitrite has strong reducing properties. In the preparation process of VB12 fermentation broth of the present invention, acetic acid, propionic acid and CO2 are produced while VB12 is synthesized intracellularly; ammonia water neutralizes the acetic acid and propionic acid produced during the fermentation process, promotes the conversion of VB12, and utilizes the weak reducing property of ammonia water itself to protect VB12 from being destroyed, thereby ensuring the yield. Although sodium nitrite has strong reducing properties, it reacts with acetic acid and propionic acid to produce nitrous acid, which in turn inhibits the conversion of VB12. In Experiment 4, although ammonia water is also involved in the conversion process, cyanamide replaces potassium ferrum, and the yield is similar to that of the impurity CN in the crude product. - The content was affected.

[0066] Example 8: Selection of flocculants during flocculation of fermentation broth and conversion broth

[0067] When vitamin B12 fermentation broth is converted and produced by ferricyanide complex, the treatment of the fermentation broth and the conversion broth before and after the conversion has a great influence on the conversion result. First, in the comparison between Examples 2, 4, and 5 and Example 1, it has been concluded that the treatment results of the fermentation broth and the conversion broth by the flocculation process of the present invention are better than those by the macroporous adsorption resin treatment process. Next, the type of flocculant used in the flocculation process of the present invention is further determined.

[0068] (1) According to the preparation method of Example 1, the flocculant in Table 7 was used in the flocculation treatment of step (1), and the total yield of vitamin B12, the purity of crude vitamin B12 and the cyanide (CN - ) content was tested, and the results are shown in Table 8.

[0069] Table 7

[0070] Serial number Flocculant Type Ratio Experiment 1 diatomite all Experiment 2 Perlite, diatomaceous earth 1：2 Experiment 3 Diatomaceous earth, polyaniline 2：0.5 Experiment 4 Perlite, diatomaceous earth, polyaniline 2：2：0.1 Experiment 5 Perlite, diatomaceous earth, polyaniline 1：4：0.1 Experiment 6 (Example 1) Perlite, diatomaceous earth, polyaniline 1：2：0.5 Experiment 7 Perlite, diatomaceous earth, polyaniline 1：4：0.5 Experiment 8 Perlite, diatomaceous earth, polyaniline 1：5：0.5

[0071] Table 8

[0072]

[0073]

[0074] (2) According to the preparation method of Example 1, the flocculation treatment in step (3) uses the flocculant in Table 9, and the total conversion rate of vitamin B12, the purity of crude vitamin B12 and the cyanide (CN - ) content was tested, and the results are shown in Table 10.

[0075] Table 9

[0076] Serial number Types of flocculants Ratio 1 Polyaluminium Ferric Chloride all 2 Polyaniline all 3 Polyaluminium ferric chloride and polyaniline 0.01：1 4 (Example 1) Polyaluminium ferric chloride and polyaniline 1：1 5 Polyaluminium ferric chloride and polyaniline 1：10

[0077] Table 10

[0078]

[0079] According to the results in Tables 8 and 10, it can be seen that the type and dosage ratio of the flocculant affect the flocculation effect. If the flocculation is not thorough, the small particles of impurities such as the fermentation liquid culture medium and the colloid cannot be completely aggregated and removed by filtration, which will affect the conversion and cause a significant reduction in the yield. If the flocculation of the conversion liquid is not thorough, the impurities cannot be removed. Although the yield is high, there are still a large number of impurities, and there is also a lot of cyanide residue, which greatly reduces the purity of the crude product.

[0080] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing vitamin B12 with cyanide removed by fermentation, characterized in that: The following steps are involved: (1) preparing vitamin B12 fermentation broth, and performing flocculation and filtration to obtain filtrate 1; (2) Vitamin B12 conversion: adding ferrocyanide complex and ammonia water to the filtrate 1 for conversion to obtain a conversion solution; (3) Crude extraction of vitamin B12: Flocculation and ultrafiltration of the conversion solution were performed to obtain filtrate 2; (4) Purification of vitamin B12: The filtrate 2 is crystallized and dried to obtain vitamin B12.

2. The method according to claim 1, characterized in that The fermentation liquid is obtained by fermenting, hydrolyzing and filtering one or more of Bacillus megaterium, Salmonella typhimurium and Propionibacterium freudenreichii.

3. The method according to claim 2, characterized in that In step (1), the flocculation adopts flocculants including perlite, diatomaceous earth and polyaniline; and the filtration adopts ceramic membrane filtration.

4. The method according to claim 3, characterized in that The flocculant dosage is 1-5% (w / v); the mass ratio of the perlite, diatomaceous earth and polyaniline is 1:2-4:0.1-0.5; the ceramic membrane is selected from titanium oxide membrane or zirconium oxide membrane, and the pore size is 0.5-1 μm.

5. The method according to claim 1, characterized in that In step (2), the ferrocyanide complex is potassium ferrocyanide; wherein the amount of potassium ferrocyanide used accounts for 0.1-2% (w / v) of the filtrate 1.

6. The method according to claim 1, characterized in that In step (2), the amount of ammonia water used is based on controlling the pH value of the system to be 6.8-8.

7. The method according to claim 1, characterized in that The vitamin B12 conversion temperature is 50-80°C, and the conversion time is 30-60 minutes.

8. The method according to claim 1, characterized in that In step (3), the flocculant includes polyaluminium ferric chloride and polyaniline.

9. The method according to claim 8, characterized in that The mass ratio of the polyaluminium ferric chloride to polyaniline is 0.05-1:1-10.

10. The method according to claim 1, characterized in that In step (3), the ultrafiltration adopts a cross-flow mode for circulating filtration; wherein the cross-flow mode conditions are: cross-flow flow rate 7-9 L / s, pressure 0.1-0.2 MPa, filtrate temperature 30-50°C; the ultrafiltration membrane material is a modified acrylic polymer with a molecular weight cut-off greater than 6 kDa.

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