CN103103235A

CN103103235A - Preparation method for vitamin B12

Info

Publication number: CN103103235A
Application number: CN2013100468799A
Authority: CN
Inventors: 冯胜利; 牛瑞昕
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-02-05
Filing date: 2010-02-05
Publication date: 2013-05-15
Anticipated expiration: 2030-02-05
Also published as: CN103103235B

Abstract

The invention relates to a method for preparing vitamin B12 by a fermenting method. The method comprises processes of fermentation, hydrolysis, filtering, cyanide conversion, crystallization, drying and the like, particularly a cyan compound in the cyanide conversion is an iron-cyan complex, wherein the reaction concentration is 200-4000 mu g/ml, the pH value of a reaction system is 3.0-8.0, the reaction temperature is 50-120 DEG C, and the reaction time is 0.5-6 hours. According to the preparation method provided by the invention, a series of disadvantages of environmental pollution, complex process, high production cost, low conversion efficiency and the like in a conventional method for generally converting the vitamin B12 by NaCN are overcome.

Description

A kind of preparation method of vitamin B12

The application is dividing an application of " a kind of preparation method of vitamin B12 ", and the application number of original application is 201010106076.4, and the applying date is 20100205, and invention and created name is " a kind of preparation method of vitamin B12 ".

Invention field

The present invention relates to a kind of method that fermentation method prepares vitamin B12, specifically, relate to a kind of extracting method of vitamin B12.

Background technology

Vitamin B12 (Vitamin B12) claim again cyanocobalamin (Cyanocobalamin), also referred to as animal protein factor(APF), is the general name that a class contains similar cyanocobalamin vitamin activity material.It is unique water-soluble vitamins that contains metal ion, is again one of VITAMIN of needed by human.

The molecular formula of vitamin B12 is C ₆₃H ₈₈CoN ₁₄O ₁₄P, its fusing point very high (320 ℃ time molten), odorless, tasteless, very easily the moisture absorption, stable in the air after the crystal suction.Vitamin B12 is water-soluble, is insoluble in ethanol, is insoluble to acetone, chloroform and ether, meets high light and can decompose, and the meeting variable color, can decompose in strongly-acid or strong basicity environment under 210～220 ℃ of high temperature.

Vitamin B12 is more special a kind of VITAMIN, does not contain vitamin B12 in most plants food, mainly is present in animal tissues.Vitamin B12 is VITAMIN essential in the tissue metabolic process, that people and other Mammals are kept growth and promote the important factor that red corpuscle is grown, has physiological action widely, as participate in body methyl conversion and folic acid metabolism, promote the formation of lipoprotein in neural myelin, keep the telotism of nervus centralis and periphery myelinated nerve fiber.Be used for the treatment of clinically pernicious anemia and recover hemopoietic function, also be used for nervous system disorders such as polyneuritis, peripheral nerve paralysis etc.

In a word, vitamin B12 is a kind of VITAMIN of needed by human, and its physiological function and trophism just day by day cause the common concern of the whole society.The application surface of China's vitamin B12 is narrower at present, is mainly pharmaceutical.Abroad, vitamin B12 is except being used for medical aspect (account for whole consumptions 40% left and right), also be widely used in nutritious supplementary and food-processing aspect (accounting for 24% left and right of whole consumptions), as vitamin oral liquid, vitamin enrichment flour, infant food etc.Therefore it is very large that the vitamin B12 of China is applied to the market development space of food service industry related products, experience that can Developed Countries, multivitamin nutritive food, the protective foodss etc. such as vitamin B12 are added in exploitation, can improve like this people's nutritional status and improve people's body constitution, can promote again development and the progress of vitamin B12 relevant food industry.

Although Woodward has completed the full chemosynthesis of Vitamin B12 with very outstanding work in 1973, at present industrial is mainly still to utilize inoculum fermentative production vitamin B12.Take the anaerobic ferment process of Xie Shi propionibacterium as example, it is as follows that it extracts flow process substantially:

At present, the research emphasis of fermentative Production vitamin B12 mainly concentrates on seed selection, optimization and the zymotechnique of bacterial strain, the aspects such as improvement of equipment.

Disclose a kind of method for preparing vitamin B12 with bacillus megaterium as patent WO2003018826 " method for preparing vitamin B12 ", related generally to the zymotechnique of bacillus megaterium under anaerobic condition.

Patent CN200510119016.5 " method of producing adenosincoenzyme vitamin B 12 by enzyme catalysis " discloses a kind of method of producing adenosincoenzyme vitamin B 12 by enzyme catalysis: utilize Triphosaden--and the vitamin B12 adenosyl transferase is produced Cobamamide, and the final concentration that hydroxyl cobalt type vitamin B12 wherein uses is 0.001-0.05mol/l.The method has the advantages that technique is succinct, with short production cycle, production cost is low.The end product Cobamamide purity of utilizing the production technique in the present invention to obtain is high, stability is strong, can be processed into multiple formulation, for the scale operation of medicinal Cobamamide provides a new way.

The people such as Nakano (Nakano K, Kataoka H, M atsum ura M.High density culture of Propionibacterium freudenreichii coupled with propionic acid removal system with activated charcoal[J] .Ferment Bioeng, 1996,81 (1): 37-41.) set up the fermentation system that is formed by rotary ceramic membrane filter, gac filling post and fermentor tank.Membrane filter and gac are loaded post and be can be used for removing propionic acid and reclaim reusable fermentation cell and substratum, the fermented liquid of Fei Shi propionibacterium in fermentor tank is first through the rotary ceramic membrane filtration, filtrate enters the filling post again, final seeing through in liquid no longer contains propionic acid, can be circulated back in fermentor tank and reuse, propionic acid and acetic acid is the recyclable byproduct that becomes also.Compare with traditional fermentation method, the utilization ratio of the nutritive substances such as Fei Shi propionibacterium and carbohydrate has all improved greatly, has reduced the waste of matrix, can further reduce costs, and is conducive to application and the popularization of actual production.

Use the fermentative Production vitamin B12, mostly refer to produce cyano group type vitamin B12 (claiming again cyanocobalamin).In fermented liquid and production vitamin B12 somatic cells, the existence form of B12 is mainly adenosine Diphosphothiamine 2 and a small amount of other type B12, for the B12 such as adenosine coenzyme are changed into cyano group B12, must use prussiate, the prussiate that generally uses at present is NaCN, it is highly toxic product, exists in process of production the poisoning potential safety hazard of worker; In the waste liquid that produces, a large amount of Cyanides is arranged, for environment protection standard, must add a large amount of NaClO to carry out wastewater treatment; The waste residue that produces also belongs to hazardous chemical, must burning disposal, and contaminate environment so not only, and increased production cost.

For the problems referred to above, although patent CN200810008004.9 " a kind of thallus extract and preparation method thereof " has realized the zero release of vitamin B12 fermentative production waste water: take vitamin B12 fermentative production waste water as raw material, through after the fermenting process of milk-acid bacteria, yeast and bacillus, it is prepared into the probiotics product, and this product is used for the pollution-less agriculture field, hewed out a kind of new purposes of vitamin B12 waste water, turned waste into wealth.But aforesaid method still can't be avoided the use of NaCN in preparation process.

Summary of the invention

The invention provides the novel method that a kind of fermentation method prepares vitamin B12, specifically, is a kind of extracting method of vitamin B12.The method refers in the prussiate conversion process of fermented liquid, the sodium cyanide that generally uses in iron cyano complex replacement prior art with stable chemical nature, in whole use procedure and production process, do not produce the material that human body, environment are harmful to, waste water and dregs need not carry out the recycling of cryanide ion, simplified production technique, reduced production cost, and the transformation efficiency of cyano group vitamin B12 is higher than the NaCN method.

The specific operation process that the iron cyano complex transforms is: add the iron cyano complex aqueous solution in the fermented liquid of vitamin B12, making its reaction density is 200-4000ug/ml.PH value with rare H2SO4 aqueous solution conditioned reaction system is 3.0-8.0, then it all is transferred in reaction flask, then splashes into 2 foam killers, and controlling temperature of reaction is 50-120 ℃, and reaction is 0.5-6 hour in the magnetic agitation reactor.

Fermented liquid in above-mentioned conversion process can be also ferment filtrate or its concentrated solution.

Iron cyano complex of the present invention preferably six cyanogen closes sour four potassium of iron (II).

As everyone knows, NaCN method conversion process is that adenosine actimide and other type B12 are first transferred to two cyanogen B12 or nitroso-group B12, sulfonic group B12, all changes at last cyano group B12 again, and because repeatedly transforming, its transformation efficiency can reduce.And the conversion process of cyano group B12 of the present invention is simple, production process safety, and working condition is gentle, environmentally safe, the prices of raw and semifnished materials of use are cheap, have higher Social benefit and economic benefit.

In experimentation of the present invention, the content of vitamin B12 is measured with high pressure liquid chromatograph.

Embodiment

Starting material and reagent:

1 fermented liquid

In this experiment, fermented liquid used refers to the denitrogenation pseudomonas (Pseudomonas denitrificans) for the production of vitamin B12, after conventional technique fermentation, hydrolysis, filtering, the filtrate of gained, the main Types of its contained vitamin B12 is the adenosine DBC.

The 2 iron cyano complex aqueous solution, the NaCN aqueous solution, sodium nitrite in aqueous solution.

Instrument and equipment

1 high pressure liquid chromatography (Lc-10A type)

Chromatographic column: C18L=30cm d=3.9mm

Moving phase: the ratio of first alcohol and water is 30%

Pressure: p=2500psi

Temperature: 40 ℃

Flow velocity: F=1ml/min

Sampling volume: 20ul

Wavelength: 361nm

2 magnetic stirring apparatuss (85-2 type)

3 full-automatic mechanicals stir stainless steel fermentation system (GUJT-10-100AUTOBI02000 type)

Embodiment 1

add the 30ml fermented liquid in the 100ml small beaker, under agitation adding 1ml concentration is the iron cyano complex aqueous solution of 20000ug/ml, the reaction density that makes the iron cyano complex is 667ug/ml, the pH value that with the quality percentage composition is 10% H2SO4 aqueous solution conditioned reaction system is 3.5 ± 0.5, then it all is transferred in the volumetric flask of 100ml, splash into again 2 foam killers, reaction is 6 hours in the magnetic agitation reactor, control 55 ± 5 ℃ of temperature, react complete, be settled to 100ml with distilled water, filter, getting filtrate is 52.97ug/ml with the content of high-pressure liquid chromatography cyano group type vitamin B12.

The comparative example 1

Add the 30ml fermented liquid identical with embodiment 1 in the 100ml volumetric flask, under agitation add successively the 3m1 Glacial acetic acid, 3ml quality percentage composition is 8% sodium nitrite in aqueous solution, and 2.0ml quality percentage composition is 2% the NaCN aqueous solution, 2 foam killers, reaction is 6 hours on the magnetic agitation reactor, control 55 ± 5 ℃ of temperature, react complete, be settled to 100ml with distilled water, filter, getting filtrate is 52.50ug/ml with the content of high-pressure liquid chromatography cyano group type vitamin B12.

Embodiment 2

be to add 7L ferment filtrate (after transforming with the NaCN method, the content of its cyano group vitamin B12 is 115.2ug/ml) in the retort of 10L to volume, then adding while stirring 70ml concentration is the iron cyano complex aqueous solution of 20000ug/ml, the reaction density that makes the iron cyano complex is 200ug/ml, the pH value that with the quality percentage composition is 10% H2SO4 aqueous solution conditioned reaction system is 7.5 ± 0.5, control 115 ± 5 ℃ of temperature of reaction, 250 rev/mins of mixing speed, reaction is 0.5 hour in retort, react complete, get the content that pure filtrate is used high-pressure liquid chromatography cyano group type vitamin B12, result is 117.59ug/ml.

Embodiment 3

The concentrated solution (after transforming with the NaCN method, the content of its cyano group vitamin B12 is 3300.32ug/ml) that adds 7L resin desorption liquid in the 10L retort, open and stir, adding 280ml concentration is that six cyanogen of 100000ug/ml close sour four aqueous solutions of potassium of iron (II), the reaction density that makes six cyanogen close sour four potassium of iron (II) is 4000ug/ml, the pH value that with the quality percentage composition is 10% H2SO4 aqueous solution conditioned reaction system is 6.5 ± 0.5, control 80 ± 5 ℃ of temperature of reaction, in retort, reaction is 1.5 hours.React complete, getting filtrate is 3368.72ug/ml with the content of high-pressure liquid chromatography cyano group vitamin B12.。

Claims

1. a fermentation method prepares the method for vitamin B12, comprise the processes such as fermentation, hydrolysis, filtration, prussiate conversion, crystallization, drying, it is characterized in that, prussiate conversion process cyano compound used is the iron cyano complex, its reaction density is 200-4000ug/ml, and in conversion process, the pH value of reaction system is 3.0-8.0, and temperature of reaction is 50-120 ℃, and the reaction times is 0.5-6 hour.

2. fermentation method as claimed in claim 1 prepares the method for vitamin B12, it is characterized in that, described iron cyano complex is that six cyanogen close sour four potassium of iron (II).