SK280146B6 - Process for separating riboflavin - Google Patents

Abstract

A process for separating riboflavin from a fermented nutrient broth subsequent to the cell cultivation of a production microorganism of Bacillus subtilis is characterised in that the fermented nutrient broth or separated biomass is subjected, in a first stage, in an aqueous medium into a contact with non-ionic or ionic tensides of a sodium laurelsulphate type, oxyethylated nonylphenol or oxyethylated p-octylphenol and/or with organic solvents selected from the grup of alkanes, cycloalkanes, alkanecarboxylic acids esters of chlorinated alkanes; the biomass is separated once again being suspended, in a second stage, to a cell concentration of 0.01 to 0.5 g of a humid mass/ml in water containing from 0.1 to 85 wt% with water-miscible organic acids and/or an additive of one or more organic aprotic solvents. Ths mixture is heated to a temperature ranging from 40 to 120 degrees Celsius in the elapse of from 0.25 to 20 hours. The mixture is, in a third stage, separated to a solid and liquid phase at a temperature of at least 30 degrees Celsius, and, finally, riboflavin separated from said phase is isolated.

Description

Technical field

The invention relates to a method for the isolation of riboflavin from fermented broth after culturing of cells of the production microorganism Bacillus subtilis 24 / pMX / 45 (Dikanskaya E.M., Balabanova, A.A., SU pat. 465 942, 1980).

BACKGROUND OF THE INVENTION

Riboflavin (vitamin B ₂ ) produces a number of microorganisms, such as Pseudomonas, Gluconobacter, Brevibacterium, Bacillus, Nocardia, Candida and Saccharomyces. Riboflavin is isolated from the fermented soil and purified in a variety of ways. According to European patent application no. 0 137 226 riboflavin is produced by some yeast using lower aliphatic compounds (C 1 to C ₄ ). After the cultivation is completed, riboflavin is extracted in an acid medium at temperatures above 50 ° C and purified. The product obtained has to be further purified and thus lost. Japanese patent specification no. 1,021,096 discloses separation of soil from solids, multi-stage isolation of riboflavin with hot water, and crystallization. The disadvantage of this process is the large volume work and loss of product during cleaning. The method of isolation and purification of riboflavin according to DOS no. 3,421,714 is carried out by dissolving riboflavin in an alkaline medium and activated carbon. After adjusting the pH to 0.8 to 6.5, the mixture is heated at 90 to 100 ° C for 10 to 80 minutes. Subsequent crystallization yields a product with a purity of 92-94% in a yield of 90-92%. According to Japanese patent specification no. The isolation and purification of riboflavin is accomplished by ion exchange, elution with a solution of inorganic salts and chromatography on polystyrene resin-filled columns. However, this process is quite costly and difficult to manufacture. DOS č. No. 3,406,319 discloses only an improved process for purifying isolated pure riboflavin by oxidation with hydrogen peroxide, nitric acid, and the like. at elevated temperature, interruption of oxidation by pouring cold water and crystallization. The purity of the product is between 94-96% with a yield of 86-94%.

SUMMARY OF THE INVENTION

The method for the isolation of riboflavin from fermented broth after culturing the cells of Bacillus subtilis 24 / pMX / 45 according to the invention consists in contacting the fermented broth or the separated biomass with water or nonionic or ionic lauryl sulfate surfactants in a first step sodium, nonylphenol oxyethylated with 9 moles of ethylene oxide or p-octylphenol oxyethylated with 9 moles of ethylene oxide and / or with organic solvents from the group of 5 to 10 carbon atoms, cycloalkanes of 5 to 7 carbon atoms, esters of 1 to 3 carbon atoms of alkanecarboxylic acids the ester group contains 1 to 3 carbon atoms or aromatic hydrocarbons based on benzene having 1 to 2 carbon atoms in the side chain or side chains, or chlorinated alkanes of 1 to 4 carbon atoms and 2 to 8 chlorine atoms, the biomass is separated again and in the second step with the suspension is a cell concentration of 0,01 to 0,5 g of wet mass / ml in water containing 0,1 to 85% by weight of water miscible organic acids having 1 to 3 carbon atoms and / or the addition of one or more organic aprotic solvents, this mixture is heated to 40 to 120 ° C for 0.25 to 20 hours, then separated into a solid and a liquid phase in a third stage at a temperature of at least 30 ° C, then the riboflavin precipitated from this phase is isolated.

Centrifugation of the soil (solid particles) is always performed on a cuvette or flow centrifuge. The surfactants used were sodium lauryl sulfate or oxyethylated nonylphenols, sorbitol monolaurate or p-octylphenol (Syntapon L, Slovafol 909 or 910, Tween 20, Triton X-100) at a concentration of 0.01 to 10% v / v or mass / volume. Organic solvents such as dichloroethane, chloroform, toluene, cyclohexane or butyl acetate at a concentration of 0.1 to 50% v / v were used to treat the cell suspension at a cell concentration of 0.01 to 0.4 g wet mass / ml. Solvents from the group of ethyl acetate, dimethylsulfoxide, dioxane, dimethylformamide or pyridine at a concentration of 0.01 to 30% v / v were used to heat the cells with formic or acetic acid. Crystallization was carried out at 5 to 10 ° C for up to 48 hours, the obtained crystals were washed with water and ethanol or acetone at a concentration of 50 to 100% by volume and dried at 30 to 120 ° C for 3 to 72 hours. The dry riboflavin product has a purity of 92-100% in a yield of 88-93%.

The riboflavin isolation process according to the invention allows riboflavin in a simple manner to be isolated from the fermented soil without further purification directly into the final product form with a purity of 92-100% in a yield of 88-93%. These values exceed those commonly reported in the patent literature and are comparable to those achieved by state of the art technologies, including purification of pure riboflavin preparation alone. The process according to the invention has also been successfully tested on a production scale.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

The wet cell paste of the Soviet strain Bacilles subtilis 24 / pMX / 45, obtained by centrifugation of the fermented soil on a Sharples centrifuge (16,000 g), was suspended in water at a final volume of 12 L with 4.2 kg. The suspension was mixed with nonylphenol oxyethylated in 9 moles ethylene oxide. (Slovafol 909) at 1% v / v and butyl acetate at 5% v / v for half an hour, and the mixture was allowed to separate until the phases were separated. The top phase was aspirated and the bottom suspension was separated on a flow centrifuge (Sharples). The resulting paste was resuspended in 19.5 L of drinking water so that the suspension contained 10% v / v formic acid and the mixture was boiled at 105 ° C for 4 hours. The mixture was then separated on a Sharples centrifuge, the supernatant was allowed to crystallize overnight. The riboflavin crystals were collected by vacuum filtration, washed with 2.5 L of deionized water and 1.26 L of 96% ethanol and dried for 6 hours at 105 ° C in a drying oven. Obtained

455 g of riboflavin with a purity of 100% in a yield of 88%.

Example 2

The 100 g paste obtained by centrifuging the fermented soil using the microorganism as in Example 1 was suspended in 400 ml of water. 100 ml of chloroform was added to the suspension, the mixture was shaken for 60 minutes and centrifuged on a cuvette centrifuge. The resulting paste was suspended in 1600 ml of 30% v / v acetic acid, 10% v / v ethyl acetate was added, and the mixture was boiled at 100 ° C for 2 hours. After separating the waste paste and the chloroform residues on a flow centrifuge (Westphalia), 998 ml of supernatant was obtained, which was concentrated twice. After crystallization in cold (5 ° C) for 12 hours, vacuum filtration, washing with 100 ml deionized water and 75% acetone 50 ml and drying at 60 ° C for 10 hours in a ventilated chamber hazel oven, 9.2 g of riboflavin were obtained. with a purity of 98.2% in a yield of 90.6%.

Example 3

The fermented soil of 3.1 L was mixed with sodium lauryl sulfate (Syntapon L) at a concentration of 1% w / v for 0.5 hour. The suspension was centrifuged on a cuvette centrifuge and the resulting paste was suspended in 2 L of deionized water so that the formic acid concentration in the mixture was 20% v / v. Dimethylsulfoxide at 5% v / v was added to the suspension and the mixture was heated at 100 ° C for 4 hours. The hot slurry was vacuum filtered using kieselguhr (Hyflo Super Cell) at 7.1 kPa, the clear filtrate was concentrated to half volume, allowed to crystallize at 5 ° C for 20 hours, after which the riboflavin crystals were collected by vacuum filtration, washed with 250 ml deionized water. and 100 µl of ethanol (96%) and dried for 20 hours at 37 ° C in a ventilated chamber hazel dryer. 23.0 g of riboflavin were prepared with a purity of 100% in a yield of 92.8%.

Industrial usability

The riboflavin (vitamin B ₂ ) prepared by the process of the invention is obtained in high quality and yield, allowing the production of larger quantities of the desired drug.

Claims (8)

PATENT CLAIMS A method for isolating riboflavin from fermented broth after culturing Bacillus subtilis producing microorganisms, comprising contacting in a first step the fermented broth or the separated biomass with non-ionic or ionic sodium lauryl sulfate surfactants in a water environment, 9 moles of ethylene oxide oxyethyl or 9-moles ethylene oxide p-octylphenol and / or with organic solvents from the group of 5 to 10 carbon atoms, cycloalkanes of 5 to 7 carbon atoms, esters of 1 to 3 carbon atoms of alkanecarboxylic acids, contains 1 to 3 carbon atoms or aromatic hydrocarbons based on benzene having 1 to 2 carbon atoms in the side chain or side chains, or chlorinated alkanes of 1 to 4 carbon atoms and 2 to 8 chlorine atoms, the biomass is separated again and in the second stage suspension to concentrate cells with 0.01 to 0.5 g of wet mass / ml in water containing 0.1 to 85% by weight of water miscible organic acids having 1 to 3 carbon atoms and / or the addition of one or more organic aprotic solvents; heated to 40 to 120 ° C for 0.25 to 20 hours, and in the third stage at least 30 ° C separated into a solid and a liquid phase, and finally the precipitated riboflavin is recovered. Method according to claim 1, characterized in that the separation of the solid particles is carried out in a cuvette or flow centrifuge. Process according to claim 1, characterized in that sodium lauryl sulphate or oxyethylated nonylphenols, sorbitol monolaurate or p-octylphenol at a concentration of 0.01 to 10% v / v or mass / volume are used as surfactants. 4. A method according to claim 1, wherein organic solvents such as dichloroethane, chloroform, toluene, cyclohexane or butyl acetate are used to treat the cell suspension with a cell concentration of 0.01 to 0.4 g wet mass / ml. 0.1 to 50% v / v. 5. A method for the isolation of riboflavin according to claim 1, characterized in that organic solvents from the group of ethyl acetate, dimethylsulfoxide, dioxane, dimethylformamide or pyridine at a concentration of 0.01 to 30% are used for heating the cells with formic acid or acetic acid. volume / volume. The process according to claim 1, wherein the crystallization is carried out at 5 to 10 ° C for 0.1 to 48 hours. 7. The process of claim 1 wherein the crystals are washed with water and ethanol or acetone at a concentration of 50-100% (v / v). A process according to claim 1, characterized in that the drying is carried out at 30 to 120 ° C for 3 to 72 hours.