CS209251B1 - Manufacturing process of l-sorbose - Google Patents

Description

(54) Method for producing L-sorbose

The invention relates to a process for the production of L-sorbose; an important intermediate in the synthesis of ascorbic acid (vitamin C).

L-sorbose is produced by fermentation-bacterial dehydrogenation of D-sorbit by suitable species of acetic bacteria, most commonly Acetobacter suboxydans.

Theoretically, 98.9 parts of sorbose are to be produced from 100 parts sorbitol. The fermentadas are generally judged to be based on the sorbose content, calculated on the basis of the determination of reducing sugars, expressed as% dry matter present in the sample, and determined refractometrically (aqueous solutions of D-sorbitol and L-sorbose of the same concentration have practically the same refractive indices). In this method can be; found the theoretical yield; not taken at; this, however, takes into account the loss of dry matter of the sugars, caused both by total dissimilization of sorbitol and by him; consumption as a carbon source for bacterial growth. According to experience, this loss is about 4-9% based on the starting amount of sorbitol.

As it has been shown (Kulhánek, Ševčíkové, Folia; microbiologica, 1962, in print), in addition to L-sorbose, D-fructose is produced in small quantities of tad and 5-keto-D-fructose (5-keto) (L-sorbose) as side metabolites which undesirably reduce the yield. These by-products are melted as sorbose in the assay, which is another source of inaccuracy in determining fermentation yield. For the purpose of determining the yield of sorbose production, only the amount of isolated sorbose relative to the starting amount of sorbitol is decisive for these reasons.

In connection with the study of the conditions for increasing yields, a novel process for the production of L-sorbose by microbial dehydrogenation of D-sorbit in a liquid nutrient broth based on nitrogen nutrients and mineral nutrient salts was developed using the enzyme system of microorganisms of the genus Acetobacter.

The principle of this process is that the microbial dehydrogenation is carried out in the presence of inhibitors that prevent further enzymatic dehydrogenation of the sorbose formed.

As enzyme inhibitors, sodium fluoride, 2,4-dinitrophenol or dithiocarbamates can be used, in a concentration of the order of 1.10 ^-3 to 1. 10 ^-4 , based on the volume of culture medium.

The beneficial effect of the addition of inhibitors on the yield increase demonstrated by paper chromatography is demonstrated by suppression of side reactions, in particular the formation of 5-keto-D-fructose (5-keto-L-sorbose).

At a suitable inhibitor concentration, the fermentation time is not adversely affected. A suitable inhibitor concentration where partial inactivation of the reaction with the constituents of the raw materials used to prepare the fermentation broth is possible must be found empirically for the given conditions (eg sodium fluoride possibility of reaction with calcium compounds of corn extract, tap water, etc.). j Inhibitors pass into the mother liquors upon isolation of sorbose and are not found in isolated sorbose.

An advantage of the process according to the invention is an increase in the recovery of isolated sorbose by up to 10% over control, at a minimally increased cost.

Example Examples:

In a 750 ml Erlenmeyer flask was placed 100 ml of broth of this composition: 0.3 g corn extract, 0.17 secondary ammonium phosphate, 18.3 g sorbitol and 16.8 mg sodium fluoride (concentration 4.10 ^-3 M). After sterilization, the soil was 94 ml (sorbitol content per 100 ml was 19.4 g). The cooled soil was inoculated with 10 ml of Acetobacter suboxydans 125 culture (collection of the Microbiol. Institute of Czechoslovak Academy of Sciences Prague), prepared on soil of the same composition but without sodium fluoride, overnight cultivation at 32 ° C (inoculum originally contained 1.9 g sorbitol). After 58 hours of incubation at 30 ° C on a reciprocating shaker, the fermentation rate was 97%, with the dry matter content decreasing by 6%. Paper chromatography showed a spot of sorbose and a very small amount of D-fructose. A total of 17.4 g of product containing 17.0 g of 100% sorbose was isolated from the solution after filtration through a pad of diatomaceous earth and concentrated in vacuo at 20-25 ° C by ¹ crystallization in several fractions. 0.7 g of reducing sugars, calculated as sorbose, remained in the mother liquor. A total of 4 ml of ¹ solution containing 0.8 g of starting sorbitol was collected for analytical control of the fermentation process. Thus, 17.0 g of 100% sorbose was isolated from a total amount of 19.3 g sorbitol (18.2 g + 1.9 g - 0.8 g), corresponding to a yield of 88%. !

In a simultaneous blank test, at; in which the broth of the same composition but without the addition of sodium fluoride, the same inoculum and the same culture conditions was used, even after 58 hours of fermentation the fermentation rate was 98%.

| Sorbose, small amounts of D-fructose and 5-keto-D-fructose were detected by chromatography. The decrease in dry matter content was 9%. Total isolated

Claims (2)

[ SUBJECT A process for the production of L-sorbose by microbial dehydrogenation of D-sorbit in a liquid nutrient medium based on nitrogen nutrients and mineral nutrient salts, by the enzyme system of microorganisms of the genus Acetobarrier, in particular Acetobacter suboxydans; characterized in that the microbial dehydrogenation is carried out in the presence of inhibitors which prevent the 15.7 g of 10% sorbose from 19.6 g of starting sorbitol (samples taken contained 0.5 g of sorbitol), ie 80%. 1.2 g of reducing sugars, calculated as sorbose, remained in the last mother liquor. Thus, the recovery of isolated sorbose was 8% using sodium fluoride. Example 2: Similarly as in Example 1. The fermentation is conducted with the addition of 2.9 mg of 2,4-dinitrophenol (final concentration of 1,7.10 ^_4 M). After 58 hours of cultivation, the fermentation rate was 98%, the dry matter loss was 7%. Sorbose and very small amounts of fructose were detected by chromatography. Isolated 16.8 g of 100% sorbose, from 19.5 g of the starting sorbitol (0.6 g of the original sorbitol was sampled). Thus, the yield of Snil was 86% based on sorbitol, which corresponds to an increase of 6% over the concurrent blank. 0.5 g of reducing sugars, calculated as sorbose, remained in the mother liquor. Example 3: 100 ml of broth containing 0.1 g of maize extract, 19.0 g of D-sorbit and 4.2 g of sodium fluoride (concentration ^1.10-3 M) inoculated after sterilization with 4 ml of Acetobacter suboxydans 125 culture, cultured for 2 days at 32 ° C on soil containing 20% sorbitol, 0.5% baker's yeast in the form of 20% autolysate, 0.2% secondary ammonium phosphate, 0.1% primary potassium phosphate and 0.02% crystals. magnesium sulfate. After 65 hours of submersible fermentation, 96% fermentation was achieved. Chromatographic analysis revealed the presence of sorbose and a small amount of D-fructose. Total isolated 16.7 g sorbose, i.e. 16.2 g 100% sorbose from 19.1 g sorbitol (19.0 g + 0.8 g - 0.7 g), i.e. 85% based on the starting sorbitol. In a blank test under the same conditions, the fermentation was 96%. Sorbose, 5-keto-D-fructose and a small amount of D-fructose were detected by chromatography. From 19.4 g sorbitol (19.0 g + 0.8 g - 0.4 g), a total of 14.9 g sorbose was isolated, i.e. 14.6 g of 100% sorbose, corresponding to a yield of 75% based on the starting sorbit. Thus, the yield increase of the isolated sorbose in the fermentation with sodium fluoride was 10%. further enzymatic dehydrogenation of the resulting sorbose. 2. A process according to claim 1, wherein the enzyme inhibitors are sodium fluoride, 2,4-dinitrophenol or dithiocarbamates, in a concentration of the order of 1. 10 ^-3 to 1. 10 ^-4 M, based on the volume of culture medium.