CH643592A5 - PROCESS FOR PRODUCING 2,5-DICETOGLUCONIC ACID. - Google Patents

Description

The present invention relates to the production of 2,5-diketogluconic acid.

2,5-Diketogluconic acid is an interesting intermediate compound for the synthesis of vitamin C. Until now, this acid has been produced by various bacterial species such as Acetobacter melanogenum, Acetobacter aurantium, Gluconoacetobacter rubigino-sus, Gluconoacetobacter liquifaciens and Pseudomonas sesami. However, the use of these microorganisms is not entirely desirable from the industrial point of view, because of the production of large quantities of brown or yellowish brown pigments as by-products of the culture, which lower the purity of the 2,5-diketo-gluconic acid produced at the same time.

U.S. Patent No. 3,790,444 relates to the production of 2,5-diketogluconic acid without concomitant production of brown pigment, by a new species called Acetobacter fragum.

The invention relates to an economical process for the production of 2,5-diketogluconic acid which uses strains of a microorganism which is easy to obtain and available to the public; this micro-organism is called Acetobacter cerinus. Two of these strains, IFO 3263 and 3266, produce 2,5-diketogluconic acid in yields of more than 95% based on glucose. They were deposited on June 4, 1982 at ATCC, Rockville, Maryland (USA) under Nos. 39140 and 39141, respectively.

2,5-Diketogluconic acid is an interesting intermediate compound to be used for the production of ascorbic acid. An aqueous solution of 2,5-diketogluconic acid can be selectively reduced to produce a mixture of 2-ketogulonate and 2-ketogluconate which can be converted into ascorbic and erythorbic acids.

2,5-Diketogluconic acid is easily prepared by bacterial action on glucose using, in accordance with the process of the present invention, strains of Acetobacter cerinus which are easy to obtain. All the indicated strains of Acetobacter cerinus made available to the public have been subjected to tests by which they have been found to give keto acids in a yield of 50 to 95% on the basis of glucose. When using Acetobacter cerinus IFO 3263 or 3266, the keto acid produced is entirely the desired 2,5-diketogluconic acid, which is obtained in yields of more than 95% on the basis of glucose. The existing strains of Acetobacter cerinus, available to the public, are as follows: IFO 3262 (ATCC 12303), 3263, 3264, 3265, 3266, 3267, 3268, 3269.

These strains of Acetobacter cerinus are grown in an environment whose main source of carbon is glucose. These microorganisms do not require expensive organic sources of nitrogen such as peptone or meat extract. When urea and inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate or ammonium phosphate are used, nicotinic acid is added as an essential growth factor.

The concentration of glucose in the medium varies between 2.5 and 20%, preferably between 10 and 12%, in order to obtain 2,5-diketogluconic acid in the most economical way. The fermentation temperature is between 20 and 35 ° C, preferably between 25 and 30 ° C, and it is very advantageously of the order of 28 ° C. The initial pH of the culture medium can range from 3.5 at 7.5 and is preferably between 5 and 6. During fermentation, the pH is maintained at around 5.5 by the addition of a sodium hydroxide solution. Calcium carbonate can be used to adjust the pH and this salt is added as a make-up to the medium after autoclaving, in an amount of 20 g / 110 g of glucose.

After inoculation, the fermentation medium is stirred using a mechanical agitator at around 1700 rpm and it is aerated at a rate of 0.5 to 1 volume of air per volume of broth per minute.

When Acetobacter cerinus IFO 3263 or 3266 is used, fermentation is carried out until 2,5-diketogluconic acid has been obtained in a yield of at least 90% on the basis of glucose (36 at 40 h).

It has been determined by paper chromatography that the conversion of glucose to 2,5-diketogluconic acid is carried out according to the following schemes:

glucose- »2-ketogluconic acid-» 2,5-diketogluconic acid glucose- »5-ketogluconic acid-» 2,5-diketogluconic acid.

Whatman # 1 and # 4 paper is used, and a solvent mixture of methyl ethyl ketone, acetone, formic acid and water is used in the ratio of 80: 6: 2: 12. The spots corresponding to the acid are localized by spraying with an ethanolic solution at 0.2% of o-phenylenediamine containing 1% of nitric acid and by heating to approximately 70 ° C (5-ketogluconic acid = blue color; 2-ketogluconic acid = yellow color; 2,5-di-40 ketogluconic acid = green color). Identification can also be carried out by high pressure liquid chromatography.

2,5-Diketogluconic acid can be separated and collected from the final fermentation broth by any conventional operation known to those skilled in the art. The fermented broth that has been filtered can be treated, for example, with borohydride and the resulting mixture of 2-ketogluconic acid and 2-ketogulonic acid can be hydro-lysed to give ascorbic and erythorbic acids.

Example 1:

N / A The following aqueous medium is prepared for inoculation:

Ingredients g / l

Glucose 25

Soluble corn extract 5

55 KH2P04 0.5

K2HP04 0.5

MgS04-7H20 0.2

CaC03 6.3

pH 6.2

60

A shake flask containing 11 of medium is autoclaved for 30 min at 121 ° C. The pH of the cooled medium is 5.0. Acetobacter cerinus IFO 3263 cells from an inclined culture on nutrient agar (5 ml of a sterile aqueous suspension 65 with a volume equal to 20 ml) are added to the flask which is then shaken on a rotary shaker. 28 ° C for 24 h.

A culture sample in growth sufficient to represent a proportion of 5% by v / v of inoculum is added to a

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643,592

fermenter with stirring of 41 capacity containing 21 of the following production medium:

Ingredients g / l

Glucose 110

Soluble corn extract 0.5

(NH4) 2HP04 0.58

KH2P04 1.5

MgS04 • 7H20 0.5

Urea 0.5

CuS04 • 5H20 1 mg

Nicotinic acid 300 y pH 6.0

The fermentation is carried out at a temperature of about 28 ° C. with stirring at 1700 rpm and aeration at a flow rate of 0.75 volumes per volume of broth per minute. After a fermentation period of approximately 20 h, sterilized glucose (55 g / l) is added. The pH is maintained at 5.5 by the addition of a sodium hydroxide solution. The fermentation is continued until the yield of 2,5-diketogluconic acid is greater than 95% on the basis of glucose.

10

Example 2:

The procedure of Example 1 is repeated using Y Acetobacter cerinus IFO 3266; comparable results are obtained.

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Claims (6)

643,592 1. Process for the production of 2,5-diketogluconic acid, characterized in that it consists in carrying out the aerobic culture of Aceto-bacter cerinv ^ ns-unjn'lieu glucose, then in collecting the acid 2,5 -di resulting ketogluconic. • - -—, - 2. Method according to claim 1, characterized in that the glucose concentration of the medium has a value of 2.5 to 20%, the temperature of the fermentation ranges from 20 to 35 ° C, the initial pH ranges from 3, 5 to 7.5, and the pH during fermentation is kept at around 5.5. 2 3. Method according to one of claims 1 or 2, characterized in that the strain of Acetobacter cerinus is the strain IFO 3263. 4. Method according to one of claims 1 or 2, characterized in that the strain & Acetobacter cerinus is the strain IFO 3266. 5. 2,5-Diketogluconic acid obtained by the process according to one of the preceding claims. 6. Method for producing a mixture of 2-ketogulonic and 2-ketogluconic acids, characterized in that a fermentation broth is prepared according to the method of claim 1, and that the 2.5 acid is subjected -diketogluconic in the broth to a selective reduction.