Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1999-04-22
2001-01-09
Peselev, Elli (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C252S364000, C435S126000
Reexamination Certificate
active
06172242
ABSTRACT:
The present invention relates to a process for the production of erythorbic acid. More particularly, the present invention relates to the recovery of erythorbic acid from aqueous solutions containing values of erythorbic acid in low concentrations, the term values of erythorbic acid as used herein referring to erythorbic acid, salts thereof, derivatives thereof and mixtures thereof.
Erythorbic acid is an isomer of ascorbic acid and is also named isoascorbic acid and D-araboascorbic acid. This acid and its salts (erythorbates) are widely used as alternatives to ascorbic acid and its salts in non-Vitamin C products. Erythorbates traditionally have two functions. First they act as antioxidants, controlling color and flavor deterioration in many foods, and as antimicrobial agents for foods. Secondly, erythorbates are used as meat-curing accelerators, speeding and controlling the nitrite-curing reaction, while prolonging color and shelf-life. Erythorbates therefore have many uses in the food industry and are used in conjunction with such comestibles as meat, fish, poultry, beverages, fruits and vegetables.
Takeshi Takahashi's U.S. Pat. No. 3,052,609 assigned to Sankyo Co., teaches a process for the production of D-araboascorbic acid which comprises subjecting one or more substances selected from the group consisting of D-glucose, D-gluconic acid, D-glucono-y-lactone, D-glucono-8-lactone, sucrose, maltose and starch to the action of an enzyme of a microorganism selected from the group consisting of
Penicillium decumbens, Penicillium chrysogenum, Penicillium chrysogenum
mut.
fulvescens
Takashima, Arima and Abe,
Penicillium meleagrinum, Penicillium cyaneofulvum
and
Penicillium notatum
in the presence of oxygen and recovering the D-araboascorbic acid which is formed.
In carrying out the process according to that invention, both a direct fermentation method and a cell suspension process may be applied.
In carrying out the direct fermentation method, too low concentration of the carbon source would result in decreased conversion to the product due to consumption of the source for the propagation of the cells. Too high concentrations would lead to lower yield due to greater conversion to by-products and to a larger amount of residual sugar. It is preferable to use a concentration between 0.5 and 10%. It is also preferred to keep the concentration between about 0.5 and 1.0% by continuously adding the material. Other substances present in the medium are organic or inorganic assimilable nitrogen sources, mineral salts and a trace of various metals. pH is usually maintained between about 3 and 7. The time required for the fermentation is from 5 to 10 days in the case of surface culture and from 3 to 7 days in the case of submerged culture. The preferred temperature in the fermentation is 26 to 28° C.
Production of D-araboascorbic acid using the intact cell or dried cell preparation is effected in a buffer solution having a pH of about 4.0 to 6.0. The concentration of the carbon source is 0.5 to 10%, the temperature between 35 and 30° C. and the time is between 50 to 80 hours. The substrate is preferably added in intervals to keep its concentration between 0.5 and 1.0%.
The isolation of D-araboascorbic acid may be performed by first removing the mycelium by means of filtration or by means of a centrifugal procedure and subsequently applying the known procedures for isolating L-araboascorbic acid to the filtrate or supernatant. For example, an adequate amount of barium acetate is added to remove phosphates and sulfates and organic impurities are removed by treatment with active charcoal, followed by adsorption of the desired product on anion-exchange resin such as Amberlite IR-4B and elution with aqueous hydrochloric acid. Furthermore, impurities are removed by means of a small amount of active charcoal and butanol and D-araboascorbic acid is crystallized by concentration in vacuum at low temperature under carbon dioxide, followed by recrystallization from solvent such as acetone or ethanolligroin.
The product concentrations, g/l, (and fermentation pH and duration) in Examples 1 to 7 of said U.S. patent are respectively: 0.5-1.2 (pH=5-6, 7 days), 2-3 (pH=5, 3 days), 4.2 (pH=5.6, 4 days), 5.3 (pH=5.3, 5 days), 5 (pH=5.6, 10 days), 1.4 (pH=5, 4 days), 10.3 (5.0 for the initial 20 hours and about 4.0 for the remaining 40 hours).
In Examples 1 to 6 the culture filtrate, after treatment with 1 g. of barium acetate and 0.1 g. of active charcoal, is adsorbed on ion-exchange resin IR-4B, followed by elution with 1 liter of 1 N-HCl. About 70% of the total content of the desired product is eluted in fractions in volume of 200-300 ml. after initiation of the elution. These fractions are shaken with butanol and, after addition of a small amount of active charcoal, are filtered to give an almost colorless transparent liquor, which is concentrated to near dryness in vacuum at temperature below 30° C. under CO
2—
, followed by several concentrations in the presence of ethanol to remove most of the water. The oily substance thus obtained is allowed to stand in a vacuum desiccator for 2-3 days to separate crystalline D-araboascorbic acid.
In Examples 7 and 8, after completion of the reaction, pH is adjusted to 2.0 with H
2
SO
4
and the mycelium is separated by filtration. The filtrate is treated with about 1 g. of barium acetate and about 0.1 g. of active charcoal per liter followed by filtration. The filtrate is passed through ion-exchange resin IR-4B pretreated with acetic acid to adsorb D-araboascorbic acid contained in the filtrate. The ion-exchange resin column is treated with 1 N-HCl and about 70% of the total content is eluted in 200-300 ml. of the first fractions from the elution. The eluate, after addition of a small amount of active charcoal, is filtered to give almost colorless transparent solution. The solution is concentrated to near dryness in vacuum under CO
2
. Repeated concentrations after addition of ethanol to the residue give an oily substance almost water free. The oily substance thus obtained is concentrated in vacuum to give crystalline D-araboascorbic acid.
The recovery yield shown in examples 3 to 8 were low, 19, 23, 20, 29, 29 and 28%, respectively. A possible explanation for that is the presence of HCl in the eluate. On concentration of the latter, HCl could cause decomposition of the product. Another disadvantage of the recovery process is the high consumption of acids and bases and the resulting formation of by-product salts.
Seven years after said U.S. patent was granted, the inventor issued an article entitled: Erythorbic acid fermentation, which was published in Biotechnology and Bioengineering Vol. XI, pages 1157-1171 (1969). (Takahashi had several earlier publications, see references in the 1969 article.) Two other related publications, by Yagi and co-workers and by Shimizu and co-workers, respectively are: Studies on Erythorbic acid production by fermentation, Part I, Erythorbic acid producing strain and cultural conditions and Part II, Erythorbic acid production by jar fermentors, published in Agr. Biol. Chem. Vol. 31, pages 340-345 and 346-352, respectively (1967). These articles describe studies directed to development of an industrial process including strain improvement, optimization of culture solution (carbon source, nitrogen source, additives, effects of iron and copper and of chelating agents), temperature, aeration and agitation.
Glucose and sucrose were found to be the most appropriate carbon source. Glucose concentration should be in the range of 8-12%. In one test the fermentation was started with 8% glucose, 8% glucose was fed on the third day and 4% glucose was fed on the sixth day. The yield in that case amounted to about 40% of the total glucose supplied. The erythorbic acid concentration in the solution reached 80 g/l. The preferred temperature is about 30° C.
In the course of typical fermentations the pH of the broth is gradually lowered along the consumption of sugar and remains in the range of 3.8-4.
Eyal Aharon Meir
Hazan Betty
Reuveni Tal
Vitner Asher
Peselev Elli
Williams Morgan & Amerson P.C.
Yissum Research Development Company of the Hebrew University of
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