CS252880B1 - A method of preparing kojic acid - Google Patents

Abstract

The present invention relates to a process for the preparation of kojic acid by transformation of carbohydrates with the aid of Aspergillus tarnaria spp. The nature of the prodrug of kojic acid consists in cultivating the Aspergillus tamarii mutant VIII (CMC F-781) or Aspergillus tamarii mutant III (CCM F-780) individually on a liquid nutrient ipod containing assimilable carbon, nitrogen and nutrient salts. In a mixture at 20 to 40 ° C, pH 1.5 to 7.0 for 72 to 240 hours, whereby the kojic acid is isolated by concentration of the ultraviolet medium followed by crystallization. The solution has a use in a fermenter for kojic acid production.

Description

(54) Method for preparing kojic acid

The present invention relates to a process for the preparation of cubic acid by the transformation of carbohydrates by means of breeding strains of Aspergillus tarnarii. The essence of the production of kojic acid consists in culturing an aspergillus tamarii mutant VIII (CMC F-781) or Aspergillus tamarii mutant III (CCM F-780) strain individually on a liquid nutrient ip containing assimilable carbon, nitrogen and nutrient salts, or In a mixture at a temperature of 20 to 40 ° C, a pH of 1.5 to 7.0 for 72 to 240 hours, wherein the kojic acid is isolated by concentrating the ultraviolet medium followed by crystallization. The solution has a use in fermentation liquor for the production of kojic acid.

The present invention relates to a process for the preparation of kojic acid by the transformation of glyeids by means of breeding strains of Aspergilluis tamarii.

Several microscopic filamentous fungi, in particular from the genus Aspergillus, such as A. flavus, A. oryzae, A. parasiticus, A. nidulans, A. candidus, A. awamoiri, A. clavatuis, A. fumigatus, A giganteus, A. chevalier, A. faciculatus, A. rugulosus [Korzybski, T., Kowszyk-Gindifer, Z., Antibiotics, PWN - Polish Scientific Puhlishers, Warszawa (1967)]. The above-mentioned disadvantages are eliminated by the method of production of kojic acid on teicite nutrient medium containing assimilable carbonate, duisil and nutrient salts, which consists in that Aspergillus tamarii mutant VIII (CCM F-781) or Aspergillus tamarii mutant III (CUM). F-780) is cultured singly or in a mixture, either surface or submerged, where glucose, fructose, sucrose, maltose, galactose, xylose, arabinose is used as the sugar source in the medium. day of these sugars, or a mixture thereof, naturally containing substrates containing these sugars, wherein the filtration ¹ is carried out at a temperature of 20 to 40 ° C, preferably at 30 ^° C, at a pH of 1.5 to 7, preferably at the beginning of 4 , 5 for submerz and 6.5 for the surface, a relative humidity for the surface of 55% to 95%, preferably 65% for 72 to 240 hours, wherein the icoic acid is isolated by concentration of ultraviolet medium followed by crystallization, optionally by extraction with ethyl acetate, or precipitation with a solution of copper sulfate, optionally by recrystallization from a decationized solution. An advantage of the process for the preparation of kojic acid according to the invention is that on a simple nutrient medium with an assimilable source of carbon and nutrient salts, it is possible by surface or submerged fermentation to obtain kojic acid in yields of up to 35 g per 100 g of culk.

Example 1

Prepare 5.3 liters of 4.5% sucrose, 4.5% glucose, 0.2% ammonium nitrate, 0.1% potassium dihydrophosphate, 0.05% magnesium sulfate heptahydrate, 0.05% potassium chloride, 0.001 percent ferrous sulfate heptahydrate, 2% corn extract with dry matter 50%. The pH of the solution was adjusted to 6.5. It is sterilized for 30 minutes at 115 ° C and poured five hot sterile aluminum pots with a diameter of 150 mm and a height of 180 mm while hot. After cooling the medium to 45 ° C, the conidiospores of Aspergillus tamarii mutant VIII in suspension with activated carbon are sprayed onto the surface of the medium at 2 x 10 ⁷ spores per dm ² of medium area. The pots are placed in a fermentation chamber in which the temperature and humidity are controlled. The temperature in the fermentation chamber drops to ° C over 12 hours and to 30 ° C over the next 12 hours. This temperature is then maintained until the end. The relative humidity at the beginning is 90 ° / o, after 24 hours after felling 70%. This moisture is maintained until cultivation is imminent. A continuous wrinkled layer of mycelia of 20 g dry matter is formed on the surface of the medium during 10 days of culture. 1 ^_1 . The concentration of formed kojic acid in the medium reached 30 g. to ^{1 of the} original volume of the medium, ie 33% of the fermentation yield. Example 2

100 ml of a medium of the composition as in Example 1 is prepared. Dissolve 50 ml into two 250 ml Erlenmeyer cells and inoculate a 0.5 ml suspension of conidium spores of Aspergillus tamarii mutant VIII in water of 10 ⁷ spores per ml. The flasks were placed on a rotary shaker with an oscillation rate of 210 rpm with an eccentricity of 8 cm and cultured for 48 hours at 32 ° C. After this time, a vegetatively inoculum is formed, which is a suspension of dowels agglomerated into spherical clusters with a diameter of 1.2-2 mra. 2.4 liters of medium containing 10% glucose, 0.1% ammonium chloride, 0.05% magnesium sulfate heptahydrate, 0.016% potassium dihydrogen phosphate, 0.007% potassium chloride, 0.002% manganese sulfate tetrahydrate, 0.001% were added. zinc sulfate heptahydrate, 0.001% ferrous sulfate heptahydrate. Sterilize at 115 ^° C for 30 minutes. After sterilization and cooling of the medium to 30 ° C, a sterile urea solution is added in a final 0.1 ° / o compression. The medium is transferred to a sterile Tabor, an atomic fermenter with a total volume of 5 liters with mechanical stirring. After aerating the medium, the contents of both vegetative inoculum flasks prepared as described above are sterile transferred and cultured at 30 ° C. The aeration rate is 0.05 to 1.0 units of air per unit of volume per minute at the beginning of 0.1 and at the end of 0.5 units of air per unit of volume per minute. The peripheral stirring speed is 3.4 m. s ^_1 .

The laminar velocity of the air flow is between 0.4 and 2.8 m. ¹ relative to the fermeintere cross-sectional unit area under turbulent mixing. Maximum production of kojic acid is achieved on day 6 of the fermentation, with a concentration of 33 g per liter of medium.

Example 3

The procedure of Example 2 is followed except that the inoculum is prepared from the coniospore producing Aspergillus tamarii mutant III. Maximum production of kojic acid was achieved on day 9 of feeder, at a concentration of

19.4 g per liter medium.

Example 1 a d 4

The procedure is as in the previous example, except that sucrose is used instead of glucose. The maximum prodrug of the -canoic acid was reached in

6.5 days and its endpoint was 37 g in liter of medium.

The importance of the invention lies in the fact that it is economically possible to obtain kojic acid which, due to its antioxidant and chelating properties as well as the biological activity properties, can be used widely in the food, pharmaceutical and chemical industries, having bearing in mind that phoic acid is not mutagenic to eupcaryotic cells [-Shibuya T. et al., J. Toxicol. 7, 4, 255-262 (1982)] and is degradable by naturally occurring microbial associations.

Claims (1)

252880 EXAMPLE 4 The same procedure as in the previous example was followed except that the glucose was used as the glucose. Maximum yield of kojic acid was reached in 6.5 days, and its endpoint was 37 g per liter of medium. The significance of the invention is that it is possible to obtain comic acid in an economical way, which, due to its amioxidative and chelating properties, as well as the biological efficacy properties, can find widespread applications in the food, pharmaceutical and chemical industries, Considering that kojic acid is not mutagenic for eufacryotic cells [Shibuya T. et al., J. Toxi-col. Sci., 7, 4, 255-262 (1982)] and is naturally degradable in the case of incipient microorganisms. SUBJECT A method of preparing kojic acid with a source of a susceptible carbon monoxide, nitrogen and nutrient salts, characterized in that Aspergillus tamarii rnutantVIII (CCM F-781) or Aspergillus tamariirnutant III (CCM F-780) is superficially and extensively treated. cultured individually, or in a mixture, where glucose, fructose, sucrose, maltose, galaclactose, mannose, xylose, arabinose, optionally these sugars, or natural substrates containing these sugars are used as the sugar source in the medium, the duties are carried out at a temperature of 20 to 40 ° C, preferably at 30 ° C, at a pH of 1.5 to 7.0, preferably at the beginning of 4.5 for the subframe and 6.5 for the relative humidity of 55 to 95 %, Preferably 70% for a surface of 72 to about 240 hours, wherein the aeration intensity at the submerge is 0.05 to 1.0 air volume per minute, preferably at 0.1 and at a at the end of 0.5 units of volume air per unit volume of medium per The circumferential velocity of the agitator is in the range of 1.0 to 5.0 m. s -1, preferably 3.4 m. s -1, wherein the laminar velocity of the air relative to the unit flat cross section of the fermenter during turbulent mixing is within the limits of 0.4 to 2.8 ms-1, wherein the kojic acid is isolated by concentration of the ultraviolet medium followed by crystallization, optionally by extraction with ethyl acetate, or by precipitation of the copper sulfate solution, optionally by recrystallization from a decationized solution.