CS276815B6 - A method of producing kojic acid by submerged fermentation - Google Patents

Abstract

The method of producing kojic acid by submerged fermentation solves the problem of regulating the level of dissolved oxygen in the medium. The essence of the solution lies in maintaining a constant level of dissolved oxygen in the medium at a value of 20 to 60% saturation, preferably 35 to 40% saturation, within 20 to 60 hours of production fermentation.

Description

The invention relates to a process for the production of kojic acid by submerged fermentation.

Kojic acid is produced in small amounts by several species of microscopic fibrous fungi. in particular the genera Aspergillus and Penicillium and bacteria of the genus Gluconobacter. Production in larger quantities, which is economically interesting for the preparation of an intermediate product for barbecue industries, is solved by several procedures dealing with the preparation of high-production mutants of the author's certificate (No. 251 532, 251 533) and using a suitable technological procedure (252 880, 252 881). The production of kojic acid according to these processes favors a submerged fermentation process, both in terms of yield and the possibility of controlling the whole process. In sterile fermenters, the batch is mixed mechanically by means of rotating stirrers and aerated with dispersed sterile air, whereby intensive metabolism takes place. ·

There is an effort to optimize the process in the direction of maximum conversion of the carbonaceous substrate to kojic acid in the shortest possible time.

The disadvantage of the prior art is the low and slow conversion of the carbohydrate source to the product. Non-optimized aeration mode in relation to production! has a negative impact on the overall economy of the process.

These disadvantages are eliminated by the process of the invention, which consists in speeding up the whole process and increasing the production of kojic acid by maintaining the level of dissolved oxygen in the fermentation medium between 20 and 60 hours of cultivation at 20 to 40% saturation. The production microorganism grows in a submerged culture, with the pO ₂ level falling to 5 to 15% saturation during the exponential growth phase, which may cause growth retardation but especially a decrease in the activity of the complex of enzymes synthesizing kojic acid. The advantage of the process according to the invention is the fact that the activity of the enzymes of the competitive metabolic pathway - glycolysis is suppressed by a _{simple regulation of the process parameter (pO 2).} Kojic acid biosynthesis is stimulated; an earlier start of production and its higher speed compared to an unregulated process increases the economics of technology.

The possibilities of using the present invention are described by the following examples:.

Example 1

Aspergillus tamarii strain CCM 781 maintained on wort agar was used to prepare the inoculum. Spores, at a concentration of 7.10 'spores per liter, are inoculated with 3 l of inoculation medium of the following composition: 14% glucose, 0.25% NH ₄ NO ₃ 0.025% MgSO ₄ 7 H ₂ 0, 0.1% starch, 0.1% KH ₂ PO ₄ , 0.0001% ZnSO _4> 7 H ₂ O, 0.0002% CuSO ₄ 5 H ₂ O, 0.00065% FeCl 3, pH of the medium 5.5. '*

Stirrer speed 300 to 500 rpm, supply air volume 1.5 l per minute, temperature 29 to 31 ° C, cultivation time 40 h. The vegetatively inoculum thus prepared is used in an amount of 5 to 10% by volume to inoculate 12 l of sterile fermentation broth.

CS 276815 B6 2

A 'media of the following composition: 10% glucose, 0.1% NH ₄ Cl, 0.05% MgSO _4> 7 H ₂ O, 0.016% KH ₂ PO ₄ 0.007% KCl, 0.002% MnSO ₄ , 7 H ₂ 0, 0.001 *% ZnSO ₄ .7 H ₂ O, 0.0005% FeSO ₄ . At 20 hours of culturing, 0.1% of urea is added. *

The actual fermentation takes place in a 20 l glass fermenter at a temperature of 30 ° C. The culture is mixed and aerated: speed: stirrers 500 rpm, aeration 12 l per minute. The maximum production was reached in 168, when the concentration reached 28 g of kojic acid per liter.

Example 2, Preparation of inoculum and fermentation medium as in Example 1. The cultivation is carried out as in Example 1, except that the dissolved oxygen level after a natural decrease to 35% saturation is automatically maintained at this value by regulating the stirrer speed in the range of 350 up to 800 rpm. and subsequently increasing the degree of aeration from 2 to 12 l per minute. The maximum concentration of kojic acid in the medium reached 36 g. 1 ^-1 for 139 hours of cultivation.

Example 3

Composition of the inoculation and fermentation medium as in Example 1. The inoculum is cultured in a 20 l glass fermenter with 12 l of medium under the same conditions as in Example 1. The production fermentation takes place in an 80 l fermenter with 70 l of medium as in Example 1, except that the dissolved oxygen level is kept constant at 40% saturation. In 129 hours, the maximum production reached 46 g of kojic acid per liter of medium. The increase in production is 15% compared to the control experiment, shortening the process by 35 hours.

Claims (3)

FOLLOW - UP A process for the production of kojic acid by submerged fermentation, characterized in that the level of dissolved oxygen is maintained at a constant level of 40% saturation between 20 and 60 hours of cultivation. End of document