JPS6036753B2 - Production method of kojic acid - Google Patents

Description

[Detailed description of the invention] The present invention relates to a novel method for producing kojic acid.

More specifically, the present invention relates to a method for producing kojic acid that can improve and stabilize the production yield of kojic acid using a kojic acid fermentation method and also shorten the culture time. The present applicant first discovered the remarkable fact that kojic acid has excellent whitening and sun protection effects, and filed a patent application for a skin-lightening cosmetic containing kojic acid as an active ingredient.

Such kojic acid is usually produced by culturing strains capable of producing kojic acid such as Asbergillus, Benicillium, Escalichia, Acetobacter, and Glyconobacter in a liquid medium using carbohydrates such as glucose as a carbon source. It is produced by so-called kojic acid fermentation.

However, in the production of kojic acid by such kojic acid fermentation, the culture period is extremely long, 7 to 10 days, and the yield of kojic acid obtained is 40 to 55% (wt%) based on sugar. The production method has many drawbacks, such as the inability to obtain kojic acid with a low and stable yield.

However, in order to eliminate the above-mentioned drawbacks, the present inventor investigated in detail the relationship between the transition of carbohydrates in conventional kojic acid fermentation, the temporal change in generated carbon dioxide, the amount of bacterial production, and the amount of kojic acid produced. As a result of extensive research, we have developed a fermentation method that produces kojic acid by culturing a strain of Asbergillus that has the ability to produce kojic acid in a medium that uses carbohydrates as a carbon source. When adding carbohydrates to the culture medium, the above-mentioned disadvantages can be eliminated, and kojic acid can be stably produced in high yield, and the cultivation time can be shortened. After discovering this fact, we have completed the present invention.

According to the method of the present invention, in conventional culture methods,
During a 17-day incubation period, kojic acid decreased to about 4% per sugar.
While kojic acid is produced at a yield of 0 to 55%, it has the remarkable effect that kojic acid can be stably produced at a high yield of about 60 to 75% based on sugar after culturing for about 5 days. sell.

In the present invention, the amount of bran added to the medium during culturing is 4 to 1 per time relative to the bran in the medium.
Preferably it is 0%. Moreover, it is preferable to add it at about 45 to 5 insect hours and about 70 to 8 feeding hours after the start of culture, but it is also preferable to add it at appropriate time intervals.
The addition may be continued sequentially for the 4th time and the 4th time. However, in the present invention, the production of cocoons is almost completed around the time of the first addition, and after that, the enzyme for producing kojic acid acts directly on the carbon source in the culture solution to produce cocoons. By adding the first time during the period when the enzyme is directly acting, the action of the enzyme is dramatically increased and the amount of kojic acid produced per sugar is increased. It is assumed that.

This increase in enzymatic action is prevented from quelling by the second addition, increasing the amount of kojic acid produced. However, in the present invention, when the amount of carbohydrates added per time is smaller than the above range, the increase in the amount of kojic acid produced is smaller than expected, and when it is larger than the above range, there are problems such as osmotic pressure. There is a tendency that should be taken into account.

Further, it is preferable to add the healthy substances in the form of an aqueous solution in order to easily and aseptically add them, but they may also be added in the form of granules or powder. The concentration of carbohydrates when adding bran in the form of an aqueous solution is appropriately determined from a range of about 4 to 10%. When the first addition of bran is done earlier than the above range, the bran is used for carbon dioxide gas production and bacterial cell production, and when it is added later than the above range, , kojic acid production will be delayed, both of which are unfavorable.

It is also unfavorable for the same machine if the second addition is made earlier or later than the above range. Bacterial strains belonging to the genus Asbergillus and capable of producing kojic acid used in the present invention include, for example, Asbergillus albus, Asbergillus candidus, Asbergillus oryzae, and Asbergillus oryzae.
Nide 1 Jurans, Asbelgillus parasiticus,
Asbergillus awamori, Asbergillus tamari,
Bacterial strains such as Asbergillus nipius, Asbergillus furanos, Asbergillus wench, Asbergillus glaucus, Asbergillus clavaitus, Asbergillus fumigatus, Asbergillus digitus, and the like can be suitably employed.

The beach composition of these strains is usually sucrose, fructose,
Approximately 5-15% carbon sources such as glucose, starch, maltose, glycerin, mannitrate, rhamnose, xylose, glyconic acid, arabinose, dihydroxyacetone, inosyte, lactose, ethanol, ammonia sulfate, veptone, sodium nitrate, baker's yeast extract , nitrogen sources such as brewer's yeast extract are approximately 0.5-1.0%, and magnesium sources such as magnesium sulfate are approximately 0.02-0.07%.
%, 0.1 to 0.3% of phosphorus and potassium sources such as potassium monohydrogen phosphate and potassium dihydrogen phosphate, and other sources such as ferric sulfate, ferric chloride, sodium chloride, calcium chloride, etc. A material containing about 0.001 to 0.005% of inorganic salt may be employed.

Furthermore, surfactants such as polyoxyethylene alkyl ether and sucrose ester are added to the base composition at a concentration of about 0.05%.
The medium according to the present invention can be obtained by adding ~0.2% and adjusting the pH to 3 to 4. Next, the method of the present invention will be explained with reference to Examples and Comparative Examples.

Example 1 Asbergil albus No. 6 (obtained from Utsunomiya University, easily available as ATCC44054 beads).

) was used to inoculate 0.2% beptone onto a potato dextrose-agar plate (manufactured by Eiken Kagaku Co., Ltd.) on a slope plate, cultured on a slope at 30°C for one week, and then cultured on a slope plate at 30°C for one week. Prepared by suspending spores. As a seed mother base, glucose 8
%, Bebutone 0.5%, potassium dihydrogen phosphate 0.15
%, magnesium sulfate heptahydrate 0.05% and surfactant Nikkor BC-1 X (trade name manufactured by Nikko Chemicals Co., Ltd.) 0.1% in the form of an aqueous solution adjusted to pH 3. , 200 of these [10 in a flat flask
00', sterilized for 15 minutes at 12100, cooled to room temperature, and then inoculated with the seed culture.

The culture was carried out in a shaking moat for 48 hours at a temperature of 3 mm, a shaking frequency of 120 times/min, and an amplitude of 5 arcs, and a seed mother was prepared. Main culture medium: glucose 9%, veptone 0.8%, potassium dihydrogen phosphate 0.15%, magnesium sulfate heptahydrate 0.05
% and Nikkor 8C-lmX (previous life) 0.1% aqueous solution adjusted to pH 3.5.
Jar fermenters (manufactured by Marubishi Rikagaku Kenkyusho Co., Ltd.) each containing 20 lotuses were filled into each tank, sterilized at 12100 for 15 minutes, cooled to room temperature, and then inoculated with the seed mother.

Culture was carried out at 32°C, aeration rate 2 sails 1/min, and rotation speed 350.
~40 ratio pm, and at 5 and 7 hours after the start of culture.
1.5% glucose aqueous solution at each hour.
5 pieces were added to each tank and cultured for 12 feeding hours. The volume of the medium after completion of the culture was 21 volumes. Comparative Example Culture was carried out for 16 hours in the same manner as in Example 1, except that the glucose concentration in the culture medium was 9.75%.

Glucose concentration, kojic acid concentration, dry bacterial cell amount, and p of soil in each culture solution in Example 1 and Comparative Example 1.
The following table shows the results of examining changes in H over time at 24 hour intervals.

The glucose concentration in the culture solution was determined by quantifying the amount of glucose using the Bertrand method and subtracting the reducing power of kojic acid from this amount.

Moreover, the kojic acid concentration was determined by measuring the color development of ferric chloride caused by kojic acid by measuring the absorbance at a 50M sail using a spectrophotometer. The dry amount of bacterial cells was determined by collecting the bacterial cells in the culture solution LOOO using quantitative oven paper, drying them at 105° C. to a constant weight, and then weighing them. pH' was measured using an M-5 pH meter manufactured by Horiba, Ltd. The graph of FIG. 1 shows the changes in glucose concentration, kojic acid concentration, dry cell mass, and pH in Example 1, and the graph in FIG. 2 shows the changes over time in Comparative Example 1. From Figures 1 and 2, it can be seen that the change in kojic acid production over time in Example 1 (Figure 1) is significantly larger than the change in kojic acid production over time in Comparative Example 1 (Figure 2). Moreover, it can be seen that the change hardly corresponds to the change in the amount of microbial cells produced or the pH, and that the change rapidly increases after the first and second additions of the aqueous glucose solution. Furthermore, the change in the concentration of glucose in the culture solution was virtually the same as that in Comparative Example 1 despite the addition of the aqueous glucose solution twice each time, indicating that the added glucose was efficiently converted to kojic acid. I understand. The yield of kojic acid based on sugar in Example 1 was 70%, and the yield of kojic acid based on sugar in Comparative Example 1 was 54%.

Example 2 As the inoculum, Asbergillus candidus M old 203 (
Inoculum culture and seed mother culture were carried out in the same manner as in Example 1 using a mutant strain in which monospores were isolated from IF06215) and the kojic acid production ability was improved by ultraviolet irradiation.

As a medium for main culture, glucose 7%, beptone 0.6%
, 0.15% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate heptahydrate, and 0.1% of Nikkor BC-1OTX (mentioned above) were used after adjusting the pH to 3.5, and the above-mentioned seed mother was used. was inoculated, and culture was started under the same conditions as in Example 1.

20 each at the 5th field hour and 7th field hour after the start of cultivation.
1.5% glucose aqueous solution was added and cultured for 12 hours. The volume of the culture solution after completion of the culture was 21 volumes. The concentration of kojic acid in the culture solution was 5.86%, and the yield of kojic acid based on sugar was 50%.

Comparative example: Glucose concentration in the medium of double culture was 8.5
%, and 12-feed culture was carried out in the same manner as in Example 2, except that no glucose aqueous solution was added during the culture.

The concentration of kojic acid in the culture solution after completion of the culture was 4.7%, and the yield of kojic acid based on sugar was 50%.

[Brief explanation of the drawing]

Figures 1 and 2 are Example 1 and Comparative Example 1, respectively.
It is a graph showing changes over time in glucose concentration, kojic acid concentration, dry cell mass, and pll in each kojic acid fermentation. 1 figure, 2 figures

Claims (1)

[Scope of Claims] 1. A fermentation method in which a strain belonging to the genus Aspergillus and capable of producing kojic acid is cultured in a medium using carbohydrates as a carbon source to produce kojic acid. A method for producing kojic acid, which comprises adding it to a culture medium. 2. The method according to claim 1, wherein the carbohydrate is glucose or sucrose. 3. The method according to claim 1 or 2, wherein the amount of carbohydrate added per time is 4 to 10% by weight based on the carbohydrate in the medium. 4. The method according to claim 1, 2, or 3, characterized in that the addition of carbohydrates is carried out about 45 to 55 hours and about 70 to 80 hours after the start of culture.