Isolation of Kojic Acid Producing Mold using Complex Carbon Sources

Introduction: An independent effort in term of pharmaceutical raw materials procurement in Indonesia, especially skin brightening agent, is required due to the high demand of brightening skin care product. One of the skin brightening agent widely used in cosmetic skin care formulations is kojic acid. This study aimed to obtain the isolate of kojic acid producing mold from nature and its optimum fermentation condition by using various complex carbon substrates. Methods: Aspergillus oryzae was used as reference. The isolates of fungi were screened with different substrates variation, namely sucrose, corn starch, cassava starch, and cellulose hydrolysate. Then, each of culture was dripped with FeCl3 1% and the most brownish-red color formed was selected as mold and media for further process. The preculture of selected isolate and A. oryzae, were inoculated into 100 ml of fermentation media respectively and incubated at room temperature, 180 RPM for 10 days. The concentration of substrate was varied to 5, 7.5, and 10%. The levels of kojic acid were determined by TLC densitometry with UV detector at 318 nm. Results: IHJ2K isolate in corn starch-yeast extract was selected as the best mold and media. However, the highest level of kojic acid was produced by Aspergillus oryzae with 10% of substrate, with kojic acid concentration of 5.22 g/L. The most efficient fermentation was obtained from A. oryzae with 7.5% of substrate, with the obtained yield of 0.53 g/g. Conclusion: Potential kojic acid producing mold namely IHJ2K was successfully isolated. The selected carbon source for optimum kojic acid fermentation condition with IHJ2K isolate was corn starch combined with yeast extract, KH2PO4, and Mg2SO4.7H2O.

compared to glucose as the carbon source was relatively similar. 9he aim of this study was to explore further the use of complex carbon such as corn starch, cassava starch, and cellulose hydrolysate as a substrate on kojic acid fermentation.In addition, the isolation of kojic acid producing mold using starch, offers the advantage of reducing the cost of raw materials for the production of kojic acid.

Screening Media
The media consisted of 5% variation of carbon sources (sucrose, corn starch, cassava starch, and cellulose hydrolysate, 10 0.5% variation of nitrogen sources (yeast extract, urea, ammonium sulfate), 0.1% KH 2 PO 4 and 0.05% MgSO 4 .7H 2 O.The media was prepared in twelve variations.The pH of media solution was adjusted to 5 with orthophosphoric acid.

Isolation of Molds
Molds were isolated from soil and rotten wood.The surface of the soil was cleaned from waste and dirt, then 100 mg of the moist layer beneath was put into the vial.A 100 mg of rotten wood were crushed in a sterile mortar.A hundred milligram of either the soil or the wood was diluted in 10 ml of aquabidest.The soil and wood suspension were diluted until 10 -9 of suspension was obtained.Both of the suspension were scratched onto potato sucrose agar (PSA) media in a petri dish and incubated at 28⁰C for 5-7 days.Mold colonies grew were isolated based on their morphology.The colony was taken with inoculating loop and scratched onto the new PSA.The new PSA contained colonies was then incubated at 28⁰C for 5-7 days.The ideal colony obtained was transferred to the culture stock tube.The procedure was repeated if the colony obtained had not met the criteria 11 The purified colonies were transferred to a test tube containing the tilted potato dextrose agar (PDA) media and stored as a culture stock for further test preparation. 12

Screening of Superior Mold and Media Combination
The mold colonies seeded on PDA were cultured into 96-wellplates containing a 200 μL of screening media, incubated for 6 days at 28⁰C.A total of 130 μL of supernatant was placed on a drop plate.The screening was done by dripping fresh FeCl 3 1%.The brownish red color formed was indicated a positive result and the most intense color was selected as the superior mold and media combination. 13croscopic and Microscopic Identification Macroscopic identification was done by observing the colony color, colony diameter, and colony texture.Microscopic identification was performed by examination and observation of mold preparations under a light microscope.Both macroscopic and microscopic observation were compared with Aspergillus oryzae (IPB Culture Collection).

Kojic Acid Fermentation
The pre-culture of the selected isolate was prepared with 50 mL of pre-culture media (superior media) in a 100 mL shake flask.Incubation was carried out by shaking at 180 rpm, 28°C for 48 h.Preparation of inoculum was also performed on Aspergillus oryzae.Fermentation was done with 100 mL media and 10% (v/v) inoculum in 250 mL shake flask.The level of carbon source was varied to 5, 7.5, and 10%.The flasks were incubated by rotary shaker at 180 rpm, 28⁰C for 10 days.At the same time, fermentation of Aspergillus oryzae was also performed.

Analytical Methods
The presence of kojic acid in the culture filtrate was determined using UV-Vis spectrophotometry and colorimetric method, and analyzed quantitatively using TLC-Densitometer (CAMAG III, Switzerland), using silica gel F 254 as stationary phase, toluene-ethyl acetate-formic acid (3:6:1) as mobile phase, and UV detector at 318 nm.The calibration curve was made using kojic acid standard solution with a range of 20-80 ppm.
Glucose concentration was determined using DNS method. 14Biomass was determined by the dry cell weight method.Culture samples were pipetted into a pre-weighed tubes and centrifuged at 7500 RPM for 15 min.Supernatant was used for kojic acid analysis, while biomass was washed with distilled water, centrifuged, and dried at 105⁰C to a constant weight.

Isolation of Molds
Eight colonies were obtained based on the morphology.There were two black-spores colonies surrounded by white mycelia named IHIT (Figure 1.a) and IHIK (Figure 1.b), which secreted yellow pigment underneath.A green colony surrounded by a white mycelia was isolated, which was IHJT (Figure 1.c).A different green colony that secreted yellow pigment underneath was also isolated and was named IHJ1K (Figure 1.d).There was also a yellowish-green colony surrounded by white mycelia named IHJ2K (Figure 1.e), a yellow-green colony isolated from wood, named IHJKK (Figure 1.f), a brownish-green that was isolated from wood called ICK (Figure 1.g), and a brown colony isolated from soil which was named ICT (Figure 1.h).

Screening of Superior Mold and Media Combination
The IHJ2K (Figure 2.e) and IHJKK (Figure 2.f) were the types of mold which produced an intense brownish color.Re-screening was performed on IHJ2K and IHJKK fermentation after two days.The results showed that IHJ2K in media containing 5% corn starch, 0.5% yeast extract, 0.1% KH 2 PO 4 , and Mg 2 SO 4 .7H 2 O 0.05% yielded the most intense brownishred color (Figure 3.a).The result showed that IHJ2K isolate contained amylase, glucoamylase, and other hydrolase enzymes that play a role in the metabolism of starch.Thus, IHJ2K could hydrolyze amylose and amylopectin into glucose, a precursor compound in the kojic acid biosynthetic pathway. 15This suggested that IHJ2K was able to produce kojic acid on complex carbon sources in a short time.Corn starch was selected as a superior carbon source due to its low percentage of amylopectin compared to cassava starch. 16The degree of polymerization (DP) of amylopectin ranged from 10 5 to 3x10 6 units of glucose.The more units required to be hydrolyzed on cassava starch the longer time required for kojic acid productivity.Meanwhile, β-cellulose had lower kojic acid productivity than corn starch due to the presence of cellulose which had not been fully hydrolyzed to glucose.Yeast extract wasalso selected considering its complex structure compared to urea and ammonium sulfate to provide a richer nutritional intake.

Macroscopic and Microscopic Identification
The macroscopic identification results showed that the growth of IHJ2K on PSA media with temperature of 28⁰C grew to 8 cm in diameter in 7 days.The colony had a yellowish green color surrounded by white mycelium and velvety texture (Table 1).IHJ2K had the same spores as Aspergillus oryzae.Aspergillus had a type of asexual spore called conidia. 17

Kojic Acid Fermentation
The UV spectrophotometric absorption spectra of the kojic acid standard and culture sample (IHJ2K in 10% corn starch) indicated the same maximum wavelength, which was 268.8 nm.The spectrocolorymetry absorption spectra of kojic acid standard and culture sample (IHJ2K in 10% corn starch) also showed the same maximum wavelength at 503,6 nm.This result showed that the IHJ2K culture contained kojic acid.The biomass produced by IHJ2K was higher compared to A. oryzae.The higher substrate concentration, the higher biomass produced by IHJ2K which could be seen in a pattern (Table 2).Meanwhile, the escalation of A. oryzae's biomass was not as patterned as IHJ2K's.Too much sugar in A. oryzae fermentation enhanced water to escape from biomass cells because of the difference in osmotic pressure between the environment outside the cell and the fluid inside the cell.Thus, the condition affected the cell to be dehydrated resulted in the inhibition of the cell growth.The differences in environmental osmotic pressure outside the could also cause plasmolysis and microorganisms cells death. 18It could be concluded that the growth of IHJ2K on the corn starch complex substrate was better than A. oryzae.The highest kojic acid level was produced by A. oryzae with 10% (w/v) of corn starch (Table 2).However, the highest yield was obtained by A. oryzae with 7.5% of corn starch.The dramatic increase in viscosity of the high concentration of starch could decrease the rate of oxygen transfer   to culture 9 leaded to the A. oryzae fermentation in 10% of corn starch was less efficient.In addition, the more level of corn starch substrate used by IHJ2K, the more the level of kojic acid was produced.
Overall, the yields obtained by IHJ2K fermentation were less than by Aspergillus oryzae.Although the IHJ2K isolate showed an increase in kojic acid concentration and its yield by increasing the substrate concentration, but it had not been able to outperform the efficiency of kojic acid productivity resulted by Aspergillus oryzae.

CONCLUSION
In conclusion, kojic acid producing mold called IHJ2K was successfully isolated.The carbon source for optimum kojic acid fermentation condition with IHJ2K isolate was corn starch combined with yeast extract, KH 2 PO 4 , and Mg 2 SO 4 .7H 2 O.

Figure 1 :
Figure 1: Mold Isolates based on their morphology.