S. HIROSHIMENSIS STRAIN WITH ANTI-TYROSINASE ACTIVITY 35 (Merck) and heated at 100°C for five minutes. The spots of A2pm appeared in a yellowish green color. The sugar content of the cells was analyzed by the same procedure as that of A2pm, but the hydrolysis and developing were carried out by using 2 N trifl.uoroacetic acid and n-butanol-distilled water-pyridine-toluene (10:6:6: 1, v/v) as solvents, respectively. The spraying reagent was acid aniline phthalate. The standard sugar solution contained 1 % (w/v) of each galactose, glucose, mannose, arabinose, xylose, and ribose. The partial gyrB gene fragment was amplified by polymerase chain reaction (PCR) using the primers: PFl (forward: 5 '-GAGGTCGTGCTGACCGTGCTGCACGCGGGCGG­ CAAGTTCGGC-3') and PR2 (reverse: 5'-GTTGATGTGCTGGCCGTCGACGTCGG­ CGTCCGCCAT-3'). Genomic DNA was extracted from seven-day cultures using the Qiagen® Genimic DNA Kit. The PCR procedure for amplifying the gyr B sequence was the same as that described by Hatano et al. (12). The amplified product was analyzed in a genetic analyzer (ABI Prism 31 O PE Applied Biosystems, USA) according to the manufacturer's protocol. The gyr B sequence of the strain TI-C3 was aligned manually against the nucleotide sequences of other whorl-forming Streptomyces strains retrieved from GenBank databases. DNA-DNA hybridization was performed by the method of Ezaki et al. (13). The experiment was performed at least five times, and the level of DNA-DNA hybridization was expressed as the mean percent of the homologous DNA binding value. FERMENTATION OF THE STRAIN TI-C3 A seed culture of the strain TI-C3 was initiated by adding 1.5 ml of a thawed spore suspension (about 108 spores per ml) into a 250-ml baffled Erlenmeyer fl.ask containing 25 ml of YMG medium. The compositions of YMG medium are 4 g yeast extract, 10 g malt extract, and 4 g glucose in one liter of distilled water. The pH of the medium was adjusted to pH 7 .2 prior to autoclaving. The fl.ask was then incubated at 30°C for two days at 180 rpm, and 2.5 ml of the seed culture was transferred into a 250-ml baffled Erlenmeyer fl.ask containing 25 ml of modified YMG medium for a secondary cultivation. For fermentation using different carbon sources, glucose in the YMG me­ dium was replaced by the desired carbon source. For fermentation using different ni­ trogen sources, both yeast extract and malt extract in the YMG medium were replaced by the desired nitrogen source. All cultivations were carried out at 30°C for 72 hours at 180 rpm. After fermentation, an equal volume of ethanol was added to each cultivation and shaken vigorously for 30 minutes at room temperature. The cell debris was removed by centrifugation at 4800 rpm. The supernatant from the extracted broth was assayed to measure the anti-tyrosinase activity. The experiments were carried out in triplicate and the mean values are shown. ANTI-TYROSINASE ACTIVITY ASSAY The modified assay method of anti-tyrosinase activity described in the literature was employed in our study (14). Each supernatant sample from the fermentation experiments was serially diluted by a reaction buffer before the assays of anti-tyrosinase activity. Then, 20 µl of each of the diluted samples was mixed with 80 µl of 0.2 mM 1-tyrosine

36 JOURNAL OF COSMETIC SCIENCE dissolved in 50 mM of potassium phosphate buffer (pH 6.8). Then, 20 µl of mush­ room tyrosinase (1000 U ml -1, dissolved in the same buffer) was added to each well to initiate the reaction. The assay mixture was incubated at 25°C for 30 minutes. The increase in absorbance at 475 nm due to the formation of dopachrome was moni­ tored in a microplate reader (microplate reader 2010, Anthos Inc., Salzburg, Austria). The percent inhibition of tyrosinase activity was calculated as follows: % Inhibition = [(A-B)/A} x 100, where A is the absorbance at 475 nm with reaction buffer instead of the tested sample and Bis the absorbance at 475 nm with the tested sample. The volume of a sample at which 50% of the enzyme activity was inhibited was obtained by linear curve fitting. One unit of inhibitory activity was defined as the amount of the sample used in the assay condition by which the enzyme activity was reduced to 50%. The anti-tyrosinase activity was expressed as the amount units in one microliter of broth samples (U/ml). RESULTS AND DISCUSSION IDENTIFICATION OF THE STRAIN TI-C3 The screening method used was adopted from our previous work (14) without any modification. Using this method, we screened nearly two thousand isolated strains in a 25 20 ■ i 1 5 � 10 u 5 0 0 18 26 30 42 54 Time (hour) 700 600 ◄ 500 400 :E 300 c 200 100 0 78 102 Figure 1. Cell growth(■) and anti-tyrosinase activity (.A) of TI-C3 culture grown in shake flasks at 30°C and 180 rpm. The media used was YMG and its compositions were as described in Materials and Methods. Samples for biomass and anti-tyrosinase activity assay were collected at predetermined time intervals. Biomass was measured gravimetrically as dry cell weight (DCW) by filtering the sample on a pre-weighed filter paper and drying at 70 ° C until constant weight was attained. The assay of anti-tyrosinase activity was done as described in Materials and Methods.