Abstract
L-Ascorbic acid is an excellent anti-oxidant, which is widely used in a number of food and cosmetic products. It plays an important role in reducing dopaquinone into dopa and inhibiting the tyrosinase activity. It can directly break down melanin biosynthesis.The main disadvantage of L-ascorbic acid for use in cosmetic preparations is its sensitivity to light and heat. In order to overcome this problem, it is important to develop stable derivatives of L-ascorbic acid. Ascorbyl glucoside is one such derivative of L-Ascorbic acid, which is produced by the glucosylation of L-ascorbic acid.In this study, the use of the culture supernatant of Aspergillus niger for glycosylation of L-ascorbic acid was investigated. The study involved the development of suboptimal fermentation conditions and using repeated batch process for obtaining large amount of culture supernatant for the production of ascorbyl glucoside.The results showed that the culture supernatant of A. niger could effectively glycosylate L-ascorbic acid to ascorbyl glucoside. The suboptimal batch culture conditions were: temp, 30oC; agitation; 630 rpm, aeration, 1 vvm, and the culture medium composition was: 6.6% yeast extract, 4.5% maltose, 0.5% K2HPO4. The conversion using supernatant of 48 h old batch culture reached to 80 %. The reaction time was 6 h at 30oC. For repeated batch culture, the suboptimal condition was found to be by using medium replacement and maltose addition alternatively in a 10 L fermenter. The temperature was 30oC and agitation was 630 rpm . The repeated volume was 3 L, and the fed-batch fermentation was repeated 7 times. The average repeated batch culture time was 24 h. The conversion of ascorbyl glucoside obtained using culture supernatant from repeated batch culture was above 80%.Identification of the glycosylation enzyme from culture supernatant by ammonium sulfate fractionation, followed by ion-exchange chromatography was also attempted. The enzyme protein was analysis by SDS-PAGE and MALDI-TOF mass spectrometry. The data from mass spectrometry was aligned with NCBI database for identifying the sequence.