Gallic acid content in green tea extract was enhanced by using a novel cell-associated tannase of Bacillus massiliensis. Biomass that contains tannase was used for this study. The activity of the cell-associated tannase was stable during 1 week of storage in the refrigerator. Response surface methodology was applied based on central composite design to determine the effects of three independent variables (pH, temperature and incubation time) and their mutual interactions. A total of 16 experiments were conducted; and a statistical model was developed, which predicted 475.74 mg/L gallic acid production at pH 6.2, 36C and incubation period of 16.71 h. The subsequent verification experiments confirmed the validity of the model. Under optimal conditions, 84.7% of the total hydrolyzable tannins were converted to gallic acid and glucose. This naturally immobilized tannase was stable enough to be used for up to 12 runs.
The current study shows that naturally immobilized tannase of Bacillus massiliensis can be used instead of artificially immobilized tannase. Such naturally immobilized tannase has many advantages as it avoids expensive and laborious isolation, purification and immobilization. Ease of separation of cell-associated enzyme from the reaction mixture and absence of any detectable extracellular tannase activity after enzymatic treatment are some of the encouraging facts. Stability during storage up to 7 days, 85% tannic acid hydrolyzing efficiency, activity at pH 3.5–8.0 and operational stability for 12 runs are some of the interesting features of this naturally immobilized enzyme. However, its application for tea treatment will be limited until Bacillus massiliensis gets “Generally Recognized As Safe” status. It can be employed, however, for production of gallic acid from agro residues and production of propyl gallate.