As an alternative, bioprocesses, such as fermentation or enzymatic hydrolysis of plant sources and their products, can release the phenolic glycosides or other conjugates and consequently, enhance the functional activity of these antioxidants. Tannin acylhydrolases, commonly referred to as tannases (E.C. 3.1.1.20), are inducible enzymes produced by fungi, yeast and bacteria. Tannases have mostly been characterised by their activity on complex polyphenolics and are able to hydrolyse the ester bond (galloyl
ester of an alcohol moiety) and the depside bond (galloyl ester of gallic acid) of substrates such as tannic acid, epicatechin gallate, epigallocatechin gallate, and chlorogenic acid (Garcia-Conesa, Ostergaard, Kauppinen, & Williamson, 2001). In this study, the activity of tannase on the extracts of green tea was investigated. The aim of this work was to evaluate the chemopreventive selleck products potential of green tea extract and EGCG after an enzymatic reaction, catalysed by
the tannase, produced by Paecilomyces variotii ( Battestin & Macedo, 2007). Green tea was purchased from local markets (Chá Leão®). Epigallocatechin gallate MEK inhibitor (EGCG, 95%), epigallocatechin (EGC, 98%), 2,2′-azobis(2-methylpropionamidine) (97%) (AAPH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), sulforhodamine B sodium salt (SRB), trichloroacetic acid, T1503 Trizma® base, thiazolyl blue tetrazolium bromide, agarose (LMP) and Triton X-100 were purchased from Sigma–Aldrich. All other chemicals
were purchased in the grade many commercially available. Fluorescein was purchased from ECIBRA, and Trolox® (97%) was purchased from ACROS Organics. Cell culture reagents were purchased from Invitrogen®. Tannase was isolated from P.variotii using a previously published procedure ( Battestin & Macedo, 2007). A 250-ml conical flask containing 5 g of wheat bran, 5 g of coffee husk, 10 ml of distilled water and 10% tannic acid (w/w) (Ajinomoto OmniChem Division, Wetteren, Belgium) was used for the fermentation process. The culture medium (pH 5.7) was sterilised at 120 °C for 20 min. After sterilisation, the flasks were inoculated with 2.5 ml (5.0 × 107 spores/ml) of the pre-inoculum suspension and incubated at 30 °C for 120 h. After fermentation, 80 ml of 20 mM acetate buffer, pH 5.0 was added, and samples were shaken at 200 rpm for 1 h. The solution was filtered and centrifuged at 9650g, for 30 min, at 4 °C (Beckman J2-21 centrifuge, Beckman-Coulter Inc., Fullerton, CA, USA). The supernatant was then treated with solid ammonium sulphate (80% saturation) and incubated overnight at 4 °C. The precipitate was collected by centrifugation (9650g for 30 min), resuspended in distilled water and dialysed against distilled water. The dialysed preparation was freeze-dried and used as crude tannase. Samples were prepared, and the enzymatic reaction, catalysed by the P.