Production of Bioavailable Flavonoid Glucosides in Fruit Juices and Green Tea by Use of Fungal r-L-Rhamnosidases ROCI ÄO GONZA Ä LEZ-BARRIO, ² LUISA M. TRINDADE, ‡ PALOMA MANZANARES, § LEO H. DE GRAAFF, ‡ FRANCISCO A. TOMA Ä S-BARBERA Ä N, ² AND JUAN CARLOS ESPI ÄN* ,² Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Campus de Espinardo, Murcia, Spain; Fungal Genomics Section, Laboratory of Microbiology, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands; and Department of Food Biotechnology, IATA-CSIC, P.O. Box 73, Burjassot, 46100 Valencia, Spain Flavonoid glucosides have been reported to be more bioavailable than their rutinoside counterparts. The aim of this study is to describe a first step in the use of R-L-rhamnosidases (RhaA and RhaB) from Aspergillus aculeatus as a way to produce functional beverages based on their potentially increased flavonoid bioavailability. Blackcurrant juice (BCJ), orange juice (OJ), and green tea infusion (GT) were incubated with either RhaA or RhaB at 30 °C for 10 h. Aliquots of controls and enzyme- treated samples were taken at different time points and analyzed by high-performance liquid chromatography-photodiode-array detector-mass spectrometry of daughter fragments (HPLC- DAD-MS-MS). Both RhaA and RhaB selectively catalyze in situ the removal of terminal rhamnosyl groups in the three beverages despite the heterogeneity of assay conditions such as different rutinosides and pH. Incubation of the three beverages with the two rhamnosidases resulted in a hyperbolic decrease in the flavonoid rutinosides (anthocyanins in BCJ, flavanones in OJ, and flavonols in GT) and a concomitant increase in their flavonoid glucoside counterparts. The time required for conversion of 50% of the rutinoside into the corresponding flavonoid glucoside ranged from 30 min (RhaB-rutin in GT) to 6 h (RhaB-delphinidin 3-rutinoside in BCJ). The results presented in this paper are a step forward in the use of enzyme-treated beverages as a source of bioavailable flavonoid glucosides. KEYWORDS: Rhamnosidase; Aspergillus; bioavailability; flavonoid; rutinoside; glucoside; functional beverage; green tea; blackcurrant juice; orange juice INTRODUCTION The health-promoting activity of polyphenols present in fruits, vegetables, and derived foodstuffs such as juices, tea, and wine has been extensively described in vitro, ex vivo, and in vivo (1-3). Flavonoids are the most abundant polyphenols in nature and have attracted great attention due to their beneficial properties. These beneficial properties included those related to the prevention of cardiovascular diseases and cancer (4-6). One important drawback is the limited bioavailability of many flavonoids. In fact, the sugar moiety has been proposed as the major determinant of the absorption of dietary flavonoids in humans (7, 8). This is the case of flavonoid rutinosides that are very abundant in many plant foods and derived products. The rutinoside moiety of the flavonoid (glucose + rhamnose) has been described to hamper its absorption in the intestine (8). On the contrary, the removal of the rhamnose group to yield the corresponding counterpart flavonoid glucoside has been reported to increase its bioavailability (8, 9). However, this is still under discussion in the case of anthocyanins (10, 11) whose bioavail- ability is lower than most of the other flavonoids (12). The enzymes R-L-rhamnosidases (Rhams) (EC 3.2.1.40) hydrolyze the terminal nonreducing R-L-rhamnose residues in R-L-rhamnosides. Rhams can be used to elucidate the structure of glycosides, polysaccharides, and glycolipids (13); to remove the bitterness from citrus juices, which is caused by the flavonoids naringin and neohesperidin (for example, by using active packaging with immobilized Rham in the inner surface of cartons (14); to hydrolyze hesperidin to yield rhamnose and hesperetin 7-glucoside, which is an important precursor in sweetener production (15); and also to enhance the aroma in grape juices and wines (16, 17). Although, the Rhams purified from Aspergillus aculeatus have been shown to be effective in removal of the rhamnose moiety from various flavonoid * To whom correspondence should be addressed: fax +34-968-396213; e-mail jcespin@cebas.csic.es. ² Department of Food Science and Technology, CEBAS-CSIC. ‡ Wageningen Univerisity. § Department of Food Biotechnology, IATA-CSIC. 6136 J. Agric. Food Chem. 2004, 52, 6136-6142 10.1021/jf0490807 CCC: $27.50 © 2004 American Chemical Society Published on Web 09/09/2004