Mechanistic Approach by Which Polysaccharides Inhibit r-Amylase/Procyanidin Aggregation SUSANA I. SOARES,RUI M. GONC -ALVES,IVA FERNANDES,NUNO MATEUS, AND VICTOR DE FREITAS* Chemistry Investigation Centre, Department of Chemistry, Faculty of Sciences, University of Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal The present work studies the inhibition of aggregation of R-amylase and procyanidin fractions by different polysaccharides (arabic gum, β-cyclodextrin, and pectins). Several analytical approaches, namely, fluorescence quenching, nephelometry, and dynamic light scattering (DLS), were used. In general, nephelometry showed that the presence of the polysaccharides in solution reduced the formation of insoluble aggregates. The fluorescence quenching measurements showed two effects: arabic gum and β-cyclodextrin reduce the quenching effect of procyanidin fractions on R-amylase fluorescence, whereas pectins do not affect the quenching of R-amylase fluorescence by procya- nidin fractions. DLS measurements have revealed that the polysaccharides studied induce a decrease in aggregates size, which probably is due to the formation of smaller aggregates resulting from the disruption and reorganization of the procyanidin fractions/R-amylase aggregates. Overall, the results obtained for arabic gum and β-cyclodextrin strongly suggest that the main mechanism by which these two compounds inhibit protein/polyphenol aggregation is by molecular association between these polysaccharides and polyphenols, competing with protein aggregation. In the case of pectins, the results obtained provide evidence that the main mechanism by which they reduce protein/polyphenol aggregation is by forming a protein/polyphenol/polysaccharide complex, enhan- cing its solubility in aqueous medium. KEYWORDS: Aggregation; R-amylase; fluorescence; polysaccharides; tannin INTRODUCTION Tannins are phenolic compounds, yielded from the secondary metabolism of higher plants, being found worldwide in many different families of plants (1 ). Bate-Smith and Swain defined the plant tannins as water-soluble phenolic compounds with molec- ular masses between 300 and 3000 Da, displaying the usual phenolic reactions [e.g., blue color with iron(III) chloride] and precipitating alkaloids, gelatins, and other proteins (2, 3). How- ever, this definition does not include all tannins. Indeed, more recently, molecules with a molecular mass of up to 20000 Da have been isolated and should also be classified as tannins on the basis of their phenolic reactions. High concentrations of tannins can be found in nearly every part of the plant, such as in wood, leaves, fruit, roots, and seed. Usually, tannins are divided in two major classes: condensed (proanthocyanidins) and hydrolyzable tan- nins. The first ones are polymers of catechin, and the latter are gallic or ellagic esters of glucose. It is assumed that these compounds have a biological role in the plant related to protec- tion against infection and herbivores (1 ). From a nutritional point of view, the interaction between tannins and enzymes, such as R-amylase or trypsin, has been shown to have harmful effects, with the inhibition of these enzymes and decrease in body weight gain (4-6). On the other hand, the interaction of R-amylase and other salivary proteins such as proline-rich proteins (PRPs) and hista- tins with tannins is thought to be responsible for the astringency sensation. This event is thought to result from the formation of protein/tannin insoluble aggregates that precipitate, reducing the palate lubrication and causing an unpleasant sensation of rough- ness, dryness, and constriction (7, 8). Astringency is often perceived as a negative attribute as in dairy products, nuts, and juices (9 ). However, in some beverages such as tea, beer, and red wine, astringency could be perceived as a positive quality factor, if not too intense. The affinities of salivary proteins to complex tannins depend on many factors and mainly on their chemical structures (10, 11). Previous works have shown that a PRP (IB8c) binds to condensed tannins much more effectively than R-amylase (12 ). This can be explained not only by their primary structure but also by the three-dimensional structure of these proteins. In fact, whereas R- amylase is a globular protein, PRPs are extended randomly coiled proteins, which offer more sites to interact with tannins. How- ever, R-amylase seems to be more specific and selective than PRPs in the aggregation with samples containing different amounts of proanthocyanidins (13 ). R-Amylase from porcine pancreas (hereafter PPA) is a single polypeptide chain of 496 residues of *Corresponding author (e-mail vfreitas@fc.up.pt; telephone +351226082858; fax +351226082959). J. Agric. Food Chem. 2009, 57, 4352–4358 4352 DOI:10.1021/jf900302r © 2009 American Chemical Society Published on Web 4/20/2009 pubs.acs.org/JAFC