Intrinsic bitterness of flavonoids and isoflavonoids and masking of their taste activity Wibke S.U. Roland Thesis committee Promotors Prof. Dr H. Gruppen Professor of Food Chemistry Wageningen University Prof. Dr G. Smit Professor of Molecular Flavour Science Wageningen University Co-promotor Dr J-P. Vincken Assistant professor, Laboratory of Food Chemistry Wageningen University Other members Prof. Dr C. de Graaf, Wageningen University Dr J. Ley, Symrise AG, Holzminden, Germany Prof. Dr W. Meyerhof, University of Potsdam, Germany Prof. Dr R.F. Witkamp, Wageningen University This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Intrinsic bitterness of flavonoids and isoflavonoids and masking of their taste activity Wibke S.U. Roland Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 25 April 2014 at 4 p.m. in the Aula. Wibke S.U. Roland Intrinsic bitterness of flavonoids and isoflavonoids and masking of their taste activity 188 pages. PhD thesis, Wageningen University, Wageningen, NL (2014) With references, with summaries in English, Dutch and German ISBN: 978-94-6173-853-0 ABSTRACT The presence of flavonoids and isoflavonoids in foods and their addition as bioactives to food products can impart unpleasant bitterness. Therefore, debittering strategies are demanded. The aim of this research was to identify human bitter receptors (hTAS2Rs) sensing (iso)flavonoids and to determine the intrinsic bitterness and structure-activity relationships of soy isoflavones, tea catechins and a large set of structurally similar (iso)flavonoids by testing for activation of hTAS2Rs in vitro. A subsequent aim was to use the bitter receptor assay for investigation of different debittering strategies. Out of all 25 human bitter taste receptors, hTAS2R14 and hTAS2R39, were activated by soy isoflavones. hTAS2R14 was only activated by isoflavone aglycones, whereas hTAS2R39 was activated by isoflavone glucosides as well. Investigation of almost 100 (iso)flavonoid aglycones for activation of hTAS2R14 and hTAS2R39 revealed that many (iso)flavonoids activated these receptors. The structural characteristics for an (iso)flavonoid to activate hTAS2R14 and hTAS2R39 were determined by 3D-pharmacophore models to be composed of two (for hTAS2R14) or three (for hTAS2R39) hydrogen bond donor sites, one hydrogen bond acceptor site, and two aromatic ring structures, of which one had to be hydrophobic. Three 6-methoxyflavanones were identified which reduced activation of hTAS2R39 by epicatechin gallate (ECG). These bitter receptor blockers were characterized as reversible insurmountable antagonists. Furthermore, complexation of epigallocatechin gallate (EGCG) with food proteins (mainly β-casein and Na-caseinates) reduced hTAS2R39 activation. A trained sensory panel confirmed reduced bitterness perception. The systematic investigation of (iso)flavonoid aglycones indicated that the substitution pattern of (iso)flavonoids is of higher importance for bitter receptor activation than the backbone structure. In case of bitter receptor antagonists, the substitution pattern as well as backbone structure revealed to be crucial for functionality. The bitter receptor assay was shown to be an appropriate tool not only for identification of bitter receptor agonists and antagonists, but also for identification of reduced receptor activation by complexing agents. TABLE OF CONTENTS Abstract Chapter 1 General introduction 1 Chapter 2 Soy isoflavones and other isoflavonoids activate the human 35 bitter taste receptors hTAS2R14 and hTAS2R39 Chapter 3 Bitter taste receptor activation by flavonoids and 59 isoflavonoids: modeled structural requirements for activation of hTAS2R14 and hTAS2R39 Chapter 4 6-Methoxyflavanones as bitter taste receptor blockers for 101 hTAS2R39 Chapter 5 Evaluation of the bitter-masking potential of food proteins 119 for EGCG by a cell-based human bitter taste receptor assay and binding studies Chapter 6 General discussion 139 Summary 161 Samenvatting 165 Zusammenfassung 171 Acknowledgments 177 About the author 181 1 Chapter 1 General introduction Chapter 1 BITTER TASTE IN RELATION TO DIETARY COMPOUNDS Bitter taste in food is mostly disliked by consumers. On the other hand, bitter taste in food can be appreciated, as long as it is moderate, in products such as bitter lemon, coffee, beer, and dark chocolate (1). Bitter tasting compounds in food are mainly derived from plants (e.g. caffeine in coffee, naringin in grapefruit, sinigrin in cabbage, hop bitter acids in beer), but they can also occur in products from animal origin (e.g. bitter peptides in cheese), due to processing (e.g. the Maillard reaction products such as quinizolate or (bis)pyrrolidinohexose reductones) and upon storage (e.g. oxidized lipids) (2-4). In this thesis, dietary compounds are defined as compounds naturally present in food raw materials, food products, and compounds (natural or synthetic) added to food products. Drugs, herbal medicine, and synthetic or natural non-consumable bitter substances are considered as non-dietary. The biological function of bitter compounds in plants is to protect the plant against pathogens, parasites and predators. Especially toxins (e.g. strychnine) are often bitter. Therefore, it has been assumed that the ability to taste bitter compounds and rejection of bitter taste has long been crucial to survival. However, a correlation between toxicity and bitter taste thresholds has not been found (1). Numerous natural bitter compounds have been associated with putative health effects. Amongst them, isoflavonoids and flavonoids play a central role. Therefore, a trend in food product development is the design of functional foods, enriched in (iso)flavonoids. However, their incorporation in, or fortification of, foods can introduce or enhance bitterness, and, therefore, debittering strategies are demanded. Investigation of bitter taste and reduction of bitter taste by sensory tests is laborious and can be influenced by many factors. A fast and objective way of investigating bitterness is the measurement of intrinsic bitterness on bitter taste receptors by the use of a cell-based assay system. This method constitutes an important part of this thesis. Intrinsic bitterness is the capacity of a compound to activate a bitter taste receptor, uncoupled from other taste qualities (e.g. sweet), senses (e.g. smell), influences (e.g. hormone levels) and interactions (e.g. with saliva). Due to its high throughput character, many (potentially) bitter compounds (even of non-food quality) can be examined in a bitter receptor assay. In this way, sensory panels can be disburdened from tasting many aversive compounds, and their services can be minimized to confirm the impact of compounds of high interest. 2 General introduction STRUCTURAL CLASSIFICATION OF FLAVONOIDS AND ISOFLAVONOIDS Flavonoids are ubiquitously occurring secondary plant metabolites that play, besides their 1 physiological role in plants as pigment and defence compounds, a role in the human diet (5). Isoflavonoids occur almost exclusively in the Leguminosae family (6). Flavonoids and isoflavonoids are classified as members of phenolic compounds. The non-(iso)flavonoid phenolics are e.g. hydroxycinnamic acids, coumarins, xanthones, stilbenes, ellagitannins, and lignans. (Iso)flavonoids consist of two aromatic rings, connected via a three-carbon bridge (C6- C3-C6). In most cases, the three carbon bridge is part of a six member heterocyclic ring, including one oxygen atom (pyran ring) (7). In Table 1, an overview of the most important dietary (iso)flavonoid subclasses is shown, including their generic numbering pattern. (Iso)flavonoids belong to the super family of phenyl benzopyrans (6), and their nomenclature is based on the variation of the C-ring, and the connection between B- and C- rings. The linkage of the B-ring to position 2 of the benzopyran describes the class of flavonoids (2-phenyl benzopyrans), whereas a linkage to position 3 describes the class of isoflavonoids (3-phenyl benzopyrans). The positions 2 and 3 are accentuated in the first structure of Table 1. In literature, isoflavonoids are often referred to as flavonoids, but due to their different substitution of the C-rings compared to flavonoids, this classification is controversial. In this thesis, a distinction between flavonoids and isoflavonoids is, therefore, made. Besides attachment to position 2 or 3, the B-ring can be connected to position 4, a class of compounds referred to as neoflavonoids (4-phenyl benzopyrans). Neoflavonoids hardly occur in the human diet. Furthermore, there are C6-C3-C6 compounds not containing the pyran C-ring, e.g. chalcones and aurones, which are referred to as minor or miscellaneous flavonoids. The backbone structures are mostly substituted by –OH, -OCH3 and O-glycoside groups, but also C-glycoside, prenylation and sulfonation are possible substitutions (5, 8). In foods, (iso)flavonoids occur as glycosides (e.g. in many fruits, seeds, and vegetables), as aglycones (e.g. in tea and in fermented soy products), or as oligomers (such as proanthocyanidins, e.g. in grapes and peanuts). This thesis focusses on the aglycones and takes two food products as examples: soybean-derived (Glycine max) products