Influence of Chemical Species on Polyphenol-Protein Interactions

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Food and Beverage Chemistry/Biochemistry

Influence of Chemical Species on PolyphenolProtein Interactions related to wine astringency Alba María Ramos-Pineda, Guy H. Carpenter, Ignacio García-Estévez, and María Teresa Escribano-Bailon J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00527 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 12, 2019

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Journal of Agricultural and Food Chemistry

Influence of Chemical Species on Polyphenol-Protein Interactions related to Wine Astringency

A.M. Ramos-Pinedaa, G.H. Carpenterb, I. García Estéveza*, M.T. Escribano-Bailóna

aGrupo

de Investigación en Polifenoles (GIP), Facultad de Farmacia, University of

Salamanca, Salamanca, Spain bSalivary

Research Unit, King's College London Dental Institute, Guy's Hospital, London,

SE1 9RT, United Kingdom

*Corresponding author: Ignacio García Estévez Phone: +34 923 294 537 e-mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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One of the most accepted mechanisms of astringency consists of the interaction between

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polyphenols and some specific salivary proteins. This work aims to obtain further insights

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into the mechanisms leading to a modulation of astringency elicited by polyphenols. The

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effect of the presence of different chemical species (present in food and beverages as food

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additives) on the polyphenol-protein interaction has been evaluated by means of techniques

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such as SDS−Polyacrylamide gel electrophoresis (SDS-PAGE) and cell cultures using a

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cell-based model of the oral epithelium. Results obtained showed that several chemicals,

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particularly sodium carbonate, seem to inhibit polyphenol binding to salivary proteins and

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to oral epithelium. These results point out that polyphenol-saliva protein interactions can be

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affected by some food additives, what can help to better understand changes in astringency

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perception.

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KEYWORDS: Astringency, Tannin, Wine, Salivary proteins, SDS-PAGE, Epithelial cell

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INTRODUCTION

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Wine polyphenols have attracted great interest in wine research and industry. They have a

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number of important functions in wine, contributing to nutrition and organoleptic properties

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such as color, taste or mouthfeel. Astringency is one of the main sensory attributes in red

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wines. It can be defined as a drying or puckering feeling in the mouth1 and plays a key role

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in the mouthfeel of wines. Even more, it is considered a quality parameter and it is an

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important property and character of a balanced wine. Condensed tannins, also called

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proanthocyanidins, have been the tannins generally considered as the main responsible for

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astringency in wine. Proanthocyanidins are polymers composed of monomeric units of

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(epi)catechin and (epi)gallocatechin.2 These compounds are extracted from both grape

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seeds and skins during red winemaking. Moreover, they can be found in commercially

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available grape seed extracts (GSEs), added to red wine during production in order improve

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their mouthfeel and structure or even to promote color stability.3

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It is well known the ability of food tannins to interact with some specific salivary proteins,

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namely proline-rich proteins (PRPs), leading to protein-tannin aggregates that could

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precipitate in the oral cavity, which is one of the main accepted mechanisms to explain oral

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astringency.4 Salivary proteins (SP) have been grouped into different classes according to

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their structure and characteristics, namely, α-amylases, mucins, carbonic anhydrases,

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statherins, P-B peptide, histatins, cystatins and proline rich proteins (PRPs). PRPs, which

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are in turn divided into acidic (aPRPs), basic (bPRPs) and glycosylated (gPRPs),5 have

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been specially studied regarding protein-tannin complexation. It has been suggested that

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their affinity for tannins is associated with their particular structure and composition, rich in



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proline (25-40% of total residues) and with a remarkable content of glycine and

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glutamine.6,7

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Several types of tannin-protein interaction have been described, being hydrogen bonds and

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hydrophobic interactions the dominant ones.4,8 These interactions can be affected by several

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factors, like environmental factors (including solvent composition, ionic strength, pH and

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temperature) or the presence of other substances such us acids, sugars, ethanol and others.

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8–10

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suggesting a negative linear relation between them.11 It seems that acidic conditions could

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result on increase in undissociated phenol groups susceptible to be involved in hydrogen

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bonds with salivary proteins, which results in an increase in astringency perception. This

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explanation appears to be consistent with other studies where the impact of pH on tannin-

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induced astringency was assessed by a trained sensory panel.12,13 Further, ionic strength and

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ethanol content have been investigating regarding their strong incident on tannin

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aggregation and hence on tannin-induced astringency.14

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However, astringency is a very complex process that is not fully understood yet. Some

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authors have proposed that astringency results from the contribution of several

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mechanisms, such as loss of saliva lubricity,15 alteration of the mucosal pellicle,16

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activation of specific taste receptors,17 direct interaction between tannins and oral

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epithelium,18 or even several of these mechanisms occurring simultaneously.19 In the last

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years, a lot of studies have been published trying to elucidate the mechanisms behind oral

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astringency,20–22 however, only very few authors have been focused on the contribution of

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the oral epithelial cells to astringency.16,18,23 The first study addressing this approach was

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carried out by Payne and co-workers,18 demonstrating that procyanidins present in wine

A number of authors have studied the relationship between pH and astringency,


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react with oral epithelial cells in vitro. After these results, subsequent studies have

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examined the role of oral epithelium considering also the effect of salivary proteins on

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these interactions.16,23 Recently, Ployon and co-workers16 have even suggested that PRPs

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play a protective role by scavenging tannins, blocking their access to the mucosal pellicle,

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highlighting the importance of considering more than one mechanism when studying

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astringency.

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Apart from that, red wine is usually a palate-pleasing accompaniment to other foods. A

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pairing selection can transform wine sensory components, sometimes with a positive

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impact enhancing specific attributes, but others with a negative impact.24 Flavour

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perception can be also strongly affected by the oral physiology, and specifically by the

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interaction of some of these compounds with saliva.25 Therefore, we could consider the

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effect of certain ingredients and additives present in foods on the perception of wine

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astringency.

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Based on these considerations, the present work aims to go further into depth on knowledge

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of the mechanisms involved in food oral astringency, considering both salivary proteins and

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epithelial cells, and its modulation by the presence of other substances used as specific

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additives in food and beverages.

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MATERIALS AND METHODS

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Chemicals (Food additives)

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12 food additives with different applications were used for the interaction assays: (1)

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ascorbic acid (VWR-BDH Prolabo Leuven, Belgium), (2) ammonium bicarbonate (MP

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Biomedicals Europe, Illkirch, France), (3) citric acid (Thermo Fisher Scientific, Pittsburgh, 5 ACS Paragon Plus Environment


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PA, USA), (4) calcium carbonate (Sigma-Aldrich, Gillingham, UK), (5) calcium chloride

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(Sigma-Aldrich, Gillingham, UK), (6) EDTA (Sigma-Aldrich, Gillingham, UK), (7)

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glycine (Sigma-Aldrich, Gillingham, UK), (8) zinc sulfate (Sigma-Aldrich, Gillingham,

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UK), (9) sodium carbonate (VWR-BDH Chemicals UK), (10) sodium chloride (Sigma-

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Aldrich, Gillingham, UK), (11) sodium bicarbonate (Fisher Scientific, Loughborough,

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UK), (12) magnesium sulfate (Sigma-Aldrich, Gillingham, UK).

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Grape Seed Tannins

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Berries of the Zalema variety were harvested at maturity and seeds were separated from the

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skins and pulp. Grape seeds were lyophilized and ground to obtain homogeneous powder.

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The resulting powder was extracted three times with ethanol/water (75:25 v/v) for 30 min in

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and ultrasonic bath. The solution was then centrifuged, and the supernatant was evaporated

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to remove the organic solvents. The aqueous grape seed extract (GSE) was frozen and

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freeze-dried. The resulting GSE powder was stored at 4 °C until analysis.

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The content of monomeric and oligomeric procyanidins was determined by HPLC-DAD-

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MS analysis. The mean degree of polymerization (mDP) was determined by acid-catalysis

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reaction in the presence of phloroglucinol according to a method described in the

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literature.26 The composition of the GSE is shown in Table SI-1 (Supplementary file). The

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mDP of the total flavanols of GSE was 2.05. The content of galloylated flavanols was

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16.67%, while the non-galloylated represent 83.33%. GSE is composed mainly of

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monomers and dimers, with a percentage of 33.85% and 47.20%, respectively.

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Saliva collection and treatment


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Unstimulated whole mouth saliva (WMS) was collected from five healthy individuals (25-

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45 years) who had no history of disorders in oral perception and were not taking any

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medication. Saliva samples were taken between 10 and 12 am at least 1 h after consuming

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any food. Samples were collected by expectorating saliva into an ice-cooled tube. All

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samples were pooled and centrifuged at 4000 g for 20 min at 4 °C to remove any insoluble

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material. The resulting supernatant was aliquoted and immediately frozen at – 80 °C, which

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is referred to as whole saliva (WS).27,28

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Binding assay (saliva-polyphenols)

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Interaction mixtures were prepared in a final volume of 20 µL, containing 11.5 µL of whole

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saliva (WS), GSE (final concentration 0.6 mg/mL) and/or chemicals (final concentration 10

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mM). Binding assays were performed in Eppendorf tubes at room temperature during 15

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min. The mixture was then centrifuged at 13000 g for 5 min. The pellet was discarded and

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lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, Paisley, UK) was added to the

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supernatant before loading the samples into the gel (1:4 LDS:sample). Control solutions of

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WS with Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma-Aldrich, Gillingham, UK)

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and WS with GSE were also included.

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SDS-PAGE electrophoresis

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of samples was

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carried out under non-reducing conditions (no dithiothreitol (DTT) and no boiling of the

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samples). 15 µL of each sample was loaded (per well) into a NuPAGE Novex Bis-Tris 4-

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12% resolving gels (Invitrogen, Paisley, UK). Electrophoresis was performed on a Bio-Rad

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MiniProtean Cell electrophoresis apparatus according to manufacturer’s instructions in



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NuPAGE MES-SDS running buffer (Invitrogen, Paisley, UK). Resolved proteins were

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stained using a solution of 0.2% w/v Coomassie Brilliant Blue (CBB) R Staining Solution

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(Sigma-Aldrich, Poole, Dorset, UK) in 25% methanol and 10% acetic acid for 1h. Gels

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were then destained in 10% acetic acid overnight. The electrophoretic gels were scanned

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using a white light transilluminator screen in a GelDocXR + Imaging System (Bio-Rad

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Laboratories, Hercules, CA, U.S.A.). Bands were analyzed using Image Lab software (v.

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5.1, Bio-Rad Laboratories, Hercules, CA, U.S.A.). Molecular masses were estimated using

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protein markers (SeeBlue Plus2 Pre-Stained Standard, Invitrogen, Paisley, UK).

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Cell culture

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TR146 epithelial cells (ATCC, Middlesex, U.K.) were used in this study as an in vitro

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model of human buccal epithelium.29 TR146 cells were grown in Dulbecco’s modified

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Eagle medium (DMEM)/F12 (1:1, v/v) supplemented with 15% Foetal Bovine Serum

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(FBS) and 1% penicillin/streptomycin (P/S) (Gibco® by Life Technologies). Cells were

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cultured in T75 flasks and the medium was changed every two days. Cells were dissociated

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with trypsin enzyme and subcultures were prepared at 80% confluence using Trypsin-

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EDTA. Culture conditions were maintained at 37 ° and 7.5% CO2.

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Interaction assays with oral cells

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TR146 cells were cultured into 96 well flat and grown to confluence before its use in an

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assay. The cell monolayers were washed twice with Dulbecco’s Phosphate Buffered Saline

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(DPBS) in order to remove residual growth medium. Stock solutions of each chemical

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compound were prepared in DPBS. Interaction assays were performed following two

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different protocols:


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Protocol A (Figure 1A): Cells were incubated for 2h with whole saliva diluted into growth

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medium (1:1, v/v) (50 µL/well) in order to deposit a mucosal pellicle on the cell’s surface.

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30

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DPBS (lines C to E, Fig. 1A) and the different chemical solutions (columns 1-12, Fig. 1A)

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were added in triplicate (final concentration GSE: 0.6 mg/mL, chemicals 1-12: 10mM) into

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a final volume of 45 µL (30 µL buffer + 15 µL GSE + chemicals), incubating for 15 min.

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After two washes with DPBS (in order to remove the unbound material) the DMACA assay

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was done. Different control conditions were also tested, in triplicate, such as the

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GSE/DPBS and saliva/DPBS with or without oral cells (lines A and B, Fig. 1A). The

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control condition named as C corresponds with GSE + DPBS with oral cells, while CS

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corresponds with GSE + saliva with oral cells.

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Protocol B (Figure 1B): Whole saliva was mixed with GSE and the chemical solutions,

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incubating during 15 min (30 µL/well saliva or DPBS + 15 µL GSE + chemicals), before

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adding to the cells. 45 µL/well of the mixture was added in triplicate (lines C-E: DPBS +

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GSE + chemicals; lines F-H: saliva + GSE + chemicals, see Fig. 1B) to attain the final

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concentration GSE: 0.6 mg/mL, chemicals 1-12: 10mM and left in contact with the

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monolayers for 15 min. After two washes with DPBS, the DMACA assay was performed.

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The same control conditions as in Protocol A were tested.

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DMACA bioassay

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4-(dimethylamino)cinnamaldehyde (DMACA) was purchased from Sigma-Aldrich as a

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chromogenic reagent for indoles and flavanols. DMACA assay was performed since this

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compound reacts selectively with catechins and procyanidins to form a blue-green product,

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allowing us to visualize and quantify the amount of procyanidins that remains bound to the

After incubation, samples were washed twice with DPBS. GSE (lines F to H, Fig. 1A) or



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epithelial cells.18,31 A 0.1% of DMACA solution was prepared in acidified methanol (0.75

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M H2SO4)18 and added to the 96 well plates after the interaction assays. Cells were

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incubated with 70 µL/well of the reagent for 20 min at room temperature. Finally, the

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absorbance of the wells at 655 nm was determined in a microtiter plate reader.

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Statistical analysis

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In order to determine statistical significance of the differences between the absorbance

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values obtained for the interaction assays, data were evaluated by a t-student test for

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independent samples, using the software packing for Windows IBM SPSS 23 (SPSS, Inc.

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Chicago, IL, USA). Differences were considered to be statistically significant when p

Influence of Chemical Species on Polyphenol-Protein Interactions

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