Flow nephelometric analysis of protein–tannin interactions

Analytica Chimica Acta 513 (2004) 97–101

Flow nephelometric analysis of protein–tannin interactions Elisabete Carvalho, Nuno Mateus, Victor de Freitas∗ Departamento de Qu´ımica, Faculdade de Ciˆencias, Centro de Investigação em Qu´ımica, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal Received 14 July 2003; received in revised form 28 August 2003; accepted 3 October 2003 Available online 3 December 2003

Abstract The interaction of tannins from different sources (grape skin, grape seed and wine) with bovine serum albumin (BSA) was studied using a continuous flow method with nephelometric detection. The tannin samples were mixed in a reaction chamber with increasing BSA concentration and the resulting tannin–BSA insoluble aggregates passed through a flow cell where the turbidity was monitored. Based on this technique, the tannin specific activity (TSA) of wine can be directly determined from different wine flows without previous dilutions or any other treatment. The maximum amount of insoluble aggregates formed with BSA and wine or grape skin tannins seemed to remain constant even with the addition of an excess of protein, whereas a decrease in the amount of insoluble aggregates with an excess of BSA was observed in the case of grape seed tannins. It should be noted that BSA was used in these experiments as a model protein and only as an analytical reagent since bovine additives are not allowed in wine. © 2003 Elsevier B.V. All rights reserved. Keywords: Nephelometry; Grapes; Procyanidin; Tannins; BSA; Wine

1. Introduction Tannins can be found in vegetal foodstuffs, particularly in fruits, cereal grains and beverages (wine, tea and beer). During foodstuff consumption, these polyphenols interact with salivary proteins forming insoluble aggregates that are supposed to obstruct palate lubrication and cause the sensation of astringency of tannin-rich food [1–4]. In general, tannin–protein associations are thought to involve cross-linking of separate protein molecules by the tannin, that acts as a polydentate ligand, on the protein surface involving hydrophobic effects and hydrogen bonding. The aromatic groups of polyphenols are supposed to be involved in a face-to-face stacking with amino acid residues of linear proteins, whereas the interaction with globular proteins probably involves only surface exposed residues. Besides protein structure, the interaction between tannins and proteins is also affected by the relative concentration of tannin and protein, by the solvent composition, ionic strength, pH and presence of other co-substrates like polysaccharides [5–10].

∗

Corresponding author. Tel.: +351-226082858; fax: +351-226082959. E-mail address: [email protected] (V. de Freitas).

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.10.010

During the last years, polyphenol complexation with proteins has been largely studied in solution by enzymatic [7], spectroscopic [11–14], calorimetric [15], colorimetric [1,16,17], chromatographic [18] and precipitation techniques [19–23]. These approaches allowed measuring the relative affinity of different polyphenols to bind proteins. One of the easiest and obvious techniques to study the appearance of insoluble aggregates is the measure of light intensity scattered by haze particles in solutions by nephelometry. The nephelometers available in the market have been developed essentially with a commercial intent to measure the haze in beverages, however, this technique was recently utilized with analytical purposes to study the effect of polyphenol structure on its ability to bind proteins [21,22]. Bovine serum albumin (BSA) has been often used as a model protein to measure the reactivity of tannins, although its globular conformation is quite different from the one of the linear proline-rich proteins (PRP) [16,24–27], which represent about 70–80 % of the total human salivary protein content [28]. In the present work, a fast and reliable flow nephelometric method has been developed in order to study the effect of increasing concentration of protein (BSA) in the reaction with different tannins solutions.

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2. Experimental 2.1. Reagents BSA fraction V (96% of purity) was purchased from Sigma (St. Louis, MO, USA). 2.2. Grape seed extract 2.2.1. Fractionation of grape seed procyanidins Condensed tannins were extracted from Vitis vinifera grape seeds tissues with an ethanol–water–chloroform solution (1:1:2, v/v/v) using a blender (Ultra-Turrax® ). The upper aqueous layer containing the polyphenols was separated from the chloroform layer containing chlorophylls, lipids and other undesirable compounds. A fraction rich in procyanidin oligomers was obtained according to the procedure described in the literature [29]: ethanol was removed using a rotator evaporator and the resulting aqueous solution containing the polyphenolic compounds was extracted with ethyl acetate followed by precipitation with hexane. The fraction of procyanidin oligomers was successively purified through different higher length TSK Toyopearl HW-40(s) gel columns in order to separate catechin monomers and polymeric procyanidins, as reported elsewhere [30]. LSI–MS analysis of the resulting oligomeric procyanidins fraction revealed that it was essentially comprised of procyanidin oligomers up to pentamers together with several galloyl and digalloyl esters: monomers gallate ([M + H]+ at m/z = 443); dimers (579); dimers gallate (731); trimers (867); dimers digallate (883); trimers gallate (1019); tetramers (1155); tetramers gallate (1307); pentamers (1443); pentamers gallate (1595). The mean molecular weight (g/mol) of the molecules in that fraction was estimated to be MW = 1002, by calculating the average of the different MW of each group of procyanidin identified. 2.3. Wine samples The Porto wine (V. vinifera, Touriga Nacional cv.) was provided by Adriano Ramos Pinto–Vinhos S.A. 2.4. Skin extract Skins of red grapes (V. vinifera) were subjected to extraction (manually stirred) with aqueous ethanol solution (40%). The solvent was partially evaporated using a rotator–evaporator at 30 ◦ C, and the sample was freezedried and stored at −18 ◦ C. 2.5. Nephelometry The online detection of protein–tannin insoluble aggregates was carried out by a HACH 2100N laboratory turbidimeter detector coupled to a PC equipped with a software for data acquisition. The optical apparatus was equipped

with a tungsten filament lamp with three detectors: a 90◦ scattered light detector, a forward-scattered light detector and a transmitted light detector. Previous calibration was performed using Gelex® secondary turbidity standard kit (HACH, Loveland, US), which consists of stable suspensions of a metal oxide in a gel. All solutions used were prepared with previously filtered (47 mm, 0.45 ␮m) ethanol–water 12% (v/v), acetate buffer solution (0.1 M, pH 5.0). All solutions were thoroughly degassed under reduced pressure to avoid bubble formation during analysis that might interfere in the turbidity values. All experiments were performed in triplicate. Results were expressed in nephelometric turbidity units (NTU).

3. Results and discussion 3.1. Evaluation of protein–tannin interactions The procyanidins affinity to bind BSA was assessed according to their ability to form insoluble aggregates in solution, which were determined directly by nephelometry. A continuous flow nephelometric analytical method was developed allowing the measurement in continuous of the scattered light resulting from the gradual formation of a cloudy precipitate of tannin–protein aggregates. The whole apparatus is composed of a liquid gradient pump system, a peristaltic pump, a mixing valve, a reactor, a laboratory nephelometer detector and a computer (Fig. 1). The liquid pump system forms a gradient of protein concentration at a constant flow rate using two solutions: A, ethanol 12% (v/v), 0.1 M acetate buffer, pH 5.0; and B, BSA in A. This solution was put in contact in a mixing valve with the sample solution (tannin extracts, wines, etc.) pumped by a peristaltic pump at a constant flow rate. The mixture passed through a reactor, which consists of a silicon tube with an internal diameter (i.d.) of 1.5 mm. The online detection of light scattering was carried out by a nephelometer detector adapted with a cylindrical glass flow cell (10 × 5 mm i.d.), especially made to fit the chamber of this equipment. The signal is sent to a computer equipped with data acquisition software. The pH was chosen at a value of 5.0 (acetate buffer), where BSA was shown to be stable and to have a strong ability to bind and precipitate condensed tannins, as previously reported [21]. The sample concentration and respective flow rate were chosen in order to obtain a turbidity within the range of 0–12 NTU. At this NTU range light absorption is negligible, the refraction index of solvated particles is small and practically identical to that of the solution, and in this case, the intensity of the scattered light is proportional to the concentration of dispersed insoluble aggregates [19]. Under these conditions, the control solutions showed that the solvent did not precipitate proteins in the absence of tannins and no precipitate was observed in the polyphenol

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Sample Turbidimeter -Flow cell

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Fig. 1. Scheme of the flow nephelometric apparatus. Carrier solution A: ethanol 12% (v/v), 0.1 M acetate buffer (pH 5.0). Protein solution B: BSA in A.

solutions without any protein, even with higher concentrations than those used in the experiments. 3.2. Grape seed extract The profiles of the appearance of the insoluble tannin– protein aggregates resulting from the interactions between grape seed procyanidin oligomers and different concentrations of BSA at pH 5.0 are shown in Fig. 2. These complexes are supposed to result from the interaction between the aromatic rings and the carbon–hydrogen skeleton of the pyranic rings of condensed tannins with surface exposed amino acid residues of BSA. The procyanidin–BSA aggregates dispersed in solution increased with increasing protein concentration up to a maximum. Further addition of BSA reduced the insoluble aggregates formation, as it was already reported in literature [3,30–33]. A mechanism has been proposed to explain the different NTU values observed with different tannin–protein ratios [32]. For a high polyphenol–protein molar ratio, only few polyphenol molecules (more reactive) should be able to bind the small number of protein molecules present, thus resulting in small

aggregates and less light scattered. With the increase in protein concentration, tannins that bind onto the protein surface can act as polydentate ligands forming large networks of protein–tannin complexes, resulting in a maximum of light scattering. For a large excess of protein, the polyphenols would be surrounded by several protein molecules, reducing the probability of forming cross-links between aggregates, thereby changing the stoicheiometry of the complex yielding smaller particles and less light scattering. The stoichiometry of the largest insoluble tannin–BSA complex can be calculated from the relative concentration of tannin and BSA at the point of maximum turbidity. The molar ratio, tannin–BSA, at this point is 11:1, a result not far from the one obtained using batch nephelometry indicating a molar ratio of tannin–BSA of 7:1 for similar procyanidin composition [30]. 3.3. Grape skin extract The graphic in Fig. 3 shows the influence of protein concentration on the amount of insoluble tannin–BSA

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Fig. 2. Flow nephelometric analysis of the formation of aggregates between grape seed tannins (2.7 × 10−4 M, 1.0 ml/min) and BSA (0.50 ml/min).

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Fig. 3. Flow nephelometric analysis of the formation of aggregates between grape skin tannins (4.23 g/dm3 , 1.0 ml/min) and BSA (0.50 ml/min).

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Fig. 4. Flow nephelometric analysis of the formation of aggregates between Porto wine tannins and BSA (2.70 ml/min). (a) Different wine flows were studied as indicated in the graph. (b) Influence of wine flow on the maximum NTU.

aggregates formed by reaction with the grape skin extract. The skin tannin–BSA aggregates dispersed in solution increased with the addition of protein up to a maximum, from which no further precipitation occurred. The reversibility of the tannin–protein aggregate formation is not as evident as it was with the grape seed extract. Grape skins contain a great structural diversity of polyphenol compounds, namely high molecular weight procyanidins and prodelphinidins [34,35] that present a strong ability to bind proteins. This higher affinity of prodelphinidins comparatively to procyanidins could be due to a higher number of hydroxyl groups in their structures, which are sites thought to strongly participate in the protein–tannin interactions [36]. 3.4. Wine Fig. 4a shows the influence of BSA concentration on the appearance of the insoluble wine tannin–protein aggregates at four different flow rates of a red wine without dilution. The maximum NTU increased linearly with wine flow rate (Fig. 4b). The general behaviour is similar to the one observed for the grape skin extract, where practically no decrease in the amount of tannin–protein aggregates was observed, even at high protein concentration. The concept of “tannin specific activity (TSA)” of wines has recently been introduced as the maximum turbidity expressed in NTU/ml of wine after reaction with proteins using a batch nephelometric method [37]. However, this flow nephelometric technique brings important improvements. The gradual increase in BSA concentration allows to determine the highest value of NTU of a sample, a difficulty that exists in the batch technique. The TSA of wines could be calculated by using the slope of the curve shown in Fig. 4b, being, in this example 36.5 NTU/(ml.min−1 ). On the other hand, wines can be directly analysed without previous dilution or any other treatment, allowing taking into account the colloidal properties of wines. It is believed that the colloidal state in which tannins are present in wines, involving other compounds such

as carbohydrates, could be determinant in their ability to bind proteins.

4. Conclusion The flow nephelometric technique described herein was shown to be a very fast and reliable method to study the interaction between tannins and proteins. The possibility of changing the flow of the different substrates involved is very functional, especially the flow of the sample solutions. This shows that the flow technique is versatile and possible to apply to complex samples such as different polyphenol extracts and beverages (red and white wines, fruit juices, beers, etc.). In wine industry, this method could also be useful to control the reactivity of enological tannins with proteins. In general, these tannins have a bitter, green astringent character and could contribute importantly to wine flavour. Thus, the type of tannins and the amount added during wine stabilization could be optimised using the present method. Further works will involve applying this technique as a nephelometric titration of tannins in solution and also to the study of the interactions of different kinds of tannins with other proteins (PRP and gelatin), and also to study the influence of some natural compounds such as carbohydrates in these interactions.

Acknowledgements This research was supported by a research project (POCTI/40124/QUI/2001) funding by Fundação para a Ciˆencia e Tecnologia (FCT), Portugal and by FEDER fundings. We thank Dr. Carlos Rocha Gomes for the development of the HACH data acquisition software. Elisabete Carvalho was supported by a grant from Fundação para a Ciˆencia e a Tecnologia (SFRH/BD/9325/2002).

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