Retention of Proanthocyanidin in Wine-like Solution Is Conferred by a

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The retention of proanthocyanidin in wine-like solution is conferred by a dynamic interaction between soluble and insoluble grape cell wall components. Keren A. Bindon, Sijing Li, Stella Kassara, and Paul A. Smith J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02900 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 16, 2016

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Journal of Agricultural and Food Chemistry

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The retention of proanthocyanidin in wine-like solution is

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conferred by a dynamic interaction between soluble and insoluble

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grape cell wall components.

4 KEREN A. BINDON1*; SIJING LI1,2; STELLA KASSARA1; PAUL A. SMITH1

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The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, SA, 5064, Australia.

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2

Australian Research Council Training Centre for Innovative Wine Production, School of

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Agriculture, Food and Wine, The University of Adelaide, PMB 1, Glen Osmond, SA 5064,

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Australia

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* Corresponding author Tel: +61-8-83136190; Fax: +61-8-83136601

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E-mail: [email protected]

15 16 17 18 19 20

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ABSTRACT

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For better understanding of the factors which impact proanthocyanidin (PA) adsorption by

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insoluble cell walls or interaction with soluble cell wall-derived components, application of a

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commercial polygalacturonase enzyme preparation was investigated in order to modify grape

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cell wall structure. Soluble and insoluble cell wall material was isolated from the skin and

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mesocarp components of Vitis vinifera Shiraz grapes.

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depolymerization of the insoluble grape cell wall occurred following enzyme application to

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both grape cell wall fractions, with increased solubilization of rhamnogalacturonan-enriched,

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low molecular weight polysaccharides. However, in the case of grape mesocarp, the

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solubilization of protein from cell walls (in buffer) was significant, and increased only

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slightly by the enzyme treatment. Enzyme treatment significantly reduced the adsorption of

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PA by insoluble cell walls, but this effect was observed only when material solubilized from

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grape cell walls had been removed. The loss of PA through interaction with the soluble cell

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wall fraction was observed to be greater for mesocarp than skin cell walls. Subsequent

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experiments on the soluble mesocarp cell wall fraction confirmed a role for protein in the

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precipitation of PA. This identified a potential mechanism by which extracted grape PA may

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be lost from wine during vinification, as a precipitate with solubilized grape mesocarp

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proteins. Although protein was a minor component in terms of total concentration, losses of

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PA via precipitation with proteins were in the order of 50% of available PA. PA-induced

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precipitation could proceed until all protein was removed from solution and may account for

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the very low levels of residual protein observed in red wines. The results point to a dynamic

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interaction of grape insoluble and soluble components in modulating PA retention in wine.

It was observed that significant

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Keywords: pectin; polygalacturonase; homogalacturonan, rhamnogalaturonan; condensed

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tannin; protein; adsorption, fining. 2 ACS Paragon Plus Environment

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Introduction

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The specific adsorption of condensed tannin (proanthocyanidin, PA) by insoluble

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grape cell wall polysaccharides and associated proteins removes, or prevents the extraction of

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PA during wine production 1-4. Modification of cell wall components by selective extraction5,

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6

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removal of pectic polysaccharides, rich in galacturonic acid, is associated with a loss in PA

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adsorption by grape cell walls. The results of a number of studies indicate that the observed

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phenomenon has less to do with the selective removal of polysaccharides with high

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selectivity for PA than the deconstruction of the supporting pectic network within the cell

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wall itself

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porosity. As such, cell wall porosity, with the resulting ability to encapsulate and facilitate

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hydrophobic interactions of PAs within cell wall cavities has been suggested to be of greater

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importance in defining PA-cell wall interaction than composition per se5, 9-12.

, or enzymatic breakdown7,

8

has been shown to alter the binding of PA. Specifically, the

5, 9-12

. Cell wall degradation can result in its collapse with a resultant loss in

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Recent studies have shown that pectic polysaccharides have the capacity to interact

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with PA in solution5 forming a stable, soluble complex. However, in circumstances where

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significant cell wall degradation has been exerted by enzymes, co-precipitation of soluble

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material and PA is observed8 and has been proposed to result from complexes formed with

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polysaccharides. Of the soluble grape-derived polysaccharides, evidence for the types of

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polysaccharides involved in the association with PAs is conflicting. For seed-derived PAs up

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to a mean degree of polymerization (mDP) 11, only the dimer of rhamnogalacturonan II (RG

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II, Mwt 10, 000 Da) was demonstrated to facilitate PA aggregation13, 14 leading to progressive

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precipitation, while arabinogalactan proteins (AGPs) and RG II monomers did not. On the

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other hand, using higher molecular mass apple PAs of mDP 30, aggregation was observed for

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AGPs with only minor effects observed for monomers or dimers of RG II13 suggesting that

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the composition of the PA as well as the polysaccharide structure is relevant. Notably, in the 3 ACS Paragon Plus Environment


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published study8 which observed co-precipitation of PA with material solubilized from skin

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cell walls in the presence of enzymes, seed PAs were used. Since treatment of grape cell

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walls with enzymes, particularly polygalacturonases, can enhance RG II extraction and

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retention15 this might account for the phenomenon observed8. However, further

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understanding of the grape to wine system using grape-derived skin PAs of a higher

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molecular mass is required, since these are compositionally more representative of the type of

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PAs found in wine16.

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Other molecular candidates for the formation of PA precipitates are proteins9, which

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in the case of grapes have been identified as pathogenesis-related (PR) proteins17. Recent

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work has shown that the loss of PA from solution as a precipitate can be significant,8

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particularly in the case of grape varieties containing elevated levels of PR protein17. In cases

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where adsorption of PA is compounded by poor extraction coefficients or removal via direct

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adsorption to cell walls in addition to loss as PA-protein precipitates, this can result in wines

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with negligible tannin concentration, such as in the case of native American Vitis species (and

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their hybrids with European species)8,

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vinifera) used in wine production have lower levels of soluble PR proteins, the total protein

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content in cell walls of skin and mesocarp is similar across a number of Vitis species, and the

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observed phenomena may be dependent not only on total protein concentration in cell walls,

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but also the degree to which that protein is solubilized during fermentation/crushing. In this

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regard, the role of cell wall deconstructing enzymes, in particular those which remain active

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in ripe grapes and during fermentation, are an important consideration.

17

. While the more common grape varieties (V.

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In V. vinifera, limited solubilization of skin cell wall-bound protein takes place during

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fermentation18, but that an apparent increase in skin cell wall-retained proteins is evident in

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fermented cell walls, potentially due to a proportional loss of cell wall polymers and soluble

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material. We have also shown that the extensive depolymerization of isolated skin cell walls


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of V. vinifera Shiraz resulted in no detectable release of protein6, suggesting that wine

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proteins in this cultivar may originate from a different berry component. An important

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correlation between juice-derived protein and wine protein17 has been demonstrated for red

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wines, as well as a stronger relationship between cell wall adsorption of PAs and protein in

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mesocarp cell walls1, 4. This effect is notably absent from skin cell wall material, for which

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PA adsorption appears to be related to the content of galacturonic acid-rich pectic material1, 4.

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Taken together, these observations suggest that the adsorption properties of skin and

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mesocarp cell walls for PAs may be conferred by different cell wall polymers, and we

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propose that differences may also exist in the nature and PA-reactivity of the soluble fraction

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from these two tissue types.

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In our early work on CWM-PA interactions, CWMs were pre-extracted under

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aqueous conditions to remove the soluble fraction, in order to explore the interaction of PA

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with insoluble CWM1, 4. Taking into consideration important new aspects raised by other

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research groups with respect to the role of the soluble CWM-derived material (PR-proteins,

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polysaccharides) and enzyme treatment in altering the CWM-PA response7, 8, 17, we aimed to

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revisit our previous results to incorporate these factors and give greater emphasis to the role

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of the soluble CWM fraction in defining CWM-PA interactions. The current study has sought

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to address the following key questions:

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1. Does the quantity, composition and molecular mass of soluble polysaccharide

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material from Vitis vinifera (cv. Shiraz) skin and mesocarp cell wall material

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differ, and how is this impacted by the action of pectolytic enzymes?

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2. Does PA interact differently with the soluble and insoluble fractions of cell

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walls, and does this have implications for PA extraction and retention in wine,

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in particular when pectolytic enzymes are used?



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3. Under which conditions does PA desorption or precipitation by the soluble

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cell wall fraction occur, and is this impacted by pectolytic enzyme

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application?

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Materials and methods

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Instrumentation. An Agilent model 1100 HPLC (Agilent Technologies Australia Pty Ltd,

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Melbourne, Victoria, Australia) was used with ChemStation software for chromatographic

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analyses.

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Preparation of juice, an ethanol-insoluble juice fraction, and acetone-insoluble grape

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cell wall material. For preparation of juice (soluble) polysaccharides, Vitis vinifera L. cv.

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Chardonnay grapes (1 kg) were obtained at 22 °Brix, pH 3.8 and crushed with addition of

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200 ppm SO2. The juice and solids were separated from skin and seed material by filtering

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through a coarse porcelain Buchner funnel and the juice was then centrifuged at 1730 g for 5

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min. Four volumes of 96% v/v ethanol were added to 40 mL of juice, and precipitation took

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place at 4 °C overnight, then centrifuged at 10000 g for 20 min, and the pellet retained. The

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pellet was reconstituted in 30 mM citric acid, and dialyzed against a 34 mm width, 3.7

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mL/cm, 3500 Da molecular weight cut-off membrane (SnakeSkin Dialysis Tubing®, Thermo

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Scientific, Rockford, IL, USA). Dialysis took place at 4 °C against four changes of Milli-Q

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water over 24 h. The sample in Milli-Q water was then frozen at -80 °C, lyophilized, and

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recovered gravimetrically (860 mg) as dry material. This material was found to convert 67%

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by mass to component monosaccharides following hydrolysis in 2 M trifluoroacetic acid

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(TFA), as described below.

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For preparation of grape cell wall fractions (acetone-insoluble), skin and mesocarp

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components were dissected from V. vinifera L. cv. Shiraz grape samples at a soluble solids

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levels of 23 °Brix and frozen whole at -80 °C until processed. The minimum sample size


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processed was 50 berries, and samples were prepared in triplicate. In order to collect both

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soluble and insoluble cell wall components, skin and mesocarp were extracted twice in 70%

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v/v aqueous acetone containing 0.01% v/v TFA over a 48 hour period. This approach differs

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from our previous published work which sought to examine only insoluble cell walls4. While

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in extraction solvent, skin and mesocarp material was homogenized using an Ultra-Turrax

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T25 high-speed homogenizer with a S25N dispersing head (Janke & Kunkel GmbH & Co.,

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Germany). Extraction was performed on a Ratek suspension mixer (Ratek Instruments Pty

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Ltd, Boronia, Victoria, Australia). Extracts were then centrifuged at 1730 g for 5 min,

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supernatants were discarded, and the 70% acetone insoluble residues retained. Cell wall

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material (CWM) was prepared from residues by extraction with slow shaking for 30 min in

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25 mL Tris-HCl equilibrated phenol pH 6.7 (Sigma-Aldrich, St. Louis, MO, USA), and the

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phenol-insoluble CWM residue recovered following filtration on glass microfiber. The CWM

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was then washed twice with 80% v/v ethanol, and then acetone to remove phenol. Samples

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were then extracted with slow shaking for 30 min in 1:1 v:v methanol:chloroform, and the

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CWM was washed twice with methanol and twice with acetone. CWM was recovered by

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filtration on glass microfiber, and solvent removed under vacuum. Yields of purified cell wall

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material were recorded and were 6.5 ± 0.27 and 11.5 ± 0.56 mg/berry for mesocarp and skin

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CWM respectively. CWM from the respective tissue types was pooled by extraction replicate

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and manually ground to a fine particle size with a mortar and pestle, passed through a 0.5 mm

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mesh, and frozen at -20 °C until used.

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Enzyme treatment of grape polysaccharide and cell wall components. An enzyme

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preparation typically used in red winemaking was sourced in powder form from a

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commercial supplier (Laffort, Burwood Ave, Woodville North, SA, Australia). The

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preparation was Lafase He Grand Cru with a reported polygalacturonase (PGU) activity of

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8600 units/g. The cinnamoyl esterase activity of the commercial enzyme preparation was 7 ACS Paragon Plus Environment


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reported to be less than 0.5 units/1000 PGU units. In addition to PGU activity, the preparation

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was reported to have a side-activity of arabinase, for which an enzyme activity estimate was

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provided. A preliminary experiment was performed on triplicate 20 mg samples of purified

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Chardonnay juice polysaccharide to determine the kinetics of galacturonic acid release from

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polysaccharide, in addition to other monosaccharide sugars, if any. Polysaccharides were

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reconstituted in 1.5 mL of 50 mM citrate buffer, pH 3.4 containing 1 mg/L of enzyme.

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Samples were placed on a Ratek suspension mixer (Ratek Instruments Pty Ltd, Boronia,

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Victoria, Australia) and held at 17 °C. Monosaccharide release was monitored over a 5 day

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period as described below. The kinetic study indicated the enzyme preparations contained

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primarily polygalacturonase activity, with minor arabinase activity, and no other significant

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release of monosaccharide sugars (Supporting information S1). A plateau in galacturonic acid

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release occurred at Day 4 with the concentration maintained to Day 5. Minor

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depolymerisation of galacturonic acid was found in a control sample containing only buffer

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which increased to day 5 (Supporting information S1). Therefore, all subsequent enzyme

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treatments were performed with a 4 day incubation period.

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Thereafter, 20 mg samples of Chardonnay polysaccharide or 30 mg purified Shiraz skin

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or mesocarp CWM were prepared in citrate buffer, or buffer containing PGU enzyme and

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incubated under the conditions described above. Experiments were performed in duplicate.

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After incubation, the samples were centrifuged at 21000 g and the supernatant and pellet

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were recovered. The pellet was washed 3 times with 1.5 mL of Milli-Q water and centrifuged

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between washes. Pellets were then dried by solvent exchange with acetone, air-dried and the

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gravimetric recovery recorded. The supernatant was analyzed directly for monosaccharide

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composition as described below. An aliquot of supernatant was precipitated in 5 volumes of

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ethanol at 4 °C overnight, then centrifuged at 21000 g. The precipitate was recovered in water

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and dialyzed against 4 changes of Milli-Q water at 4 °C using molecular weight cut-off 3500 8 ACS Paragon Plus Environment

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Da in 1 mL Pur-a-Lyzer dialysis tubes (Sigma-Aldrich, St. Louis, MO, USA). After dialysis,

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each sample was transferred to a fresh screw-cap centrifuge tube, and the dialysis tube

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washed with an additional 800 µL of Milli-Q water to ensure that any insoluble

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polysaccharide residue was recovered. Thereafter, the dialyzed samples were frozen at -80

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°C, lyophilized, and the gravimetric recovery recorded.

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Hydrolysis of cell walls and polysaccharides. Lyophilized soluble polysaccharide fractions

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were hydrolyzed in 2 M TFA at 100 °C in a dry bath for 3 h. Hydrolysates were cooled on

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ice, and thereafter concentrated under vacuum at 30 °C in a Heto vacuum centrifuge (Heto-

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Holten A/S, Allerod, Denmark). For whole, untreated CWM, buffer-extracted or enzyme-

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treated CWM, samples were hydrolyzed in 12 M H2SO4 for 3 h at room temperature, then

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diluted to 1M H2SO4 with Milli-Q water and placed at 100 °C in a dry bath for a further 3 h.

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Samples were cooled on ice, and neutralized with 4 M NaOH. Hydrolysates were analyzed

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for monosaccharides as described below.

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Monosaccharide composition of cell wall fractions. For the analysis of monosaccharides an

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adaptation of the method of Honda et al. (1989)19 with the modifications reported previously6

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was used. Samples containing monosaccharides were diluted in Milli-Q water, with ribose

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addition as an internal standard (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration

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of 0.3 M. The derivatizing reagent was 0.5 M of methanolic 1-phenyl-3-methyl-5-pyrazolone

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(PMP) in 1 M NH4OH. For the derivatization, 25 µL of sample was mixed with 96.2 µL of

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derivatizing reagent and placed in a heating block at 70 ºC for 1 h. After this step, the

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samples were cooled on ice and neutralized with 25 µL of 10 M formic acid. Then, the

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samples were extracted twice with dibutyl ether (Sigma-Aldrich, St. Louis, MO, USA) and

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the upper layer was discarded. The excess of dibutyl ether was dried under vacuum at room

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temperature. PMP-monosaccharide derivatives were quantified by HPLC using a C18 column

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(Kinetex, 2.6 µm, 100 Ǻ, 100 × 3.0 mm) protected with a guard cartridge (KrudKatcher Ultra 9 ACS Paragon Plus Environment


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HPLC in-line filter, 0.5 µm) (Phenomenex, Lane Cove, NSW, Australia). The mobile phases

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were solvent A, 10% (v/v) acetonitrile in 40 mM aqueous ammonium acetate, and solvent B,

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70% (v/v) acetonitrile in Milli-Q water. The following linear gradient was used: for solvent A

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(with solvent B making up the remainder) 92% at 0 min; 84% at 12 min; to 0% at 12.5 min;

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0% at 14 min, then returning to the starting conditions at 14.5−18.5 min, 92%. A flow rate of

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0.6 mL/min was used with a column temperature of 30 °C. The PMP-monosaccharide

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derivatives were identified at 250 nm using commercial standards (Sigma-Aldrich, St. Louis,

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MO, USA).

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Size exclusion chromatography of polysaccharides solubilized from cell walls. For

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analysis of polysaccharide molecular mass distribution, dried, dialyzed polysaccharide

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samples were resuspended to approximate a gravimetric concentration of 2 mg/mL in 0.1 M

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sodium nitrate and 25 µL was injected onto a BioSep Sec 2000 column (300 x 7.8 mm,

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Phenomenex, Lane Cove, NSW, Australia) fitted with a guard column of the same packing

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material. Analysis was performed by HPLC–SEC in 0.1 M sodium nitrate at a flow rate of 1

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mL/min. Polysaccharide elution was monitored by refractive index detection over 14 min.

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Column calibration was carried out with Shodex P-82 Pullulan Standards (Phenomenex, Lane

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Cove, NSW, Australia) from ≅ 5 kDa to ≅ 800 kDa. A fourth order polynomial was fitted

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with the cumulative mass distribution at 50% for each standard in order to determine

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molecular mass.

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Binding of proanthocyanidin and enzyme-treated skin and mesocarp cell walls.

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Proanthocyanidin (PA) was purified as a dry powder from immature Cabernet Sauvignon

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grape skins (veraison) as described previously20 and characterized by phloroglucinolysis21, 22

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using (-)-epicatechin (Sigma Aldrich, St. Louis, MO, USA) as the quantitative standard to

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determine subunit yield (70%), composition and a mean degree of polymerization (mDP), of

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21 units. Compositional information for the PA isolate is shown in Supporting information 10 ACS Paragon Plus Environment

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S2. Having a high relative subunit yield of 70% conversion by mass, the PA was deemed to

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be of sufficient purity for the experiment. Duplicates were performed for each experimental

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treatment, for which the overall design and analysis steps which is outlined in Figure 1. For

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each treatment, a 10 mg sample of dry skin or mesocarp CWM was weighed into 1.5 mL

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screw-cap centrifuge tubes, and reconstituted in 1.3 mL of citrate buffer alone (control), or

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containing PGU and allowed to react under the conditions described previously. Thereafter,

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the samples were centrifuged at 21000 g for 20 min. To control and enzyme-treated CWM

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samples, a 200 µL aliquot of ethanol containing 10 mg/mL of purified PA was added to

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determine the interaction of PA in the presence of both soluble and insoluble CWM-derived

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material. To determine the interaction of PA with the only material solubilized from enzyme-

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treated CWM, the supernatant was removed from a further treatment. To the insoluble CWM

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residue, and a fresh aliquot of 1.3 mL buffer was applied. A 200 µL aliquot of the ethanol/PA

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solution was added to both the supernatant and CWM residue samples. Samples were

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vortexed, and the interaction of PA and CWM, or enzyme-solubilized material was

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performed for a further 2 h at 17 °C on a suspension mixer. A standard blank of the PA

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solution without CWM was included. After interaction, all treatments were centrifuged at

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21000 g for 20 min, and the supernatants recovered in a fresh 10 mL centrifuge tube.

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Following interaction of enzyme-solubilized material and PA, a precipitate was observed to

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form which was recovered following centrifugation. The CWM residues and precipitates

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were extracted in 1 mL 13% v/v aqueous ethanol for 18 h, re-centrifuged, and the

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supernatants recovered in a 10 mL centrifuge tube. Supernatants recovered from the binding

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assays, as well as the 13% ethanol extracts of insoluble materials were transferred to 10 mL

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centrifuge tubes and 5 volumes of absolute ethanol were added. Solutions were retained at 4

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°C for 18 h, centrifuged at 1730 g for 10 min and the ethanolic supernatant and the pellet

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formed recovered. The pellets together with 13% ethanol-extracted CWM residues were 11 ACS Paragon Plus Environment


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extracted twice over a 24 h period in 1 mL aliquots of 70% v/v aqueous acetone, centrifuged

270

at 21000 g for 20 min, and the supernatants recovered and pooled. All ethanolic and 70% v/v

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aqueous acetone supernatants were concentrated under nitrogen at 30 °C and thereafter

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recovered directly in N, N-dimethylformamide (DMF) containing 1% v/v glacial acetic acid,

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5% v/v Milli-Q water and 0.15 M lithium chloride. Samples were directly analyzed by GPC22

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with the modifications previously described20. PA quantification was determined using the

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GPC peak area of the original PA at concentration levels which covered the range observed

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in the experiment. This approach was based on the observation previously reported23 that

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increased sensitivity and a wider detection range is found using this approach. For calibration

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of the GPC method to determine relative molecular mass distribution of the PA recovered in

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samples, preveraison skin PA fractions of known molecular mass were used as standards for

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GPC calibration24. For calibration, a second order polynomial was fitted with the PA elution

281

time at 50% for each standard.

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Precipitation of proanthocyanidin by material solubilized from mesocarp cell walls. A

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subsequent experiment was designed to investigate the formation of insoluble precipitates

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upon addition of PA to material solubilized from mesocarp cell walls. The experiment was

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repeated as described above, with the following modifications. CWM was prepared in buffer

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only, or buffer containing PGU, and the supernatants recovered after centrifugation. To

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supernatants, PA was added at 2 mg/mL and at 1 mg/mL, with buffer/ethanol concentrations

288

kept constant. Precipitates formed were recovered and directly extracted in two 1 mL aliquots

289

of 70% v/v aqueous acetone over 24 h. PA concentration in acetone extracts, as well as that

290

which remained soluble after formation of the precipitate, was analyzed by GPC as described

291

above. The precipitate residue following acetone extraction was lyophilized and diffuse

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reflectance MIR spectra of the dry material were obtained using a Spectrum-One

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(PerkinElmer, Wellesley, MA, USA) Fourier transform mid-infrared spectrometer (FT-IR),


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with comparison to known standards forming part of the AWRI Commercial Services FTIR

295

database. FT-IR analysis indicated an absence of tartaric acid, potassium hydrogen tartrate,

296

pectin, cellulose, based on spectra from commercial standards (Sigma-Aldrich, St. Louis,

297

MO, USA). FT-IR spectra of purified rhamnogalacturonan standards from soybean and

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potato (Megazyme, Bray, Co. Wicklow, Ireland) and calcium tartrate (synthesized standard)

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were also poorly related to those of the precipitate. Spectra from the precipitate were strongly

300

aligned with those of white wine protein (Supporting information S3) using a proteinaceous

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deposit isolated from a white wine as the standard (tested positive for protein via solubility in

302

0.1 M sodium hydroxide and staining with nigrosine). Similarity with the FTIR spectra of the

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grape skin tannin used in the current study (Supporting information S3) were also observed.

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Further analysis was performed to quantify protein, and possibly polysaccharide

305

associated with PA in precipitates. Sodium dodecyl sulfate polyacrylamide gel

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electrophoresis (SDS–PAGE) of cell wall extracts and the respective soluble and insoluble

307

material recovered after interaction with PA was performed with NuPage 12% Bis–tris and

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an XCell SureLock Mini Cell (Invitrogen, Mulgrave, Vic, Australia) following the

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manufacturer’s instructions, with 50 mg Na2S2O5 added to prevent cysteine oxidation.

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Aqueous samples were prepared by precipitating proteins with four volumes of cold ethanol

311

and the pellet was collected by centrifugation (14,000 g, 15 min, 4 ºC), before reconstitution

312

in loading buffer (Invitrogen NuPage recipe) with 3% 2-mercaptoethanol. Insoluble

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precipitate material was directly reconstituted in loading buffer. The standard used to

314

determine molecular weight was the BenchMark™ Protein Ladder (Invitrogen, Mulgrave,

315

Vic, Australia). Proteins were stained with Pierce Imperial Protein Stain (Quantum Scientific,

316

Sydney, NSW, Australia) according to the manufacturer’s microwave instructions. SDS-

317

PAGE analysis confirmed the presence of proteins in mesocarp extracts (Supporting

318

information S4) but resolubilization of precipitate material was not achieved. The presence of



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protein in mesocarp extracts (data not shown) was also confirmed using the Bio-Rad protein

320

assay kit (Bio-Rad Laboratories, Gladesville, NSW, Australia) with bovine serum albumin

321

(Sigma-Aldrich, St. Louis, MO, USA) as the quantitative standard, but could not be detected

322

in precipitates due to insolubility. Therefore, to confirm the presence of protein in both

323

extracts and precipitates, duplicate samples of the dried insoluble precipitates, as well as

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dried pellets prepared from soluble fractions upon addition of excess ethanol were hydrolyzed

325

in 1M H2SO4 at 100 ºC in a dry bath for 3 h. After cooling on ice, an aliquot was neutralized

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with NaOH and the remainder of the hydrolysate retained. Hydrolysates were analyzed for

327

amino acids by the method of Dukes and Butzke (1998)25 with appropriate sample blanks and

328

using L-isoleucine (Sigma-Aldrich, St. Louis, MO, USA) as the quantitative standard. For the

329

experimental samples, the 3 h, 1 M H2SO4 hydrolysis was found to give an equivalent yield

330

of amino acids to hydrolysis in 6 M HCl for 20 h, and was the preferred method for protein

331

quantification since polysaccharide content could also be quantified as monosaccharide units.

332

Monosaccharides were assayed in the neutralized aliquot as described above.

333

Desorption of cell wall-bound proanthocyanidin. A further experiment was designed to

334

determine whether re-extraction of CWM-bound PA was affected by the presence of material

335

solubilized from CWM. In order to produce larger volumes of CWM-soluble material, a

336

scaled-up extract (77 mg) of mesocarp CWM in either buffer or buffer-PGU (10 mL) was

337

prepared under the conditions described previously. Samples of 10 mg mesocarp CWM were

338

prepared in buffer only, or buffer containing PGU, and the supernatants removed after

339

centrifugation. The CWM residues were resuspended in ethanolic citrate buffer (13% v/v, pH

340

3.4) containing 2 mg/mL PA and allowed to react, then centrifuged and the supernatant

341

removed. The insoluble CWM-PA residue was extracted for 18 h at 17 °C in 1.3 mL of the

342

scaled-up buffer or buffer-PGU extracts of CWM, each which had been heated to 75 ºC for


Page 15 of 49


343

10 s to inactivate the enzyme and cooled prior to addition. Samples were centrifuged, and PA

344

was quantified in supernatants and residues as described above.

345

Statistical analysis. Data were analyzed by one-way analysis of variance (ANOVA) using

346

the JMP 7 statistical software package (SAS, Cary, NC, USA). ANOVAs were followed by a

347

post hoc Student’s T-test to determine differences between means.

348

Results and discussion

349

Solubilization of grape cell wall components by extraction in buffer or pectolytic

350

enzyme application. The recovery of buffer-soluble polysaccharide (as component sugars)

351

from mesocarp and skin CWM was comparable, at 0.21 and 0.36 mg respectively (Table 1).

352

Enzyme application significantly increased the yield of polysaccharide from both CWM

353

types in comparison with that extracted in buffer. The release of polysaccharide CWM by

354

enzyme was greater for skin than for mesocarp. Given that the total contribution of skin

355

CWM on a per berry basis was 11.5 ± 0.56 mg compared with 6.5 ± 0.27 mg for mesocarp

356

CWM, this indicates that under winemaking conditions involving skin contact, skin cell walls

357

should contribute most significantly to soluble polysaccharide release. These findings are in

358

agreement with those previously reported, that skin contact during red winemaking imparts

359

the bulk of grape-derived polysaccharide26,

360

converted to monosaccharides (≈ 15%) following mild or harsh acid hydrolysis was half that

361

of skin CWM (Table 1), suggesting a significant contribution of non-polysaccharide material

362

to the solubilized fraction, in agreement with previous results4.

27

. The degree to which mesocarp CWM was

363

What was surprising, was the extent of polysaccharide degradation to galacturonic

364

acid exerted by the application of pectolytic enzyme. Other authors have noted the extensive

365

depectination of cell walls conferred by enzyme application18,

366

determined quantitatively. Calculated from data shown in Table 1, the recovery of

367

monosaccharides (>76% as galacturonic acid) in buffer solution after enzyme application was

28

but this had not yet been



Page 16 of 49

368

between 33 and 43% of total recovered monosaccharides from CWM with 45 to 51%

369

remaining in the insoluble CWM fraction. Compositional investigation of the acetone-

370

insoluble grape CWM before, or following extraction in buffer; or treated with the

371

commercial enzyme preparations showed that enzyme application caused a loss of

372

galacturonic acid, rhamnose and arabinose in both tissue types studied (Figure 2).

373

Consequently, CWM residues were enriched in glucose (cellulose), xylose and mannose

374

following enzyme treatment.

375

Monosaccharide profiles of recovered soluble polysaccharides indicated that while a

376

substantial portion of CWM-derived galacturonic acid was converted by enzymes to the

377

monomeric form for both tissue types (Table 1) a significant amount was retained (Figure 3).

378

The soluble polysaccharides of enzyme-treated CWMs also had proportional increases in

379

rhamnose together with galacturonic acid relative to the buffer treatment, suggesting the

380

presence of rhamnogalacturonan, an expected result15,

381

enzyme-solubilized polysaccharides revealed that these were primarily in the lower molecular

382

mass range ≅ 6000 Da and were similar, regardless of the tissue type or enzyme preparation

383

used (Figure 4). Buffer-soluble polysaccharides were enriched in galactose and arabinose

384

(Figure 3) and had a higher molecular mass average (Figure 4, ≈ 77 – 104 kDa), more

385

representative of arabinogalactan proteins. The absence, or proportional reduction of a higher

386

molecular mass polysaccharide fraction in the enzyme treatments suggests that this was

387

partially depolymerized, yielding lower molecular mass fractions. However, due to the

388

increase in total soluble polysaccharide concentration with enzyme application (Table 1) the

389

large increase of lower molecular mass material was likely to be primarily as a result of

390

extraction from the grape cell walls, and we note that the peak is within the range reported

391

previously for the rhamnogalacturonan (RG) II monomer

392

walls contain both RGI and RGII, with RGI being more abundant27 the possibility exists that

18, 26

. Size exclusion analysis of the

27, 29

. However, since grape cell


Page 17 of 49


393

both fractions were extracted by enzyme. RGI is also known to be extracted in significant

394

quantities from plant cell walls by pectolytic enzyme but has been observed to be

395

polydisperse, and of a higher molecular mass (36 – 200 kDa),30 however the size properties

396

of grape-derived RGI are unknown. This may be due to its limited importance, as it is usually

397

not retained in wine,29 possibly due to the action of glycanases during crushing and

398

fermentation. Our study did not further attempt to determine the potential contributions of

399

RGI or II to the enzyme-solubilized polysaccharide fraction since it was beyond the scope of

400

the primary objective of the study: to explore the interaction of soluble and insoluble cell wall

401

materials with PA.

402

Effect of cell wall depolymerization on the adsorption of proanthocyanidin. Some recent

403

research has attempted to unravel the effects of cell wall depolymerization on the resulting

404

interaction with PA, yielding key observations6-8. In those studies, the removal of the pectic

405

fraction from skin CWM by the application of chelating agent (CDTA) or enzyme application

406

(pectolytic mixture, PGU or cellulase) was found to reduce the adsorption of PA relative to

407

the native CWM. In the case of enzyme treatments, it was interesting to note that the type of

408

enzyme used for depolymerization was less important in defining the PA-binding effect than

409

the loss of pectic material itself, which can be facilitated by cellulase activity even more

410

effectively than for pectin-specific enzymes8. Our current results using skin PA support these

411

previous observations7, 8, and we noted that total PA retained in supernatants was higher

412

following interaction with PGU-treated CWM relative to the buffer-treated native CWM

413

(Table 2). This observation was confirmed by analysis of the insoluble PA-CWM complex,

414

whereby PA desorbed in dilute ethanol, and 70% v/v aqueous acetone was lower for PGU-

415

treated skin CWM compared to buffer-extracted skin CWM. As evidenced by GPC analysis

416

of the PA desorbed from CWMs, high molecular mass PAs were preferentially bound by skin

417

CWMs (Table 3), in agreement with previous observations6-8. A small effect was observed



Page 18 of 49

418

whereby the molecular mass of bound skin CWM PAs was slightly greater following

419

enzymatic CWM depolymerization.

420

For the PGU treatments of skin CWM, the recovery of PA in the supernatant (Table

421

2) was higher when the PA-CWM interaction was performed with fresh buffer (i.e. in order

422

to remove the effect of the soluble fraction on PA-CWM binding). However, when skin

423

CWM-PA complexes from these treatments were analyzed there was no difference in the

424

amount of bound PA. It was of interest to note that the total recovery of PA, relative to the

425

control was lower when PA was added directly to the PGU-treated supernatant, suggesting a

426

fraction of PA was rendered non-extractable in 70% v/v aqueous acetone. This was not

427

observed when PA was added to buffer solution containing only enzyme (data not shown)

428

indicating the loss in PA recovery was due to interaction with material solubilized from

429

CWM, and not with the enzyme itself.

430

The reduction in PA recovery relative to the control was observed to be even greater

431

in the same experiments performed on mesocarp CWM, relative to skin CWM. For mesocarp,

432

total PA remaining in solution after interaction with CWM was not different between the

433

buffer and PGU treatments. Irrespective of this, a strong effect of PGU treatment on insoluble

434

mesocarp CWM-PA complexes was found, and the amount of PA extractable in 70% v/v

435

aqueous acetone was reduced in PGU-treated CWM. This was also observed when the PGU-

436

soluble material was removed and replaced with fresh buffer prior to interaction of enzyme-

437

treated mesocarp CWM with PA, suggesting that CWM depolymerization reduced PA

438

adsorption. To further understand the lack of a PA reduction effect in the analysis of the

439

supernatants, we noted that in the interaction of the PGU-solubilized supernatant with PA, a

440

visible precipitate formed. PA could only be partially re-extracted from this precipitate in

441

70% v/v aqueous acetone. The molecular mass of the recovered PA from this precipitate was

442

high, in the order of 30,000 g/mol (Table 3). The resulting PA recovery following the


Page 19 of 49


443

interaction was low, even in CWM samples which had not undergone PGU treatment, and

444

due to this we considered that further experiments were required to understand our

445

observations for mesocarp CWMs.

446

To confirm whether enzymatic depolymerization of mesocarp CWM reduced PA

447

adsorption, the experiment was repeated in the absence of the soluble fraction (Table 4). For

448

this experiment, the supernatant was removed from buffer- and PGU-treated mesocarp

449

CWMs and replaced by fresh buffer containing PA. In this instance, reduced PA adsorption

450

by CWM following PGU treatment was evident in both the soluble and insoluble (PA-bound)

451

components despite the fact that the total PA recovery was still low (74%, not different

452

between treatments) and the effect on the bound PA molecular mass was unchanged (data not

453

shown) from that observed in the preliminary experiment (Table 3). This result suggests that

454

PGU treatment reduced PA adsorption by mesocarp CWMs, but it is noteworthy that a

455

significant fraction of PA could not be re-extracted from the insoluble PA-CWM complex,

456

irrespective of whether enzyme- or buffer-treatments were applied.

457

To determine whether the soluble fraction itself might affect the re-extraction of

458

CWM-bound PA, a desorption study of CWM-bound PA was also performed, in this instance

459

with buffer only, buffer-soluble fraction or the PGU-soluble fraction. This was a different

460

approach from desorption in 13% v/v aqueous ethanol as for the first experiment (Table 2),

461

aiming to understand whether association of CWM-bound PA and solubilized CWM

462

components might occur. It was found that PA was less effectively removed from PGU-

463

treated CWM than buffer-treated CWM (Table 4). While this might suggest that associations

464

between PA and CWM were rendered non-reversible (in buffer) by PGU treatment, it is also

465

important to qualify that the overall amount of PA bound in the PGU-treated mesocarp CWM

466

was lower. Importantly, the presence of buffer soluble material was found to exert a limited

467

effect on PA re-extraction from grape PA-CWM complexes, which differs from previous



Page 20 of 49

468

results on apple which showed that polysaccharides from cell walls could facilitate PA

469

desorption from PA-CWM complexes11. Overall, re-extraction of PA bound to insoluble

470

grape CWM was very low, as observed by others7, 8 and was only marginally increased by

471

buffer-soluble polysaccharide in native CWM but not in enzyme-treated CWM. This result

472

indicates that the nature of the soluble CWM fraction is not likely to affect tannin

473

resolubilization after it has been adsorbed by CWM.

474

To provide context for our observations regarding mesocarp CWM from these

475

experiments, we note that in our early work on CWM-PA interactions, the CWMs used had

476

been pre-extracted under aqueous conditions to remove the soluble fraction4, 24. Under those

477

conditions, insoluble mesocarp CWM was consistently found to remove more PA from

478

solution than skin CWM. For the current dataset which explored the interaction with whole

479

(soluble and insoluble) CWM, the observed difference in PA adsorption by skin and

480

mesocarp CWMs could not be well qualified, particularly due to the differences in total PA

481

recovery between tissue types. In the case of mesocarp CWM, this points to an important

482

interaction between the soluble and insoluble CWM components which defines how PA is

483

retained or lost from solution.

484

The data presented (Table 4) have shown that the soluble grape fraction had a limited

485

effect in facilitating desorption of CWM bound PA, and as such it was of greater relevance to

486

explore the conditions under which the CWM soluble fraction caused the precipitation and

487

loss of PA from solution. Given the more significant effect on PA precipitation observed in

488

the mesocarp extracts, a follow up experiment was performed using PGU-soluble and buffer-

489

soluble supernatants, with PA addition at two concentrations of 1 and 2 mg/mL (Table 5).

490

The results obtained were similar to those observed previously (Table 2) with a similar low

491

total PA recovery (76%) from the control which was not significantly different between

492

treatments. Surprisingly, there was no significant effect of enzyme treatment on the


Page 21 of 49


493

precipitation of PA, indicating that the component facilitating the precipitation event was

494

readily soluble from the mesocarp CWM in buffer. In terms of the amount of PA precipitated

495

under different PA concentrations, it was noteworthy that while slightly less PA was

496

precipitated at the lower PA addition, the recovered concentrations in precipitates were close,

497

regardless of enzyme treatment. As a result, 15-16% of PA was precipitated from the 2

498

mg/mL addition, and a higher proportion (24-30%) was removed from the lower addition.

499

This indicates that under conditions where a low PA concentration exists in wine, a greater

500

proportion of otherwise soluble PA might be lost via precipitation with material solubilized

501

from CWM during crushing and fermentation, an important observation.

502

Identification of compounds which confer the proanthocyanidin precipitation

503

response. A similar observation was recently reported for extracts of skin CWMs isolated

504

from cv. Monastrell grapes8 and based on the observation that greater precipitation was

505

observed for extracts derived from enzyme addition (PGU, cellulase) which were enriched in

506

pectic polysaccharides. Considering the evidence which exists for polysaccharide-PA

507

interactions5, 14, 31 a logical consideration would be that the loss of PA from solution might be

508

through interaction with the soluble polysaccharide component, followed by precipitation.

509

Our current results have indicated that the yield of polysaccharide (in buffer) from skin CWM

510

was similar to mesocarp CWM (Table 1), and increased in the presence of enzyme.

511

Regardless of whether CWM origin was mesocarp or skin, when extracted in buffer or in the

512

presence of enzyme, the polysaccharides recovered were compositionally similar and of

513

equivalent molecular mass (Figures 3 and 4). A further observation was that the increase in

514

polysaccharide yield associated with the enzyme addition, and the corresponding reduction in

515

the average polysaccharide molecular mass did not affect the PA precipitation response,

516

either positively or negatively.



Page 22 of 49

517

Noting that a recent report has also highlighted the importance of protein, in particular

518

pathogenesis-related proteins in conferring the PA-adsorption properties of mesocarp cell

519

walls1, and likewise precipitation of PA in wines,17. Although the phenol extraction step of

520

the cell wall preparation would be expected to remove adventitious (cytoplasmic) proteins

521

from the cell walls, it was conceivable that a fraction of cell wall-associated protein may have

522

been solubilized by the mild chelating activity of citrate buffer, or the more complete

523

depolymerization exerted by enzymes. We therefore considered that quantification of protein

524

in mesocarp extracts might be an important consideration in addition to polysaccharide.

525

Preliminary investigation of the tannin precipitates or the supernatants recovered following

526

precipitation indicated an absence of protein detectable either using the Bradford32 assay or

527

SDS-PAGE. On the other hand, the original CWM extracts from mesocarp (either in buffer or

528

in the presence of enzyme) were found to contain protein by the Bradford method (data not

529

shown), being slightly higher in the enzyme solubilized extract. SDS-PAGE evaluation of the

530

proteins indicated low concentrations of protein present within the molecular mass range of

531

grape chitinase and thaumatin-like PR proteins33 at 20-25 kDa (Supporting information S4)

532

and a more dense band at 10-15 kDa of an unidentified protein, possibly a lipid transfer

533

protein34 which was elevated in the enzyme treatment. SDS-PAGE analysis of the PGU

534

enzyme mixture itself indicated that the enzyme proteins were below detectable levels at the

535

concentrations applied in the treatments, and were of a higher molecular mass than the grape-

536

derived proteins.

537

Considering the low PA recoveries and the absence of detectable protein in PA-

538

precipitates (or supernatants) in the experiments it was likely that the precipitate had been

539

rendered resistant to solubilization even in 70% v/v aqueous acetone or denaturing reagents 2

540

mercaptoethanol/lithium dodecyl sulfate (as part of Invitrogen NuPage recipe) respectively.

541

Since preliminary investigation of the precipitate composition by FT-IR (data not shown, see


Page 23 of 49


542

supporting information S3) suggested the presence of both PA and protein based on a

543

comparison with spectra of pure compounds (skin PA and white wine protein), an alternative

544

protein quantification approach was followed using harsh acid hydrolysis of samples, with

545

quantification as non-proline amino acids. Although the approach excluded the quantification

546

of proline, it nonetheless confirmed the presence of a significant amount of protein in ethanol

547

precipitates (Figure 5A) and PA precipitates from mesocarp, with a minor amount remaining

548

soluble (Figure 5B). The protein quantity precipitated was similar between the two PA

549

additions tested (1 and 2 mg/mL) and between buffer- and enzyme-solubilized mesocarp

550

extracts. However, based on the initial assessment of protein in the two CWM treatments it

551

was evident not all protein was accounted for in the enzyme-treated samples following

552

precipitation, again likely to be due to limited solubility even under harsh acid-hydrolysis

553

conditions. Nonetheless, the results provide strong evidence for the presence of significant

554

quantities of protein in PA precipitates from mesocarp. In an assessment of PA precipitates

555

from skin PGU extracts, however, only trace amounts of protein were recovered following

556

the same hydrolysis approach at 2.25 µg/mL and was not detected in PGU-only controls. We

557

had previously shown that protein in solubilized fractions of the same skin cell walls was

558

below detection limit using a standard analytical method for protein6. Although this lower

559

level of protein in skin extracts by comparison with mesocarp may account for the relatively

560

reduced precipitation of PA (Table 2), it was nonetheless important to consider the results of

561

other work on Vitis vinifera cv. Monastrell grape skins8 suggesting that polysaccharide-PA

562

interactions may underpin the PA precipitation response.

563

Therefore, an assessment of polysaccharide distribution in the precipitates following

564

PA addition was required. Using the same acid hydrolysates for which protein was assayed as

565

amino acids, monosaccharide recovery was determined. In PA precipitates from the skin

566

PGU extract, insoluble polysaccharide was found to be associated with precipitates at 14.2



Page 24 of 49

567

µg/mL, a significantly greater concentration than the proteins (2.25 µg/mL) recovered in

568

these samples. The carbohydrate-rich material recovered had a molar percentage composition

569

as follows (data not shown) of glucose (39%) > galactose (20%) > glucuronic acid/rhamnose

570

(8%) > xylose (7%). This demonstrates that while some polysaccharide material may have

571

been associated with PA precipitates in skin extracts, this was not necessarily the galacturonic

572

acid-rich pectic material proposed to be responsible for PA precipitation proposed previously

573

for the Monastrell grape skin study8. Given the extremely small precipitation response in skin

574

extracts, and low recoveries of both protein and polysaccharide, the phenomenon was not

575

further investigated in skin CWM extracts.

576

In mesocarp extracts on the other hand, PA precipitate material was found to contain

577

99.5% recovered in the supernatant (soluble, non-precipitated)

578

fraction (data not shown). Supernatant-associated polysaccharides were found to have

579

equivalent concentration and composition (independent of enzyme or buffer treatment) to that

580

reported in the initial analysis (Table 1, Figure 3) and only trace amounts of xylose and

581

arabinose were associated with the precipitate. Together with the observations that protein

582

was associated with the PA precipitate, the polysaccharide recovery data suggests that trace

583

amounts of cell wall protein, and not polysaccharide, were the principal molecular candidates

584

associated with PA precipitation in mesocarp extracts. This result strongly supports the recent

585

observations for certain non-vinifera grape varieties, which have high levels of protein

586

associated with mesocarp cell walls, with concomitant low levels of extractable (retained) PA

587

in finished wines1 suggesting that a similar phenomenon may exist in vinifera varieties albeit

588

of a smaller magnitude. In the follow-up study by the same group,17 the retention of protein in

589

finished wines from non-vinifera grape varieties studied was associated with the ongoing

590

precipitation of grape tannin added post-vinification. The absence of this occurrence in their

591

finished vinifera wines was proposed to be due to pre-fining of protein by PA in the earlier


Page 25 of 49


592

stages of vinification. In that case, protein levels in vinifera varieties were expected to be at

593

concentrations low enough to be precipitated via interaction with PA and mostly removed

594

from the wine. Nevertheless, we note that soluble complexes of protein, PA and potentially

595

polysaccharide have been observed in the vinifera variety Pinot noir35. While some of this

596

protein may be yeast-derived mannoprotein or grape arabinogalactan protein, our current

597

results suggest that a small amount of grape-derived protein (possibly including PR protein)

598

may remain in stable solution with tannin (5-9%), an effect which appears to be somewhat

599

independent of PA concentration.

600

Partitioning of soluble proanthocyanidin after the interaction with cell walls.

601

Considering the substantial change in soluble polysaccharide concentration and composition

602

after cell wall depolymerization, a further relevant aspect to study was the partitioning of PA

603

between complexed and free (unbound) states. A previous publication from our group has

604

shown that in Pinot noir wines, up to 33% of wine PA may be associated in a complexed

605

form (with polysaccharide, protein)35. The approach by which the complexed PA is measured

606

involved precipitation in excess ethanol, which excludes the estimation of secondary colloidal

607

interactions (hydrophobic interaction, stacking). A similar approach to that reported

608

previously35 was followed but PA in complexed form was estimated following extraction in

609

acetone after precipitation in ethanol. This is because we had previously shown that

610

complexed PA was effectively re-solubilized using this approach35. For the addition of PA to

611

mesocarp or skin CWM prepared in buffer, similar concentrations of total PA remained in

612

solution, albeit slightly higher for skin CWM, as discussed previously (Table 2). Within this

613

soluble fraction, 22% and 18% were in complexed form respectively (Table 2), and the

614

complexed PA was of a higher molecular mass than unbound PA (Table 3), in agreement

615

with our previous observations35. In the instance where PGU-depolymerized CWM was

616

reconstituted in fresh buffer containing PA, the PA retained in solution was found not to form



Page 26 of 49

617

complexes, independent of the origin of the CWM. This, together with the observation that

618

enzyme addition to PA alone did not produce complexes, indicated that material solubilized

619

from the CWM facilitated complex formation. What was unexpected in the results was that

620

while PGU-treated CWM extracts from mesocarp consistently formed soluble PA complexes,

621

and these complexes were greatly reduced in the same treatments of skin CWM. The very

622

low concentration of complexes formed in the PGU-treated skin CWM extracts were

623

associated with far higher molecular mass PA than the corresponding treatments in mesocarp

624

(Table 3) possibly indicating that a different mechanism was associated with complex

625

formation.

626

We have previously reported an absence of detectable protein in extracts of the same

627

skin CWM used in this study6 suggesting that protein was not likely to be the factor in

628

complex formation with PA in the buffer-extracted skin CWM sample. We do recognize,

629

however, that trace amounts of protein may have been present in the PGU extracts, since a

630

loss in PA recovery was observed, but this was not investigated further. In this instance,

631

therefore, the similarity in released polysaccharides between mesocarp and skin cell wall

632

buffer extracts (Table 1) with similar molecular mass average (Figure 4) infers

633

polysaccharide as the likely molecular candidate for complex formation. We note that the

634

binding affinity between whole, soluble polysaccharide extract from plant cell walls for PA is

635

observed to be higher5 than when sub-fractions (fragments) of defined polysaccharide classes

636

interact with the same PA13,

637

dimensional structure (size, porosity) is important in defining the degree of PA association

638

with polysaccharides. The reduction in complex formation in PGU-treated skin CWM

639

extracts could have been associated with the reduction in polysaccharide average size, despite

640

the net increase in polysaccharide concentration and pectin associated with CWM

641

depolymerization. However, it was notable that despite the similarity in polysaccharide

31

, under the same conditions, suggesting that the three-


Page 27 of 49


642

composition and size between skin and mesocarp PGU extracts, the formation of complexes

643

still occurred in the PGU-treated mesocarp CWM extracts, and was in fact increased in the

644

extract in which CWM was absent. In the case of mesocarp, therefore, differences in

645

polysaccharide structure or composition may exist which were not characterized by our

646

methods, or a different molecular candidate may have been involved in the interaction.

647

Speculatively, the very small proportion of residual protein remaining in stable solution with

648

PA may have been in a complexed form. To address this question further using the suite of

649

techniques available, the partitioning of PA in free and complexed form was explored where

650

additions of PA were made at two concentrations (1 and 2 mg/L) to mesocarp extracts in

651

buffer or PGU, for which data was presented previously (Table 5). In that instance, the

652

distribution of PA (free or complexed) was the same irrespective of whether mesocarp had

653

been treated with enzyme or not (data not shown). The proportion of PA in complexes was

654

not changed between treatments and was ≅ 28%, similar to what was observed in the

655

preliminary experiment (Table 2). This finding suggests that complex formation in mesocarp

656

extracts was not altered by PGU treatment, and was therefore not affected by the observed

657

changes in polysaccharide concentration, size distribution or composition. Based on this

658

observation, it is more likely that another factor, possibly residual soluble protein, underpins

659

the response by mesocarp material.

660

These results raise some key questions regarding the colloid state of PA in the

661

presence of grape-derived soluble materials. While we have observed a strong precipitation

662

effect for protein and PA, yet the association of PA and polysaccharide remains less clear

663

since complexes may not necessarily precipitate, potentially interacting stably (colloids) in

664

aqueous solution. Notably, the effect of polysaccharide molecular mass and three-

665

dimensional structure in defining interactions with PA should be an important consideration

666

for future studies, in addition to structural and compositional differences. Furthermore, the



Page 28 of 49

667

effect of trace quantities of protein in solution (ng-µg quantities per mg PA) will need further

668

clarification for its potential role in determining both PA stability in solution, and PA

669

colloidal state.

670

Practical implications of proanthocyanidin precipitation during winemaking. The study

671

of PA-cell wall interactions in grapes was initiated following the recognition that these may

672

determine PA extractability and retention in wine36-38. Adsorption by cell wall fragments

673

released during crushing and fermentation may remove solubilized PA from wine when

674

settled as lees. However, PA-cell wall interactions in the grape skin (possibly seed, mesocarp)

675

may also prevent or limit PA extraction. As a result, predicting PA extractability and

676

retention in wine from a grape-based measure has proven challenging, since multiple levels

677

exist at which interactions can occur. This means that extraction outcomes do not reflect a

678

linear relationship between grape and wine PA concentration. Recently, our group has

679

identified an approach by which wine tannin concentration could be predicted from a grape

680

sample39 by gently crushing the grapes and extracting with a dilute ethanol solution. It was

681

found that tannin extracted via this process was strongly related to tannin concentration in

682

finished wines, and was primarily skin-derived. We proposed that this approach performs

683

well because tannin-cell wall interactions which would have occurred under winemaking

684

conditions were mimicked during the procedure. However, until now we had not considered

685

that other factors, namely soluble material from grapes, may also strongly impact the grape-

686

wine PA relationship. Noting this, protein-PA interactions may also have occurred during the

687

extraction of grapes via the published39 approach.

688

From the model studies performed, we note that a sizeable portion of soluble PA was

689

lost from solution as a precipitate with mesocarp-derived protein. Considering that mesocarp

690

treatments were associated with a loss in total PA recovery, losses could approach 50% if this

691

was factored into the calculation. Based on our cell wall yield results, total protein released


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692

from mesocarp during standard winemaking (without enzyme) would be in the order of 63

693

µg/mL, an amount which may be greater than the protein concentrations often used as wine

694

fining agents for PA40. Taken together with the potential contributions of juice-soluble PR-

695

proteins, which were not quantified in this study but have been noted to be ≈ 30 µg/mL in

696

Shiraz grapes33 this represents a significant PA-fining capacity derived from native grape

697

proteins. To follow on from the observations for certain non-vinfera grape varieties17 that

698

high concentrations of PR-proteins (up to 700 µg/mL) in finished wines can remove a

699

significant fraction of added PA as a precipitate, our work shows that albeit at far lower

700

protein concentrations, red vinifera varieties may also present PA losses via precipitation

701

with proteins during crushing and winemaking. This also might account for the generally low

702

levels of grape-derived PR protein recovered in finished red vinifera wines, which for Shiraz

703

in particular are

Retention of Proanthocyanidin in Wine-like Solution Is Conferred by a

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