Grape skins as supplements for color development in Pinot noir wine

Journal Pre-proofs Grape skins as supplements for color development in Pinot noir wine Angela M. Sparrow, Robert G. Dambergs, Dugald C. Close PII: DOI: Reference:

S0963-9969(19)30593-9 https://doi.org/10.1016/j.foodres.2019.108707 FRIN 108707

To appear in:

Food Research International

Received Date: Revised Date: Accepted Date:

8 May 2019 18 September 2019 21 September 2019

Please cite this article as: Sparrow, A.M., Dambergs, R.G., Close, D.C., Grape skins as supplements for color development in Pinot noir wine, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres. 2019.108707

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier Ltd.

Full Title Grape skins as supplements for color development in Pinot noir wine Author names and affiliations Angela M Sparrowab,*, Robert G Dambergsac and Dugald C Closea a

Tasmanian Institute of Agriculture, University of Tasmania, Hobart, Tasmania, Australia.

Angela Sparrow, principal author, conducted the research during her PhD candidature. Robert Dambergs and Dugald Close had a supervisory role for research and manuscript preparation. *

Corresponding author: Angela M Sparrow

b

Permanent Address: 353 Rosevears Drive, Lanena, Tasmania 7275, Australia

Email: [email protected] Additional authors: c

Robert G Dambergs: Wine TQ, PO Box 3 Monash SA 5342, Australia

Email: [email protected] a

Dugald C Close: Tasmanian Institute of Agriculture, Private Bag 98, Hobart, Tasmania, 7001

Australia. Email: [email protected]

1

Abstract A particular challenge to making wine from Pinot noir grapes is the delicate flavor, light color and poor ageing potential of the wine. Conventional Pinot noir must preparations were compared with those made using a skin-based supplement to assess the impact on nonbleachable (sulfur resistant) pigments in the wine. When supplemented with either fresh grape pomace of Pinot noir, Pinot gris or Chardonnay grapes; Pinot noir grape marc or a commercial liquid grape skin extract, the additional seeds and pulp from the supplements were shown to compromise the development of stable pigments in the wine. To compare the relative merits of tannin derived from grape skins and seeds, the supplements used in a parallel experiment were the skins alone of the same three grape varieties and at six months bottle age, the stable pigment concentration was found to exceed the amount attributable to the supplement. A third experiment used fermented grape skins as the supplement, with 85% of the supplementary anthocyanin recovered as stable pigment complexes in the wine. Notably, this series of experiments showed that supplements containing grape seeds appeared to compromise non-bleachable pigment formation in the wine while skin only supplements stimulated their development.

Keywords: anthocyanin; grape marc; non-bleachable pigment; seed; skin; tannin. 1. Introduction Anthocyanin pigments and tannins are major components of red wine and their relative concentrations have been correlated with measures of wine quality (Cozzolino, Cynkar, Dambergs, Mercurio, & Smith, 2008; Holt, Francis, Field, Herderich, & Iland, 2008; Kassara & Kennedy, 2011). The different properties, localizations and extractability of skin and seed tannins influence the contribution of each tissue source to phenolic attributes of the wine (Vidal, Francis, Guyot, Marnet, Kwiatkowski, Gawel, & Waters, 2003; Cortell, Halbleib,

2

Gallagher, Righetti, & Kennedy, 2005; Koyama, Goto-Yamamoto & Hashizume, 2007; Sparrow, Dambergs, Bindon, Smith, & Close, 2015). Consequently wine quality is governed by the extraction of skin and seed tannins in optimal proportions (Bautista-Ortin, FernandezFernandez, Lopez-Roca, & Gomez-Plaza, 2007; Sparrow, Dambergs, Bindon, Smith, & Close, 2015; Sparrow, Holt, Pearson, Dambergs, & Close, 2016; Sparrow & Smart, 2017). All phenolic compounds are unstable and undergo numerous enzymatic and chemical reactions during winemaking and aging, making the effect of a supplement difficult to predict. Many of the changes in appearance and sensory properties during aging of red wine have been ascribed to anthocyanin-tannin reactions (Cheynier, Duenas-Paton, Salas, Maury, Souquet, Sarni-Manchado, & Fulcrand, 2006). Flavanols are the building blocks of both wine pigments and tannin, therefore the quantity and source of the flavanols influence the degree of polymerization of proanthocyanidins (condensed tannins) and the astringency and bitterness of red wine (Singleton 1992; McRae, Schulkin, Kassara, Holt, & Smith, 2013). Both the grape variety and the tissue source (skin or seed) determine the amount and structure of the flavanols that occur in the wine matrix (Mattivi, Vrhovsek, Masuero, & Trainotti, 2009; Sparrow, Dambergs, Bindon, Smith, & Close, 2015; Soares, Brandão, Mateus & de Freitas, 2017). The concentration of stable, non-acylated forms of anthocyanin in Pinot noir grapes is low when compared to other red wine grape varieties, which generally have a greater range and concentration of anthocyanins (Mazza, Fukumoto, Delaquis, Girard, & Ewert, 1999). Pinot noir grapes also have an unusual distribution of tannins within the berry, with seed tannins being considerably higher in concentration than skin tannins (Mattivi, Vrhovsek, Masuero, & Trainotti, 2009; Kennedy, 2008; Downey, Harvey, & Robinson, 2003; Sparrow, Dambergs, Bindon, Smith, & Close, 2015). In addition, the procyanidins and proanthocyanidins found in grape seeds have a different subunit composition from those occuring in grape skins, which

3

in turn impacts the structural configuration of the proanthocyanidins and consequently wine quality (Rousserie, Rabot and Geny-Denis 2019). To balance these characteristics of Pinot noir grapes, supplementary sources of grape pigments or tannins are sometimes included with the fermenting must to improve the wine quality parameters and color stability of the wine. Food processing legislation however, places constraints on the addition of supplementary compounds to wines, a condition that is supported by the market demand for products containing only locally produced, naturally occurring raw materials. The use of enotannins provides one option (Soares, Brandão, Mateus & de Freitas, 2017), these being commercially available products that consist of tannins extracted from oak wood or other suitable plant products such as grape seeds or skins and are sometimes used in red winemaking to improve aging characteristics such as texture, depth of color and wine color stabilization, attributes that are particularly vulnerable in Pinot noir wines (Crespy, 2003). However, the effectiveness of enotannins is not guaranteed; in a recent report on the influence of winemaking supplements on tannin composition and sensory properties of Shiraz wines (Li, Bindon, Bastian, Jiranek and Wilkinson 2017), the effect of a commercial, grape derived enotannin was found to be negligible. The main difference between white and red grapes is the color of their skins. The red skin pigment is due to the presence anthocyanins (flavan-3-ols) found in the skin cells (Nel, 2018). Many commercial wineries produce a number of wine styles from different grape varieties, such that a range of grape solids; stalks, seeds and skins are produced as by-products. In the case of white or sparkling wines, fresh juice is pressed off the grapes prior to fermentation leaving the fresh grape solids (the pomace: composed of skins, pulp and seeds), whereas grape marc (fermented skins and seeds) is a common waste product of red wine production. While these by-products are normally either composted, sent off-site for tannin extraction or

4

used as stock-feed, both are readily accessible sources of grape phenolics that might be used to supplement new wine (Somers, 1971; Muhlack, Potumarthi, & Jeffery, 2018). Our previous investigations showed that source of grape tannin (skin or seed) has a significant impact on the non-bleachable pigment concentration of Pinot noir wine (Sparrow, Dambergs, Bindon, Smith, & Close, 2015, Sparrow, Smart, Dambergs, & Close 2016; Sparrow & Smart 2017). Furthermore, in a recent review on the practical interventions influencing the sensory attributes of red wines and their relationship to the phenolic composition of grapes, Harrison (2018) points out that the ability to influence the relative extraction and subsequent reaction of phenolic components from the skin and seed of grapes is important in the production of full color development, optimal aromatic quality and sustained aging potential of the wine. Consequently, the purpose of this investigation was to compare the effect of stable color development in Pinot noir wines made with a conventional Pinot noir must and those made using skin-based tannin supplements, sourced from either fresh red or white grape pomace, Pinot noir marc, or a commercially available grape skin extract. This latter treatment allowed the comparison of a commercially available grape skin tannin supplement with those that are readily available at low cost. To further examine the effect of fresh grape skins as a supplement in the absence of seeds and of pulp, a second experiment was conducted using only the skins of the grape varieties that had been selected for the pomace supplement investigation. Acknowledging that it is much simpler to separate skins from grape marc, than from fresh grapes, a third experiment was conducted to compare the impact of fresh grape skin and fermented skin supplements on Pinot noir color development. The detailed comparisons described in these experiments were made possible using micro-vinification techniques (Sparrow & Smart, 2015) and provided

5

valuable insights on aspects of color development in Pinot noir wine during the first six months of wine maturation. 2. Materials and Methods The grape varieties chosen as supplementary tannin sources were those readily available at the time that Pinot noir was harvested in northern Tasmania during the 2012 vintage. Pinot gris is a genetic mutant of Pinot noir, distinguished by having only one skin cell layer that contains color pigments rather than the two cell layers found in Pinot noir (Lecas & Brillouet, 1994). Chardonnay, the second variety used, has no red pigment, but is a source of both skin and seed tannins (Yilmaz & Toledo, 2004). The quantity of supplement added for each treatment (20% w/w) was deemed sufficient to emphasize any effect that the supplement might have on the wine. 2.1 Grape sampling and replication. Grapes of Vitis vinifera cultivars Pinot noir clone 115, D4V2 and G5V15, Pinot gris clone D1V7 and Chardonnay clone 96 were harvested during March 2012 from a 14-year-old vineyard located in northern Tasmania, (41.2oS; 146.9oE) that lies within the wine region of southern Australia. The vines of each cultivar were own rooted, drip irrigated and trained to vertical shoot position with planting density of 2500 vines/ha. For each of the three experiments conducted, either three or four replicates were prepared as detailed in the following sub-sections. Prior to fermentation, 100 berries from each clone, variety and replicate were selected at random to characterize fruit composition. The berries were crushed by hand in a zip-lock bag and transferred to a sieve (mesh size 0.5 mm) to prepare clear grape juice. Total soluble solids in the grape juice (oBrix) was measured using a hand-held refractometer, the pH of the juice was measured using a Metrohm pH meter/autotitrator and titratable acidity was determined by titration with 0.333 M NaOH to an end point of pH 8.2 and reported as g/L tartaric acid.

6

2.2 Treatments for winemaking. The three separate experiments were undertaken to assess the influence of fresh or fermented grape skin supplements, that either retained or excluded the grape seeds and pulp. The treatment preparation for the three experiments are summarised schematically in Figure 1. 2.2.1 Experiment 1 Fresh grape solids, fermented grape solids or liquid grape skin extract used as tannin supplements. Sixty kilograms of grapes (Pinot noir, clone 115) were divided into four 12 kg replicates to provide the base must for the winemaking experiments. Grapes from each replicate were further divided into six 1.5 kg batches to make six treatments, then destemmed by hand to yield one kilogram of berries for each treatment. The berries were placed in a ziplock bag, crushed by hand then transferred to a 1.5 L Bodum® coffee plunger for fermentation. An extra five kilograms of Pinot noir clone 115, plus five kilograms each of Pinot gris and Chardonnay grapes were harvested on the same day as the grapes used for the base must. Fresh grape pomace of each variety (Pinot noir, Pinot gris and Chardonnay) was prepared by removing stalks from randomized bunches to yield one kilogram of berries which were pressed in a basket press to recover 500 mL of juice. The fresh grape solids were removed from the press and 100 g of this was added to each one-kilogram batch of berries that made up the Pinot noir base must such that grape pomace supplements equivalent to 200 g of fresh berries (20% (w/w)) were included in the fermenter. Pinot noir marc (80 g marc equivalent to 200 g of fresh berries) and a liquid grape skin extract (LGS) added at the highest rate recommended by the manufacturer (20 mL/L of must) were also compared as sources of supplementary skin tannins. Treatments for this experiment were: 1) Pinot noir with no supplement (control); 2) Pinot noir plus 20% (w/w equivalent) Pinot noir pomace supplement; 3) Pinot noir must plus 20% (w/w equivalent) Pinot gris pomace supplement; 4) Pinot noir must plus 20% (w/w

7

equivalent) Chardonnay pomace supplement; 5) Pinot noir must plus 20% (w/w equivalent) Pinot noir marc supplement; and 6) Pinot noir must plus 20 mL/L LGS supplement. 2.2.2 Experiment 2 Fresh grape skins as tannin supplements. To compare the relative contribution of skin and seed tannins to color development in the wine, the base must for experiment 2 used the same batch of fruit as for experiment 1 (Section 2.2.1), while the tannin supplement included the skins only of the three grapes varieties used in experiment 1 (Pinot noir, Pinot gris and Chardonnay). Again, Pinot grapes (clone 115) were used for the base must by removing the stalks from four one-kilogram replicates with the berries of each replicate divided into four 200 g treatments. The berries were placed in a ziplock bag, crushed by hand, then transferred to a 450 mL fermentation vessel. For each of the cultivars selected as tannin supplements, skins were removed from 40 g of fresh berries and added to fermentation vessel. Treatments for this second experiment were: 1) Pinot noir must with no supplement (control); 2) Pinot noir must plus 20% (w/w equivalent) Pinot noir skin supplement; 3) Pinot noir must plus 20% (w/w equivalent) Pinot gris skin supplement and 4) Pinot noir must plus 20% (w/w equivalent) Chardonnay skin supplement. 2.2.3 Experiment 3 Fresh vs fermented Pinot noir skins as tannin supplements. The third experiment was conducted to compare fresh Pinot noir grape skins with fermented skins of that variety; the latter being a by-product of red winemaking and more readily separated from fermented grape marc than are skins from fresh grapes. Experiment 3 was conducted 10 days after the first two experiments (Sections 2.2.1 and 2.2.2) and consequently a different clone of Pinot noir (G5V15) was used for the base must and the fresh skin supplement. Six kilograms of grape bunches were divided into four replicates of 1.5 kg; stalks were removed and the berries of each replicate divided into three 200 g batches. Each batch was placed in a ziplock bag, hand-crushed and transferred to a 450 mL fermentation vessel. Fresh Pinot noir grape skins for the supplement were prepared as in experiment 2, treatment 2 (Section 2.2.2).

8

Whereas, for preparation of the fermented Pinot noir skin supplement, entire skins were selected from the grape marc prepared previously, and the seeds removed. The fresh berry weight (1.29 g /berry) was used to calculate the number of fermented grape skins equivalent to 40 g fresh berries (31 skins = 14 g) so that the number of fermented skins added was equivalent to the treatment with the fresh skin supplement in this experiment. There were three treatments for this experiment: 1) Pinot noir must with no supplement (control); 2) Pinot noir base must plus 20% (w/w) fresh Pinot noir skin supplement; 3) Pinot noir base must plus 14 g fermented Pinot noir skin supplement (equivalent to 20% (w/w) fresh skins). 2.3 Quantification of phenolic components in base must and skin tannin supplements. At harvest, a second 200 g of berries from each clone, variety and replicate were isolated and frozen for color and tannin analyses. The frozen whole berries were subsequently thawed overnight at 4oC and homogenized at 8000 rpm for 20 seconds in a Retsch Grindomix GM200 homogenizer, with an S25 N-18G dispersing element (Janke & Kunkel GmbH & Co, Germany) fixed with a floating lid. One gram of the homogenate was extracted in acidified 50% (v/v) ethanol for the determination of grape color (Iland, Bruer, Wilkes, & Edward, 2004) and tannin concentration (Dambergs, Mercurio, Kassara, Cozzolino, & Smith, 2012). A modification of these methods was used to determine the color and tannin concentration of the pomace supplements. In order to avoid extraction of phenolics during the homogenization step, the supplements were reconstituted in sufficient sucrose buffer to achieve the same volume and density as the thawed berries. The sucrose buffer was prepared as an osotonic medium consisting of 22% (w/v) sucrose and 8 g/L tartaric acid dissolved in distilled water and adjusted to pH 3.3 with 5 M sodium hydroxide. The buffer was introduced to the pomace sample in the bowl of the homegenizer, and thoroughly homogenized at 8000 rpm for 20 seconds such that the calculation of phenolic composition of the pomace supplements mimicked that for whole berries. For analysis of the phenolic composition of the Pinot noir

9

grape marc, the marc samples were reconstituted: 40 g marc:110 mL in model wine solution prior to the homogenization step. The model wine solution consisted of a saturated solution of potassium hydrogen tartrate in 12% (v/v) ethanol, adjusted to pH 3.4 as described by Mercurio, Dambergs, Herderich and Smith (2007). The liquid grape skin (LGS) extract (GrapEX™ Tarac Technologies, Nuriootpa, Australia) being a source of both grape tannin and anthocyanins, was used as the fifth supplement in experiment 1 (Section 2.2.1). The phenolic composition of the reconstituted supplements and the LGS were analysed in the same way as the homogenized berries. 2.4 Preparation of Pinot noir grape marc. To prepare the grape marc and fermented skins used in experiments 1 and 3, 10 kg of mature Pinot noir grapes (clone D4V2) were harvested one week prior to the winemaking experiments. Grapes were destemmed and crushed in a Marchisio Grape Crusher/Destemmer™ (1000 kg/h) and fermented for 7 days in a 20 L food grade plastic bucket using submerged cap maceration (Sparrow & Smart, 2015). The end of fermentation was confirmed at less than 2 g/L of residual sugar using Clinitest™ reagent tablets (Bayer Australia Ltd.). The wine was pressed in a flatbed press at 200 kPa of pressure to produce 4 kg of grape marc. 2.5 Estimation of tannin and anthocyanin in the supplements. While recognizing that the methodology used to determine the phenolic composition of fruit requires extraction in 50% ethanol, and is therefore likely to be greater than the extraction that occurs in fermenting wine, the fruit composition data (Table 1) was used to provide an estimate of the amount of tannin and anthocyanin contributed by the supplements (Table 2). In the case of the skin-only supplements, the estimate was calculated by comparing the ratio of fresh grape skins to total berry weight as observed previously (Sparrow, Dambergs, Bindon, Smith, & Close, 2015). The experiments described in this report were undertaken in the following year using Pinot noir grapes of similar ripeness and berry weight, harvested from the same vineyard and using

10

identical extraction methodologies and vinification protocols. For this reason, the ratio of percentage fresh weight of the grape skins to weight of whole berries (29:100) and the ratio of tannin concentration (mg/g) of fresh skins to whole berries (4.3:4.6) previously calculated (Sparrow, Dambergs, Bindon, Smith, & Close, 2015), were used to estimate the amount of grape phenolic material contributed by the skin-only tannin supplements (Table 2). The estimates were subsequently used to gauge the proportion of phenolic material added to the wine via the supplement. 2.6 Vinification protocol. The wine was made using submerged cap micro-fermentation techniques described previously (Sparrow & Smart 2015; Sparrow, Dambergs, Bindon, Smith, & Close, 2015). To each fermentation vessel 50 mg/L SO2 was added in the form of potassium metabisulfite and the musts refrigerated overnight at 4oC. The following day the grape must preparations were allowed to equilibrate at 25oC then inoculated with 300 mg/L RC212 yeast solution (Lallemand Australia Pty Ltd. Adelaide, Australia) and fermented at 25C (±1C). On day 3 of the fermentation, 300 mg/L of diammonium phosphate was added to each fermentation vessel to provide 60 mg/L yeast assimilable nitrogen. The one-kilogram ferments conducted in 1.5 L Bodum® coffee plungers were pressed after eight days skin contact at which time they were tested for dryness using Clinitest™ tablets (< 2 g/L residual sugar). The wine was pressed by depressing the plunger to a specified mark on the fermentation vessel and 500 mL of wine was recovered. For experiments 2 and 3 that used 450 mL fermentation vessels, the wine tested dry after six days of skin contact and was pressed using a plunger with a mesh sieve (mesh size 1mm) fixed at the base, to recover 100 mL of wine. The wine was stored at 4C for 14 days, at which time it was racked and a further 80 mg/L SO2 added. Following 4 weeks storage at 12C the wine was decanted under CO2 cover into 25 mL amber glass bottles and a 10 mL sample of the wine taken for phenolic

11

analysis. Following six months storage at 12C a fresh bottle was opened and a further wine sample taken for phenolic analysis. 2.7 Analysis of grape extracts and wine Frozen samples were thawed at room temperature and clarified by centrifugation at 4500 g for 5 min using an Eppendorf 5417C centrifuge with an Eppendorf F45-30-11 rotor (Crown Scientific, Melbourne, Australia). Extracts and wines were diluted with 1 M HCl and spectral analysis of the phenolic composition of the samples performed using a UV-VIS Spectrophotometer (Model Genesys™ 10S Thermo Fisher Scientific Inc., Madison, WI, USA) scanning at 2 nm intervals for wavelengths 200 to 600 nm. Total tannin concentration was determined using the method of rapid tannin analysis described and fully validated relative to methyl cellulose precipitation, by Dambergs, Mercurio, Kassara, Cozzolino and Smith (2012). All samples were analyzed for wine color using a modification of the Somers assay described by Mercurio, Dambergs, Herderich and Smith (2007). The model wine buffer for color analysis was prepared using 5 g/L potassium hydrogen tartrate in 12% (v/v) ethanol. Assays were performed following a 1:10 dilution of wine in either model wine buffer containing 0.1% (v/v) acetaldehyde or model wine buffer containing 0.375% (w/v) sodium metabisulfite. At six months bottle age, the proanthocyanidin (PA) composition of a sub-set of wine samples from experiment 1 (Section 2.5.1) was determined using the methyl cellulose precipitable tannin assay (MCPT) described by Sarneckis, Dambergs, Jones, Mercurio, Herderich and Smith, (2006). Subsequently, tannin was isolated using solid-phase extraction according to the conditions described by Kassara and Kennedy (2011) and reconstituted in methanol to give a concentration of 10 g/L based on the initial wine tannin concentration determined by MCPT. Tannin subunit composition was determined using acid catalysis in the presence of excess phloroglucinol (phloroglucinolysis) (Kennedy & Jones 2001).

12

2.8 Statistical analysis. Mean and standard deviation for fruit composition characters in each experiment were calculated. The phenolic composition of wines from each experiment assessed at two time points was analyzed using Repeated Measures ANOVA with treatment as the main plot and time as the sub-plot (GenStat Release 13.1 Copyright 2010, VSN International Ltd). For each type of statistical analysis, ANOVA was followed by post-hoc analysis using Fisher’s Protected Least Significant Difference test. 3. Results 3.1 Grape and supplement composition. The composition of each grape variety and clone used for the base must and for the tannin supplements was determined from the sample taken from each treatment replicate prior to the commencement of the winemaking experiments (Table 1). The berry weight, sugar content and pH were similar for all three varieties used in experiments 1 and 2. The total phenolic concentration of Pinot noir clone 115, used for both the pomace addition and base must, was significantly higher than for the Pinot gris and Chardonnay clones. Pinot noir clone 115 grapes were 14% higher in tannin than Pinot gris and 48% higher in tannin than Chardonnay grapes. The fresh grapes of Pinot noir clone D4V2 that were used to prepare grape marc, contained 7.8 mg/g of tannin and when reconstituted in model wine solution with the equivalent volume of liquid to that of fresh grapes, the grape marc contained 5.2 mg/g of tannin indicating that approximately 30% of the tannin (2.6 mg per gram of grape tissue) had been extracted during the initial fermentation, whereas approximately 75% of the anthocyanin had been extracted (Table 1). The anthocyanin and tannin concentrations of the LGS were significantly higher than that of the fresh berries and the reconstituted grape marc, however the anthocyanin/tannin ratio for this supplement was similar to that of the fresh berries from the Pinot noir clones, and the quantity added to the base must for the winemaking experiment an effective comparison with the pomace supplements (Table 2).

13

Subsequent to the winemaking experiments, data from the grape composition analyses were used to estimate the quantity of tannin and anthocyanin added to the ferments through the supplements prior to fermentation. These estimates showed that the tannin contributed by the Pinot noir and Pinot gris pomace in experiment 1 (Section 2.5.1) was 30% greater than that from either the Chardonnay pomace or the Pinot noir marc which in turn were 65% greater than the contribution from the LGS supplement (Table 2). The Pinot noir pomace was estimated to contribute four times as much anthocyanin as either the Pinot gris or Chardonnay pomace and 2.5 times more than the LGS. For experiment 2 (Section 2.5.2), the Pinot noir and Pinot gris skins were estimated to contribute 30% more tannin than the Chardonnay skins and the Pinot noir skins contributed six times more anthocyanin than did the Pinot gris skin (Table 2). The fresh Pinot skins used in experiment 3 (Section 2.5.3) were estimated to contribute four times more tannin and anthocyanin than did the fermented Pinot noir skins (Table 2). 3.2 Phenolic composition of wine. The phenolic composition of the wines was measured at the time of bottling (50 days post-inoculation) and again, six months after bottling (230 days post-inoculation). 3.2.1 Experiment 1. Fresh grape solids, fermented grape solids or liquid grape skin extract as tannin supplements. Interactions between grape tannin supplements and bottle age were observed for total phenolics (p = 0.016, LSD = 3.39) and sulfur dioxide resistant wine hue (p = 0.026, LSD = 0.105), for the remaining parameters the effects of treatment and time were independent but nonetheless significant (Table 3). 3.2.1.1. Tannin: Analysis at 50 days post-inoculation showed that the tannin concentration of wines supplement with Pinot gris pomace had increased by 20% while Chardonnay pomace, while the Pinot noir marc and LGS effected a 30% increase. This increase represented the recovery of 5% of the tannin estimated to be in the Pinot gris and Chardonnay pomace

14

supplements, 10% from the Pinot noir marc supplement and 30% from the LGS supplement (Tables 2 and 3). During the six months of bottle aging the tannin concentration of all wines decreased by approximately 10%, and the wine made with Pinot noir pomace supplement was no longer significantly different from the control (Table 3). 3.2.1.2 Pigments: The pigment of the wines increased significantly with the addition of any of the supplements in this experiment. Specifically, at 50 days post-inoculation, the Pinot gris pomace conferred a 10% increase in the anthocyanin concentration of the wine relative to the control treatment, both Pinot noir pomace and Pinot noir marc an 18% increase, and the LGS supplement a 45% increase. The increases reflected the recovery of 75% of the anthocyanin estimated to be in the Pinot gris pomace, 20% from the Pinot noir pomace, 80% from the Pinot noir marc and 100% from the LGS supplement. As the wine aged for six months, the situation changed such that at 230 days post-inoculation only the wines made with Pinot noir marc and the LGS supplements maintained a significantly higher concentration of anthocyanin than the control wines. There was no significant difference in the ratio of anthocyanin to tannin observed for any of the treatments. Only the wine supplemented with LGS had a higher concentration of non-bleachable pigment than the control wine at each of the time periods (average 60%: Table 3). 3.2.1.3 Wine color density: Each of the supplements were found to increase the wine color density relative to the control treatment at both time intervals measured. The Pinot noir pomace and Pinot noir marc supplements brought about an average increase of 14%, while the LGS supplement increased this parameter by almost 50% (Table 3). 3.2.1.4 Hue and hue SO2: Lower hue values reflect more blue coloration in the wine. The Pinot noir marc and LGS supplements alone significantly reduced the wine hue relative to the control wine and did so by 5% and 10% respectively when analyzed at six months bottle age. The LGS supplement had an even greater impact on the stable wine hue parameter (hue SO2)

15

reducing it to ~15% below that of the control wine. By contrast the wine made with the Chardonnay pomace supplement had higher hue than the control indicating that the lack of anthocyanin in this supplement became more apparent as the wine aged (Table 3). 3.2.1.5 Proanthocyanidin composition of wine. Examination of the Proanthocyanidin (PA) composition of the wines aged for six months in the bottle showed that there was no significant difference in mean degree of polymerization (mDP), nor the total PA concentration among any of the wines. The percentage of trihydroxylated PAs was less in wines made with supplements regardless of the source of supplement, with Pinot noir marc and Chardonnay pomace having the lowest percentages relative to the control (Table 4). Each of the grape pomace supplements conferred a significantly higher percentage of galloylated PAs relative to the control wines: Pinot noir 14%, Pinot gris 7% and Chardonnay a 12% increase, while the Pinot noir marc effected a 20% increase, by contrast the percentage of galloylated PAs in wines made with LGS supplement was 14% less than for wines from the control treatment. As a consequence, the ratio of tri-hydroxylated to galloylated PAs was highest for the control and LGS treatments. Only wines made with LGS supplement had significantly lower mass conversion percentage (% MC) than the control wines; whereas for the remaining treatments, % MC was higher than for the control treatment. 3.2.2 Experiment 2 Fresh grape skins as tannin supplements. This experiment provided insights into the tissue source of the tannins conferred by the pomace supplements used in experiment 1 (Section 2.5.1), as the skins alone were used for the fresh grape tannin supplement. Notably, the difference in ferment size between the experiments 1 (Section 2.5.1) and 2 (Section 2.5.2) had no significant impact on the phenolic parameters of the control wines at p ≤ 0.05 (Tables 3 and 5). When Pinot noir base must was supplemented with 20% (w/w) of skins isolated from either Pinot noir, Pinot gris or

16

Chardonnay grapes, the only parameter with a significant interaction between the type of supplement and bottle age, was non-bleachable pigment (p = 0.033; LSD = 0.237), the focal parameter of this investigation which was apparent when fresh Pinot noir skins were added as the tannin supplement (Table 5). 3.2.2.1 Tannin: There was a significant increase in wine tannin (average 26%) for all treatments that included a skin supplement relative to the control wine (Table 5). The increases reflected the recovery of 15 to 20% of the tannin estimated to be in the skin supplements (Tables 2 and 5). During the 6 months of bottle aging the tannin concentration of all wines increased, this increase being 2.6-fold higher in wines made with skin supplements than for the control wine. 3.2.2.2 Pigments: The Pinot noir skin supplement had the greatest impact on the pigment regime of the wine: while no significant increase in anthocyanin concentration was attributed to the supplement , during six months of bottle aging a 25% reduction in anthocyanin concentration was observed across all treatments, whereas the non-bleachable pigment content of the wines increased 2.7-fold (Table 5). Following six months of bottle aging the non-bleachable pigment concentration of wine treated with Pinot noir skins had increased by 42% relative to the control wine. 3.2.2.3 Wine color density: Fifty days post-inoculation the color density of wines supplemented with Pinot noir skins were found to be ~8% higher than control wines. Following six months of bottle aging this parameter increased by 50% across all treatments (Table 5). 3.2.2.4 Hue and hue SO2: The inclusion of skin supplements in the wines made no significant difference to wine hue, however, there was a small but significant age-related increase (12%) in red coloration observed in wines of all treatments. However, it was only the Chardonnay

17

skin supplement that conferred more red, stable hue to the wine, as indicated by the 6% increase in hue SO2 relative to the remaining treatments. 3.2.3 Experiment 3 Fresh vs fermented Pinot noir skins as tannin supplements. In this experiment interactions between the Pinot noir skin supplements and bottle age were observed for non-bleachable pigment concentration (p = 0.037, LSD =0.048) and wine hue (p = 0.005, LSD = 0.028). Conspicuously, both types of Pinot noir skin supplement conferred significant effects on all of the wine phenolic parameters evaluated (Table 6). 3.2.3.1 Tannin: The greatest impact on phenolic composition of the wine made with supplements was the 45% increase in tannin concentration which represented approximately 30% recovery of the tannin estimated to be in the fresh skin supplement and 100% recovery of the tannin from the fermented skin supplement (Tables 2 and 6). 3.2.3.2 Pigments: Significant increases in anthocyanin concentration were observed with the inclusion of Pinot noir skin supplements: 20% with fresh skin supplement and 10% with fermented skin supplement (Table 6). The higher concentration of anthocyanin in the wine reflected a recovery rate of 15% of the anthocyanin estimated to be in the fresh Pinot noir skin supplement and 65% from the fermented Pinot noir skin supplement (Tables 2 and 6). Markedly, the concentration of non-bleachable pigment increased by 17% relative to the control wines with the inclusion of either fresh or fermented skin supplements (Table 6). 3.2.3.3 Wine color density: The fresh skin supplement increased wine color density by 20%, while the fermented skin supplement realized a 10% increase (Table 6). 3.2.3.4 Hue and hue SO2: Of particular note were the values for hue and hue SO2 that were significantly lower than the control wine for wines treated with fresh skin supplement (5% lower) or fermented skin supplement (10% lower), these values being indicative of more blue-purple tones in the wine. 4. Discussion

18

This investigation explored options for the inclusion of easily obtainable sources of supplementary grape skin tannins, with a view to improving the development of stable color compounds in Pinot noir wine. The influence of different grape tissues on the development of color stable pigments (nonbleachable pigments) in Pinot noir wine that were observed during the this study are supported by the findings of Zerbib, Cazals, Enjalbal & Saucier (2018), who isolated and identified flavanol glycosides from grapes that are known to form the backbone of proanthocyanidins (condensed tannins); they found these to be in greater concentration in grape seed tissues than skin tissues. Their wine quantification studies identified flavanol dimers derived from wine proanthocyanidins and a subsequent report showed that the concentration of flavanol dimers was higher in grape skin tissue than in grape seeds (Zerbib, Mazauric, Meudec, Le Guernevé, Lepak, Nidetzky, Cheynier, Terrier & Saucier, 2018). More recently, Li and Sun (2019) reviewed a range of new methods for separating polymeric polyphenols in grapes and wine and concluded that current methods of wine analysis confirm that a higher proportion of wine polymeric polyphenols are derived from grape skins than from grape seeds and also that the former are correlated with superior color stability. The work of Teng, Hayasaka, Smith & Bindon (2019) involved the extraction and isolation of skin and seed tannins from frozen grape tissues. After combining tannins of similar molecular mass from each tissue source with anthocyanins in model wine solution they found that seedderived tannins formed polymeric pigments more readily than did skin-derived tannins. Moreover, they noted a higher precipitation rate of seed-derived tannin-pigment complexes relative to their skin-derived counterparts, an effect they attributed to the greater mass of the seed-derived polymeric pigments. They concluded that in model wine solution, skin-derived pigments were more stable than seed-derived pigments. Considering these recent reports, it is conceivable that the high concentration of tannin formed during six months of bottle aging in

19

wines made with grape skin supplements (Section 3.2.2.1) consisted of skin-derived tanninpigment complexes. The Pinot noir marc supplement used in experiment 1 (Section 2.5.1) contained skins and seeds but no pulp, and was shown to have a high concentration of extractable pigment that became incorporated into the wine during the second alcoholic fermentation, as foretold by the work of Somers (1971) and reported by Pinelo, Arnous and Meyer (2006). However, whilst the total anthocyanin concentration increased, relative to the control wine, there was no apparent increase in the concentration of non-bleachable pigment. In light of the report by Teng, Hayasaka, Smith & Bindon (2019), it seems plausible that there may have been competition between seed-derived and skin-derived tannins to form polymeric pigments with anthocyanins and the subsequent precipitation of the larger seed-derived polymeric pigment complexes. Alternatively, the higher proportion of seeds associated with the grape marc may have restricted the development of non-bleachable pigments (polymeric pigments) either skin-derived or seed-derived, an effect attributed to phenolic components from the seeds in former investigations (Kovac Alonso, & Revilla 1995; Neves, Spranger, Zhao, Leandro, & Sun, 2010; Sparrow, Dambergs, Bindon, Smith, & Close, 2015). The influence of seeds in these experiments is highlighted in part by the maceration technique selected; unlike maceration techniques that involve punch-down or pump-over of the must, the submerged cap maceration technique is non-invasive and involves no agitation of the pomace cap during fermentation. Furthermore, microvinification experiments conducted in parallel to those reported here and using the same source of fruit, demonstrated that on average 65% of the seeds were retained in the grape marc at pressing (Sparrow, 2015). The presence of tri-hydroxylated PAs in wine are indicative of phenolic extraction from grape skins as they do not occur in grape seeds (Mattivi, Vrhovsek, Masuero, & Trainotti, 2009; Sparrow, Dambergs, Bindon, Smith, & Close, 2015). PA analysis of the wines made with

20

pomace supplements (Section 3.2.1.5) confirmed that a higher proportion of wine tannin was derived from the seeds of the supplement rather than from the skins. When analyzed at the time of bottling, the tannin concentration of wines treated with either grape pomace or skin-only supplements was higher than the control wine made without supplements an increase that reflected the entire 20% (w/w) supplement size and was independent of the grape variety (Sections 3.2.1.1 and 3.2.2.1). The apparent loss of tannin between bottling and six months bottle age observed in the pomace supplement treatments suggested that either fining of some tannins by colloidal material or precipitation of polymeric pigments had taken place (Hanlin, Hrmova, Harbertson, & Downey 2010; Bindon & Smith, 2013, Teng, Hayasaka, Smith & Bindon 2019). During six months of bottle aging the tannin concentration of wines supplemented with fresh skins only, increased by an average of 2.7-fold across the three grape varieties compared to the control wine, making it apparent that the tannin loss observed in the pomace treatments over the same time period was concomitant with the inclusion of pulp and seed in the supplement. The loss of tannin and poor development of non-bleachable pigments in Pinot noir wines observed when grape pulp alone was added as a supplement has been reported previously (Sparrow, Dambergs, Bindon & Close, 2015), suggesting that for experiment 1 (Section 3.2.1) grape pulp had a major impact on the concentration of tannin and its derivatives in the wine. Not surprisingly, Pinot gris skin-only supplements increased the tannin concentration of the wine more than the anthocyanin concentration, and the treatment confirmed that the formation of non-bleachable pigment was limited by the concentration of anthocyanin in the supplement rather than by the concentration of skin tannins, supporting the review findings of Boulton in 2001. By a process of elimination, we conclude that the higher concentration of non-bleachable pigment (42%) in wines made with fresh Pinot noir and Pinot gris skins

21

(20%) represented all of the anthocyanin that was added through the supplement while the Pinot noir skin supplements appeared to trigger further development of non-bleachable pigment from phenolic components extracted from the base must. The effect of fermented grape skin on the development of non-bleachable pigment formation supplements (17% increase) was less than that of fresh skins, and yet the increase represented 85% recovery of the anthocyanin added through the supplement and greater wine color density. The investigation showed that at 50 days post inoculation, there was a high concentration of anthocyanin added through the LGS supplement as well as a high concentration of nonbleachable pigment. Considered together with the high tannin concentration of the wines made with this supplement, it is reasonable to predict that non-bleachable pigments would continue to develop as this wine matures. The review of co-pigmentation of anthocyanins and their role in red wine color, (Bolton, 2001), reported that molecular associations between pigments and other organic molecules (copigments) had been found to cause a bathochromic shift in the wavelength of maximum absorbance of red wine typically by 5 to 20 nm, resulting in blue-purple tones in an otherwise red solution. However, the review warned that these copigments can be difficult to detect when the wine is diluted in model wine buffer for analysis, as was the case for the experiments reported here, so an alternative explanation is required to explain the low hue values found in the wines treated with some of the skin derived supplements. As early as 1971, polymeric pigments were found to be much less effected by low pH (Somers, 1971); and more recently, blue-purple tints in red wine, together with enhanced color density and resistance to sulfite bleaching have been attributed to the formation of pyranoanthocyanins (Marquez, Serratosa, & Merida, 2013). Interestingly, an increase in wine color density was observed in wines supplemented with either fresh or fermented Pinot noir skins during this

22

investigation and was concomitant with a higher concentration of non-bleachable pigments and more blue tones in the wine. Studies that aim to determine the effect of specific fresh grape tissues on the tannin composition and sensory properties of wine are rare. Research methodologies developed for proanthcyanidin extraction from grape tissue often include freezing of the tissues prior extraction in strong organic solvents, making it difficult to compare the likely extraction rate of proanthcyanidin precursors from fresh grape tissues into the relatively benign environment of fermenting wine. Nonetheless, in their review of the sensorial properties of red wine polyphenols Soares, Brandão, Mateus & de Freitas (2017) observed that while seed-derived tannins impart more structure to the wine, they have also been associated with excessive astringency. Furthermore, during characterization of grape and wine proanthocyanidins from Vitis vinifera cv. Agiorgitiko, it was demonstrated that the proanthocyanidin subunit composition of wine not only resembled that of grape skins, but the abundance of epigallocatechin subunits in proanthocyanidin component of both grapes and wine was anticipated to impart a lower perception of astringency in the wine (Petropoulos, Kanellepoulou, Praskevopoulus, Kotseridis & Kallithraka, 2017). Clearly, the sensory evaluation of wines made with grape skin supplements from a range of sources remains an important area of enquiry. 5. Conclusion The unique composition of grape skins when used as sources of supplementary tannins in wine, together with the influence of seeds and grape pulp, had a significant impact on the phenolic parameters of the wines made during this study. The investigation emphasized the importance of choosing a maceration technique and wine tannin supplement that maximise the extraction of the preferred tannins. Significant benefits to both wine hue and color stability were derived from the addition of a commercial liquid grape skin extract and from

23

both fresh and fermented Pinot noir skins, the latter being readily available to any winery where red wine is made. While grape marc with its associated skins may be more readily accessible than fresh grape skins, the extra seed associated with the marc was found to be detrimental to the development of pigments associated with stable wine color, consequently the omission of seeds from a grape-derived supplement may be advantageous.

24

Acknowledgements This work was conducted at the University of Tasmania, during the research doctorate of the principal author, Angela Sparrow. The work was funded by an Australian Postgraduate Award, the Australian Grape and Wine Research and Development Corporation [GWR PhD 1107], and the Australian Wine Research Institute. The authors would like to thank the tannin research team at Australian Wine Research Institute for conducting the proanthocyanidin composition analyses, and Brown Brothers, Kayena vineyard for the donation of grapes and access to the micro-winery facility at Kayena, Tasmania.

25

References Bautista-Ortin, A. B., Fernandez-Fernandez, J. I., Lopez-Roca J. M., & Gomez-Plaza, E. (2007). The effects of enological practices in anthocyanins, phenolic compounds and wine colour and their dependence on grape characteristics. Journal of Food Composition and Analysis 20, 46-552. Bindon, K. A., & Smith, P. A. (2013). Comparison of the affinity and selectivity of insoluble fibres and commercial proteins for wine proanthocyanidins. Food Chemistry 136, 917928. Boulton, R. (2001). The copigmentation of anthocyanins and its role in the color of red wine: A critical review. American Journal of Enology and Viticulture 52, 67-87. Cheynier, V., Duenas-Paton, M., Salas, E., Maury, C., Souquet, J. M., Sarni-Manchado, P., & Fulcrand, H. (2006). Structure and properties of wine pigments and tannins. American Journal of Enology and Viticulture 57, 298-305. Cortell, J. M., Halbleib M., Gallagher A. V., Righetti T. L,. & Kennedy J. A. (2005). Influence of vine vigor on grape (Vitis vinifera L. Cv. Pinot noir) and wine proanthocyanidins. Journal of Agricultural and Food Chemistry 53, 5798-5808. Cozzolino, D., Cynkar, W. U., Dambergs, R. G, Mercurio, M. D., & Smith, P. A. (2008). Measurement of condensed tannins and dry matter in red grape homogenates using near infrared spectroscopy and partial least squares. Journal of Agricultural and Food Chemistry 56, 7631-7636. Crespy, A. (2003). Etude de l’elimination de composes soufrés par tanisage avec des tannins de pépins de raisin. Revue des Oenologues 30, 38-39. Dambergs, R. G., Mercurio, M.D., Kassara, S., Cozzolino, D., & Smith, P. A. (2012). Rapid measurement of methyl cellulose precipitable tannins using ultraviolet spectroscopy with

26

chemometrics: application to red wine and inter-laboratory calibration transfer. Applied Spectroscopy 66, 656-664. Downey, M. O., Harvey, J . S., & Robinson, S. P. (2003). Analysis of tannins in seeds and skins of Shiraz grapes throughout berry development. Australian Journal of Grape and Wine Research 9, 15-27. Hanlin, R. L., Hrmova, M., Harbertson, J. F., & Downey, M. O. (2010). Condensed tannin and grape cell wall interactions and their impact on tannin extractability into wine. Australian Journal of Grape & Wine Research 16, 173-188. Harrison, R. (2018). Practical interventions that influence the sensory attributes of red wines related to the phenolic composition of grapes: a review. International Journal of Food Science and Technology 53, 3−18. Holt, H. E, Francis I. L., Field, J., Herderich, M. J., & Iland, P.G. (2008). Relationships between berry size, berry phenolic composition and wine quality scores for Cabernet Sauvignon (Vitis vinifera L.) from different pruning treatments and different vintages. Australian Journal of Grape and Wine Research 14, 191-202. Iland, P. G., Bruer, N., Wilkes, E., & Edward, G. (2004). Anthocyanins (colour) and total phenolics of grape berries. Chemical Analysis of Grapes and Wine: Techniques and Concepts: Winetitles (1st ed). Broadview, Australia. Kassara, S. & Kennedy, J. A. (2011). Relationship between red wine grade and phenolics. 2. Tannin composition and size. Journal of Agricultural and Food Chemistry 59, 8409-8412. Kennedy, J. A. (2008). Grape and wine phenolics: Observations and recent findings. Ciencia e investigación agraria 35, 107-120. Kennedy, J. A. & Jones, G. P. (2001). Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. Journal of Agricultural and Food Chemistry 49, 1740-1746.

27

Kovac, V., Alonso, E., & Revilla, E. (1995). The effect of adding supplementary quantities of seeds during fermentation on the phenolic composition of wines. American Journal of Enology and Viticulture 46, 363-367. Koyama, K., Goto-Yamamoto, N., & Hashizume, K. (2007). Influence of maceration temperature in red wine vinification on extraction of phenolics from berry skins and seeds of grape (Vitis vinifera). Bioscience, biotechnology and biochemistry 71, 958-965. Lecas, M. & Brillouet J. M. (1994). Cell-wall composition of grape berry skins. Phytochemistry 35, 1241-1243. Li, S., Bindon, K., Bastian, S.E.P., Jiranek, V. & Wilkinson, K.L. (2017). Use of winemaking supplements to modify the composition and sensory properties of Shiraz wine. Journal of Agriculture and Food Chemistry 65, 1353-1364. Li, L. & Baoshan S. (2019). Grape and wine polymeric polyphenols: Their importance in enology. Critical Reviews in Food Science and Nutrition 59, 563-579. Marquez, A., Serratosa, M. P., & Merida, J. (2013). Pyranoanthocyanin derived pigments in wine: Structure and formation during winemaking. Journal of Chemistry 2013. Mattivi, F., Vrhovsek, U., Masuero, D., & Trainotti, D. (2009). Differences in the amount and structure of extractable skin and seed tannins amongst red grape varieties. Australian Journal of Grape and Wine Research 15, 27-35. Mazza, G., Fukumoto, L., Delaquis, P., Girard, B., & Ewert, B. (1999). Anthocyanins, phenolics, and color of Cabernet Franc, Merlot, and Pinot Noir wines from British Columbia. Journal of Agricultural and Food Chemistry 47, 4009-4017. McRae, J. M., Schulkin, A., Kassara, S., Holt, H. E., & Smith, P. A. (2013). Sensory properties of wine tannin fractions: Implications for in-mouth sensory properties. Journal of Agricultural and Food Chemistry 61, 719-727.

28

Mercurio, M. D., Dambergs, R. G., Herderich, M. J., & Smith, P. A. (2007). High throughput analysis of red wine and grape phenolics-adaptation and validation of methyl cellulose precipitable tannin assay and modified Somers color assay to a rapid 96 well plate format. Journal of Agricultural and Food Chemistry 55, 4651-4657. Muhlack, R. A., Potumarthi, R., & Jeffery, D. W. (2018). Sustainable wineries through waste valorisation: A review of grape marc utilisation for value-added products. Waste Management 72, 99-118. Neves, A. C., Spranger, M. I., Zhao, Y. Q., Leandro, M. C., & Sun, B. S. (2010). Effect of addition of commercial grape seed tannins on phenolic composition, chromatic characteristics, and antioxidant activity of red wine. Journal of Agricultural and Food Chemistry 58, 11775-11782. Petropoulos, S., Kanellopoulou, A., Paraskevopoulos, I., Kotseridis, Y., & Kallithraka, S. (2017). Characterization of grape and wine proanthocyanidins of Agiorgitiko (Vitis vinifera L. cv.) cultivar grown in different regions of Nemea. Journal of Food Composition and Analysis 63, 98-110. Pinelo, M., Arnous, A., & Meyer, A. S. (2006). Upgrading of grape skins: Significance of plant cell-wall structural components and extraction techniques for phenol release. Trends in Food Science and Technology 17, 579-590. Rousserie, P., Rabot, A., & Geny-Denis, L. (2019). From Flavanols biosynthesis to wine tannins: what place for grape seeds? Journal of Agriculture and Food Chemistry 67, 13251343 Sarneckis, C., Dambergs, R. G., Jones, P., Mercurio, M., Herderich, M. J., & Smith, P.A. (2006). Quantification of condensed tannins by precipitation with methyl cellulose: development and validation of an optimised tool for grape and wine Analysis. Australian Journal of Grape and Wine Research 12, 39-49.

29

Singleton, V. L. (1992). Plant polyphenols: synthesis properties significance. R. W. Hemingway, P. E. Laks, & S. J. Branham (Eds.), Tannins and the qualities of wines. (Vol. 59). New York: Plenum. Somers, T. C. (1971). The polymeric nature of wine pigments. Phytochemistry 10, 21752186. Soares, S., Brandão, E. Mateus, N. and de Freitas V. (2017) Sensorial properties of red wine polyphenols: Astringency and bitterness. Critical Reviews in Food Science and Nutrition 57, 937-948. Souquet, J. M., Cheynier, V., Brossaud, F., & Moutounet, M. (1996). Polymeric proanthocyanidins from grape skins. Phytochemistry 43, 509-512. Sparrow, A. M. (2015). Comparative analysis of wine tannins from Pinot Noir grapes. Doctoral dissertation, University of Tasmania. Sparrow, A. M. & Smart, R. E. (2015). Fermentation volume studies for red wine experimentation. South African Journal of Enology and Viticulture 36, 343-346. Sparrow, A. M. & Smart, R. E. (2017). Pinot noir wine processing and quality improved by skin fragmentation. Catalyst 1, 88-98. Sparrow, A. M., Dambergs, R. G., Bindon, K. A., Smith, P. A., & Close, D. C. (2015). Interaction of grape skin, seed and pulp tissues on tannin and anthocyanin extraction in Pinot noir wines. American Journal of Enology and Viticulture 66, 472- 481. Sparrow, A. M., Smart, R. E., Dambergs, R. G., & Close, D. C. (2016a). Skin particle size impacts the phenolic attributes of Pinot noir wine: Proof of concept. American Journal of Enology and Viticulture 67, 29-37. Sparrow, A. M., Holt, H. E., Pearson, W., Dambergs, R. G., & Close, D. C. (2016b). Accentuated Cut Edges (ACE): Effects of skin fragmentation on the composition and

30

sensory attributes of Pinot noir wines. American Journal of Enology and Viticulture 67, 169178. Teng, B., Hayasaka, Y., Smith, P., & Bindon, K. A. (2019). Grape seed and skin tannin molecular mass and composition affects the rate of reaction with anthocyanin and subsequent formation of polymeric pigments in the presence of acetaldehyde. Journal of Agricultural and Food Chemistry 67, 8938-8949. Vidal, S., Francis, L., Guyot, S., Marnet, N., Kwiatkowski, M., Gawel, R., & Waters, E. J. (2003). The mouth-feel properties of grape and apple proanthocyanidins in a wine-like medium. Journal of the Science of Food and Agriculture 83, 564-573. Yilmaz, Y. & Toledo, R. T. (2004). Major flavonoids in grape seeds and skins: Antioxidant capacity of catechin, epicatechin, and gallic acid. Journal of Agricultural and Food Chemistry 52, 255-260. Zerbib, M., Mazauric, J-P., Meudec, E., Le Guernevé, C., Lepak, A., Nidetzky, B., Cheynier, V., Terrier, N. & Saucier, C. (2018). New flavanol O-glycosides in grape and wine. Food Chemistry 266, 441−448. Zerbib, M., Cazals, G., Enjalbal, C. & Saucier C. (2018). Identification and quantification of flavanol glycosides in Vitis vinifera grape seeds and skins during ripening. Molecules 23, 2745.

31

Figure captions Figure 1. Schematic diagram of micro-vinification treatments. PN, Pinot noir; PG, Pinot gris; CH, Chardonnay; LGS, liquid grape skin.

Chemical Compounds Diammonium phosphate; CID: 24540 Ethanol; CID: 702 Methanol; CID: 887 Methyl cellulose; CID 2724070 Phloroglucinol; CID: 359 Potassium hydrogen tartrate; CID: 23681127 Potassium metabisulfite; CID: 28019 Sodium metabisulfite; CID: 656671 Sodium hydroxide; CID: 14798 Tartaric acid; CID: 875

32

Table 1 Composition of fruit and supplements

pH

Titratable acidity (g/L)

Total phenolics (AU)

Total tannin (mg/g)

Anthocyanin (mg/g)

22.1 ± 0.4

3.37 ± 0.1

11.4 ± 0.2

1.23± 0.1

7.03 ± 0.4

0.63 ± 0.1

1.33 ± 0.1

21.5 ± 0.4

3.30 ± 0.1

9.13 ± 0.1

1.42 ± 0.2

8.68 ± 1.2

0.69 ± 0.1

D1V7

1.38 ± 0.1

23.0 ± 0.3

3.25 ± 0.0

8.93 ± 0.3

1.06 ± 0.1

6.87 ± 0.5

0.12 ± 0.0

Chardonnay pomace/skin

96

1.43 ± 0.0

20.9 ± 0.1

3.19 ± 0.1

8.65 ± 0.3

0.83 ± 0.1

5.16 ± 0.6

NA

Pinot noir (pre-ferment)

D4V2

11.6

1.29 ± 0.3

7.82 ± 1.9

0.61 ± 0.2

Pinot noir marc

D4V2

NA

NA

ND

ND

0.83 ± 0.2

5.21 ± 1.2

0.14 ± 0.0

NA

NA

b

2.16

b

1129 ± 0.5

37.2 ± 3.0

4.96 ± 1.2

G5V15

1.27 ± 0.1

1.23 ± 0.05

6.84 ±0.3

0.68 ± 0.01

Clone

Berry weight (g)

Sugar (Brix)

Pinot noir base must

115

1.53 ± 0.1

Pinot noir pomace/skin

115

Pinot gris pomace/skin

Fruit/supplement

Liquid grape skin Pinot noir base/fresh skin

a

1.29

a

22.4

1.6

21.7 ± 0.3

a

b

3.15

3.33 ± 0.1

a

1.5

10.2 ± 0.1

Data are mean and standard deviation, n=4. aUnreplicated sample; bCertified product analysis; NA, not applicable; ND, not determined

33

Table 2 Estimate of phenolic addition to wine with each supplement. Supplement type

Tannin Anthocyanin concentration (g/L) concentration (g/L)

Experiment 1 Pinot noir pomace

3.12

0.24

Pinot gris pomace

2.74

0.04

Chardonnay pomace

2.06

0.00

Pinot noir marc

2.08

0.06

Liquid grape skin

0.74

0.10

P value

<0.001

<0.001

LSD 0.05

0.622

0.043

Pinot noir skin

0.86

0.24

Pinot gris skin

0.75

0.04

Chardonnay skin

0.56

0.00

P value

0.028

<0.001

LSD 0.05

0.196

0.059

Experiment 2

Experiment 3

34

Pinot noir skin (fresh)

0.85

0.27

Pinot noir skin (fermented)

0.19

0.06

P value

<0.001

0.001

LSD 0.05

0.244

0.086

One-way ANOVA, mean, n=4. P-values at p ≤ 0.05. LSD, least significant difference.

35

Table 3 Experiment 1: Effects of five supplement types on Pinot noir wine phenolic parameters at two time periods: bottling (50 days postinoculation) and six months (230 days post-inoculation). Nil (control)

Pinot noir pomace

Pinot gris Chardonnay Pinot noir Liquid pomace pomace marc grape skin

p-value (trt)

Total phenolics (AU)

37.8 d

44.0 ab

42.2 c

43.2 b

44.3 a

46.3 a

<0.001

Anthocyanin concentration (g/L)

0.31 d

0.37 b

0.34 c

0.33 cd

0.36 bc

0.44 a

<0.001

Non-bleachable pigment (AU)

0.36 b

0.40 b

0.38 b

0.38 b

0.40 b

0.60 a

Tannin concentration (g/L)

0.71 d

0.91 b

0.85 c

0.92 ab

0.92 ab

Wine color density (AU)

3.73 f

4.27 c

3.98 d

3.93 e

Huea

0.63 a

0.62 a

0.62 a

Hue SO2b

1.81 a

1.85 a

Total phenolics (AU)

36.0 c

Anthocyanin concentration (g/L)

Phenolic parameter

p-value (time)

LSD0.05 (time)

Wine at 50 days post-inoculation -

-

-

-

<0.001

-

-

0.93 a

0.031

-

-

4.35 b

5.57 a

<0.001

-

-

0.63 a

0.60 a

0.59 b

0.019

-

-

1.84 a

1.86 a

1.76 a

1.52 b

<0.001

-

-

36.2 c

39.5 b

37.9 bc

43.7 a

44.9 a

0.004

0.23 c

0.24 c

0.25 c

0.23 c

0.30 b

0.35 a

0.017

Non-bleachable pigment (AU)

0.58 b

0.63 b

0.58 b

0.57 b

0.56 b

0.91 a

Tannin concentration (g/L)

0.69 c

0.67 c

0.79 b

0.77 b

0.92 a

0.92 a

Wine at 230 days postinoculation

36

<0.001

1.24

<0.001

0.025

0.010

<0.001

0.059

0.026

0.001

0.105

Wine color density (AU)

4.10 d

4.65 b

4.30 c

4.26 c

4.41 b

6.03 a

0.039

0.004

0.369

Huea

0.73 a

0.72 a

0.71 a

0.73 a

0.68 b

0.66 b

0.019

<0.001

0.026

Hue SO2b

1.52 b

1.56 b

1.53 b

1.62 a

1.59 b

1.33 c

0.026

<0.001

0.098

Repeated measures ANOVA. At each time interval, numbers within a row with different letters are significantly different at p ≤ 0.05; NS, not significant. aHue (ratio A420:A520 in model wine solution); bHue SO2 (resistant to sulfur dioxide bleaching). LSD, least significant difference.

37

Table 4 Proanthocyanidin composition of Pinot noir wine made with three skin supplement types at six months bottle age (230 days postinoculation). Must addition

mDPa

Nil (control)

7.70

5.81

60. 6

24.9

2.53

9.84

Pinot noir pomace

7.42

7.63

67.2

23.4

2.88

8.13

Pinot gris pomace

7.47

7.25

64.1

22.5

2.70

8.33

Chardonnay pomace

6.88

7.68

72.0

21.9

2.82

7.77

Liquid grape skin

6.97

4.68

49.1

23.2

2.22

10.4

Pinot noir marc

6.23

7.22

72.9

19.5

3.04

6.44

P-value (trt)

NS

0.02

0.04

0.03

0.002

0.01

-

1.91

0.15

0.03

0.003

2.07

LSD (Trt)

Total PA g/Lb %MC g/Lc

%tri-OHd PA %galle PA tri-OH/gallf

One-way ANOVA. P-values at p ≤ 0.05; amDP, Mean degree of polymerisation; bTotal proanthocyanidin subunits after depolymerisation; cMC, Mass conversion: percentage recovery of tannin subunits by phloroglucinolysis based on the MCPT tannin concentration; dpercentage of PA subunits that are tri-hydroxylated; epercentage of PA subunits that are galloylated subunits; fratio of tri-hydroxylated to galloylated subunits. NS, Not significant.

38

Table 5 Experiment 2: Effects of three skin supplement types on Pinot noir wine phenolic parameters at two time periods: bottling (50 days postinoculation) and six months (230 days post-inoculation). Phenolic parameter

Nil Pinot noir (control) skins

Pinot gris skins

Chardonnay skins

p-value (trt)

p-value (time)

LSD0.05 (time)

Wine at 50 days post-inoculation Anthocyanin concentration (g/L)

0.30

0.32

0.29

0.28

NS

-

-

Non-bleachable pigment (AU)

0.41

0.44

0.44

0.40

NS

-

-

Tannin concentration (g/L)

0.48 b

0.63 a

0.61 a

0.58 a

0.015

-

-

Color density (AU)

3.85 b

4.14 a

3.84 b

3.68 b

0.025

-

-

Hue

0.65

0.66

0.66

0.67

NS

-

-

Hue SO2

1.7

1.72

1.69

1.76

NS

-

-

Anthocyanin concentration (g/L)

0.22

0.25

0.19

0.22

NS

<0.001

0.019

Non-bleachable pigment (AU)

1.03 bc

1.46 a

1.20 ab

0.84 c

0.01

<0.001

0.133

Tannin concentration (g/L)

0.62 b

0.83 a

0.75 a

0.68 b

0.004

<0.001

0.044

Color density (AU)

5.85

6.15

6.06

4.85

NS

<0.001

0.429

Huea

0.73

0.73

0.74

0.75

NS

<0.001

0.011

Hue SO2b

1.18 b

1.12 b

1.11 b

1.30 a

0.044

<0.001

0.054

Wine at 230 days post-inoculation

39

Repeated Measures ANOVA. At each time period numbers within a row with different letters are significantly different at p ≤ 0.05; LSD, least significant difference; NS, not significant. aHue (ratio A420:A520 in model wine solution); bHue SO2 (resistant to sulfur dioxide bleaching).

40

Table 6 Experiment 3: Effects of fresh and fermented Pinot noir skin supplements on wine phenolic parameters at two time periods: bottling (50 days post-inoculation) and six months (230 days post-inoculation). Nil

Fresh

Fermented

(control)

Pinot noir skins

Pinot noir skins

p-value

p-value

LSD0.05

(trt)

(time)

(time)

Phenolic parameter Wine at 50 days post-inoculation Anthocyanin concentration (g/L)

0.18 c

0.22 a

0.20 b

0.004

-

-

Non-bleachable pigment (AU)

0.30 b

0.35 a

0.31 b

0.006

-

-

Tannin concentration (g/L)

0.48 b

0.72 a

0.73 a

0.009

-

-

Color density (AU)

2.65 b

3.12 a

2.84 b

0.008

-

-

Hue

0.76 a

0.74 ab

0.71 b

0.012

-

-

Hue SO2

1.94

1.90

1.87

NS

-

-

Wine at 230 days post-inoculation Anthocyanin concentration (g/L)

0.15 b

0.18 a

0.16 b

0.006

<0.001

0.336

Non-bleachable pigment (AU)

0.48 b

0.60 a

0.56 a

0.013

<0.001

0.024

Tannin concentration (g/L)

0.60 b

0.83 a

0.83 a

0.003

<0.001

0.027

Wine color density (AU)

2.99 b

3.62 a

3.37 a

0.005

<0.001

0.080

Hue

0.85 a

0.80 b

0.81 b

0.025

<0.001

0.006

41

Hue SO2

1.59 a

1.47 b

1.43 b

0.015

<0.001

0.033

Repeated Measures ANOVA. At each time period numbers within a row with different letters are significantly different at p ≤ 0.05; NS, not significant. aHue (ratio A420:A520 in model wine solution); bHue SO2 (resistant to sulfur dioxide bleaching).

42

GA

43

Highlights    

44

Demonstrates the impact of tannin supplements on wine color stability; In six months, 20% more grape skins increased stable pigment concentration by 40%; Up to 85% of anthocyanin from recycled grape skins retained as stable wine pigments; Color benefit of grape skin supplement compromised when seed equivalent included.