Food Chemistry 157 (2014) 385–392

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Effect of fining on the colour and pigment composition of young red wines Gustavo González-Neves a,⇑,1, Guzmán Favre a,1, Graciela Gil b a b

Facultad de Agronomía, Universidad de la República, Avda. Garzón 780, C.P. 12900 Montevideo, Uruguay Instituto Nacional de Vitivinicultura, Dr. Pouey 463, Las Piedras, Uruguay

a r t i c l e

i n f o

Article history: Received 21 November 2013 Received in revised form 12 February 2014 Accepted 15 February 2014 Available online 24 February 2014 Keywords: Tannat Fining Anthocyanins Tannins Wine

a b s t r a c t This work aimed to evaluate the effect of four fining agents on the colour and pigment composition of red wines of Tannat. The wines were analysed 15 days after fining and immediately after separation of sediments and bottling. Colour was evaluated by spectrophotometry and polyphenols were analysed by spectrophotometry and HPLC–DAD. The colour intensity of wine was significantly decreased by bentonite and egg albumin. The most remarkable effects on wine phenolic composition were produced by bentonite and gelatin, which significantly decreased anthocyanin and tannin concentrations, respectively. Results show that each fining agent has very different impact on the wine attributes, and their effects depended as well on the composition of the clarified wine. The use of non-traditional agents of fining, as vegetable proteins, may have less impact on the colour and anthocyanin content of red wines. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The colour and limpidity are the first sensory attributes of wines that are appreciate by consumers, predisposing their acceptance or rejection. A suitable wine stabilisation and limpidity is progressively obtained after winemaking due to physical and chemical phenomena that determine the precipitation of unstable compounds and the sedimentation of the clouding particles. This process is often improve by using different agents that will interact with the components of wine because the natural clarification, in addition to being slow, may not be enough for proper clarity and stability of the wine. Clarification using fining agents can reach a better limpidity in less time and may improve the stability of the wines (Sims, Eastridge, & Bates, 1995). Additionally, protein based fining agents can determine some declines in astringency and bitterness of wine due to its interaction with tannins (Karamanidou, Kallithraka, & Hatzidimitrou, 2011; Oberholster, Carstens, & Du Toit, 2013; Tschiersch, Pour Nikfardjam, Schmidt, & Schwack, 2010). Nevertheless, the interactions between protein based fining agents (like albumin and gelatin) and polyphenols can affect colour of young red wines due to the precipitation of pigments (Castillo-Sánchez, Mejuto, Garrido, & García-Falcón, 2006; ⇑ Corresponding author. Tel.: +598 24002663. 1

E-mail address: [email protected] (G. González-Neves). Tel.: +598 23563294.

http://dx.doi.org/10.1016/j.foodchem.2014.02.062 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

Castillo-Sánchez et al., 2008) even though the impact reported in some cases was slight (Stankovic, Jovic, & Zivkovic, 2004). In the case of other fining agents as bentonite, important declines in the colour intensity of wines were reported (Patil, Kaur, & Sharma, 2012; Stankovic, Jovic, Zivkovic, & Pavlovic, 2012). Polyphenols are the most important secondary metabolites and the major bioactive compounds synthesized in the berries. Several polyphenols extracted from grape skins and seeds along winemaking have an important role in sensory properties, as anthocyanins, pigments responsible for the colour of young red wines, and tannins, responsible for astringency and bitterness of wine (Cheynier et al., 2006; Fulcrand, Dueñas, Salas, & Cheynier, 2006). The most commonly used fining agents perform their tasks by attracting the positively and negatively charged particles in the unclear wine since they also also are charge carriers. Examples include bentonite (negatively charged), gelatin (positively) and egg white (positively). The emergence of bovine spongiform encephalopathy (BSE) has given considerable interest in the replacement of animal-derived protein in food processing and the alternative use of plant-derived proteins (Bindon & Smith, 2013; Chagas, Monteiro, & Ferreira, 2012). Additionally, food allergy and food intolerance based on hidden food ingredients increasingly raises public awareness (Tschiersch et al., 2010). Since 1999, many investigations have been carried out with wheat prolamins, commonly called gluten, as white musts and wines clarifying agents. Different experimental procedures were established to compare gluten efficaciousness

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with usual fining agents (Marchal, Marchal-Delhaut, Lallement, & Jeandet, 2002). At present, a wide variety of commercial preparations of plant-derived proteins from soy, gluten wheat, rice, potato, lupine or maize had been studied and proposed for oenological use with the name generically of ‘‘vegetable proteins’’ (Bindon & Smith, 2013; Chagas et al., 2012; Marchal et al., 2002; Tschiersch et al., 2010). This work aimed to evaluate the effect of different fining agents on the colour and pigment composition of red wines of Tannat. In order to evaluate these effects there were four small-scale experiences, contrasting four treatments of clarification with a witness. 2. Materials and methods 2.1. Chemical and fining agents Folin–Ciocalteu reagent and vainillin were purchased from Sigma–Aldrich (Switzerland). Sodium carbonate anhydrous was from Carlo Erba (Italy). Clorhidric acid was from J.T. Bakker (Mexico) and ethanol from Dorwil (Argentine). Water for high-pressure liquid chromatography (HPLC) analyses was nanopure. The anthocyanin standard was malvidin glucoside chloride from Extrasynthèse (France). Formic acid and methanol HPLC grade were from Sigma–Aldrich (Switzerland). The fining agents evaluated were bentonite, gelatine, vegetable protein and egg albumin. Bentonite (Bentogran) was from AEB (Italy) and Gelatin (Gelita Gold Strength/200 Bloom) was from Gelita (Spain). Egg albumin was added as fresh egg whites. Vegetable proteins used were gluten proteins. Abastecimientos S.A. (Uruguay) sponsored gelatin and vegetable protein. The company did not provide additional information about these products. 2.2. Wine treatments Four different wines were used in the clarification trials, including three wines of two months of age (wines 1–2 m, 2–2 m, 3–2 m) and one wine aged 14 months (wine 4–14 m). Each wine was clarified with the same four fining agents. The corresponding untreated wines were employed as a control (C) in each assay. The doses employed of each fining agent are the usually used in wineries. In each case, the doses were 50 g/HL for bentonite (B), 15 g/HL for gelatine (G), 15 g/HL for vegetable protein (VP), and 10 egg whites/HL for egg albumin (EA). Wine 1–2 m was elaborated in 2010, wine 4–14 m was elaborated in 2011, and wines 2–2 m and 3–2 m were elaborated in 2012. All wines were produced employing Tannat grapes grown in the south of Uruguay. The harvest was made according to the relationship between sugars contents, total acidity and pH of musts. The grapes employed to produce the wine 1–2 m had 20.8 Brix, 83.6 meq/L of total acidity and pH 3.44; for wine 2–2 m they had 21.2 Brix, 65.3 meq/L and pH 3.41; for wine 3–2 m they had 21.0 Brix, 73.4 meq/L and pH 3.30; and for wine 4–14 m they had 24.2 Brix, 89.8 meq/L and pH 3.35. These analyses were carried out using an Atago N1 refractometer (Atago, Japan) and a Hanna HI8521 pH metre (Hanna instruments, Italy), respectively. At harvest, the clusters were transported in plastic boxes (20 kg each one) to the winery. The bunches of grapes were destemmed and crushed with an Alfa 60 R crusher (Italcom, Italy), and the barrelling was in stainless-steel tanks (100 L capacity each). Potassium metabisulfite (50 mg SO2/100 kg of grapes) was added and dry active yeast (20 g/HL Saccharomyces cerevisiae, Natuferm 804; OenoBioTech, France) was inoculated in all the musts. The sulphur dioxide additions and yeast inoculations were realised immediately

after the crushing of grapes. Wines were made by classical fermentation on skins for 8 days. Two pumping over followed by punching the cap were carried out daily along the skin contact. The temperatures of fermentation were comprised between 23 and 26 °C. At devatting, free-run juice was obtained and the marc was pressed with a stainless steel manual press. In all cases, free-run juices and press juices were mixed. The wines were maintained in the stainless-steel tanks, where the fermentations were completed, until racking. The wines were stabilized with dioxide additions (50 mg SO2/L) realised at the end of malolactic fermentation. Finally, the wines were kept in glass recipients of 10-litre capacity, closed with cork stoppers, until fining. Before clarification, we proceeded to standardize the total volume of wine that was used in each trial, then dividing it into 10-litre containers, which hosted the addition of clarifiers. Fining was made in 2010 for wine 1–2 m and in 2012 for the others three wines. Fining treatments were performed in two replicates (two 10-litre containers of each wine for each fining agent). The wines were racked 15 days after fining and bottled in 750 mL green glass bottles, closed with cork stoppers. Immediately, the wines were analysed.

2.3. Analysis of turbidity of wines Turbidity of wines was analysed before fining and 15 days after, following a separation of the sediment and bottling. Turbidity was measured by nephelometric analysis performed by an Oakton TN-100/T-100 turbidity metre (Oakton Instruments, USA). Measurements were realised according to the methodology proposed by the turbidity metre manufacturer. Briefly, a calibration was made using standard solutions of 0.02, 20, 80 and 800 NTU, provided by the manufacturer. After that, the turbidity index of the wines were measured by duplicate. Results are expressed in NTU (Nephelometric Turbidity Unit).

2.4. Spectrophotometric analysis of wines Analyses of colour and polyphenol composition were realised 15 days after fining. Polyphenol indexes and colour were analysed by spectrophotometric methods. The colour of the wines was evaluated with the indexes proposed by Glories (1984): colour intensity (CI) and hue. Also, the CIELAB parameters brightness (L⁄), chromaticity (C⁄), redness (a⁄) and yellowness (b⁄) were determined, using the D65 illuminant and a 10° observer according to Ayala, Echávarri, and Negueruela (1997). Co-pigmentation indexes according to Boulton (2001) were measured and ‘‘colour due to anthocyanins’’ (CA), ‘‘colour due to co-pigmentation’’ (CC) and ‘‘colour due to polymers’’ (CP) were calculated. Total polyphenols were analysed with Folin–Ciocalteu reagent, according to the method proposed by Singleton and Rossi (1965), catechins according to Swain and Hillis (1959), and proanthocyanidin content was measured according to Ribéreau-Gayon and Stonestreet (1966). The DMACH index was measured and tannin polymerisation index was calculated as the relationship between the DMACH index and the proanthocyanidin content according to Vivas, Glories, Lagune, Saucier, and Augustin (1994). The wines were centrifuged for 3 min at 3000 rpm before spectrophotometric analysis. The measurements were carried out using a Cole Parmer S2100-UV+ (Cole Parmer, USA) and a Shimadzu UV-1800 (Shimadzu, Japan) UV–VIS spectrophotometer, employing glass cells with a 1 mm path length for the colour analyses and glass cells with a 1 cm path length for the polyphenol analyses. All analyses were performed by duplicate.

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2.5. HPLC–DAD analysis of pigments Anthocyanins and derivative pigments were analysed by HPLC–DAD, according to Revilla, Pérez-Magariño, González-Sanjosé, and Beltrán (1999). Briefly, after filtration through Sartorius filters (Sartorius, USA) (0.45 lm diameter), the samples were injected directly into a chromatographic system equipped with two pumps Waters 510 and 515, a Rheodyne 7725i injector (20 lm loop) and a photodiode detector Waters 2996 (Waters Corp., USA). The system was controlled with Millennium 32 Software (Waters Corp., USA). A Luna C18 reverse phase column, 5 lm, 150  4.6 mm (Phenomenex, USA) was used as the stationary phase, with a mobile phase flow rate of 0.8 mL/min. The solvent A was an aqueous solution (10%) of formic acid, and solvent B was an aqueous solution of methanol (45%) and formic acid (10%). A gradient was established from 35% to 95% B for 20 min, from 95% to 100% B for 5 min, isocratic 100% B for 5 min. Two replications of the analyses were performed in all cases. The identification of the compounds was carried out taking into account the spectrum of each and the retention time of each peak. Previously, the identification was confirmed (González-Neves et al., 2007) using a chromatographic system with a mass spectrophotometer (Hewlett Packard 1100 Series LC–MS) as a reference. Revilla et al. (1999) described the chromatographic conditions performed in this case. A photodiode array detector was coupled directly to the sprayer needle where ions were generated by atmospheric pressure chemical ionisation (APCI) or electrospray ionisation (ESI) in both positive and negative ionisation modes. The separation carried out by H.P.L.C. allowed the quantification of the non-acylated glucosides of delphinidin, cyanidin, malvidin, petunidin and peonidin, the acetylated glucosides of the same anthocyanidins and the coumarylic glucosides of delphinidin, cyanidin, malvidin and petunidin. Derivative pigments from anthocyanins identified and quantified were peonidin-3-O-glucoside pyruvate; vitisin B; vitisin A; malvidin-3-O-acetylglucoside pyruvate; malvidin-3-O-glucoside-etyl-catechin; malvidin-3-O-coumarylglucoside pyruvate; malvidin-3-O-glucoside-4-vinylcatechol; malvidin-3-Oglucoside-4-vinylphenol. The concentration of each pigment was calculated using a calibration curve with malvidin glucoside chloride (Extrasynthese, France) and the results are expressed in mg/L of malvidin-3-O-glucoside. The total amounts of malvidin, petunidin, delphinidin, peonidin and cyanidin and those of non-acylated, acetylated and coumarylated glucosides were calculated. The total anthocyanin content of the wines was calculated considering the sum of all the anthocyanins quantified.

2.6. Statistical analyses Analyses of variance and media separation by Tukey at 5% were performed using the Statgraphics Plus package, version 4.1 (Statistical Graphics Corporation, USA).

3. Results and discussion 3.1. Turbidity of wines After fining, the turbidity of the wines was compared with those registered before treatment (Fig. 1). All fining treatments increased the limpidity of wines but every fining agents made different effect in each wine, in agreement with the results reported by several studies (Cosme, Ricardo-Da Silva, & Laureano, 2009; Maury, Sarni-Manchado, Lefebvre, Cheynier, & Moutounet, 2003; Oberholster et al., 2013).

Fig. 1. Turbidity of wines before and after fining. BF = before fining; B = wine after 15 days of treatment with bentonite; VP = wine after treatment with vegetable proteins; EA = wine after treatment with egg albumin; G = wine after treatment with gelatin. NTU = Nephelometric Turbidity Unit.

A higher degree of clarification was obtained in young wines than in older one, except when gelatin was the clarificant agent. It can be assumed that the 14 months aged wine, in spite of having a greater turbidity than some of the younger wines, had a greater degree of stability achieved naturally, which may explain the lower impact of the clarification on their limpidity. Bentonite showed the best clarification effect in all two months aged wines whereas gelatin done the best limpidity in the older wine. Bentonite decreased turbidity 96.3% to 99.1% in the wines of two months of age, and 71.3% in the wine of 14 months, compared with wines before fining. Egg albumin 92.6–97.6% and 70.4%, respectively; vegetable proteins 68.0–94.7% and 51.2%. Instead, gelatin decreases turbidity 67.7–94.9% in the wines of two months and 91.8% in the wine of 14 months. 3.2. Colour and pigment contents of wines Table 1 shows the chromatic properties of wines. Most fining treatments decreased the colour intensity compared to the control wines. Bentonite had the highest impact on the colour of wines (Table 1), affecting their intensity and quality. Decreases in colour intensity ( 11.1% to 17.4% in relation to control wines) were accompanied by increases of hue (+1.2 to +6.0) in the wines clarified with this agent. The loss in redness and the increase in yellowness, compared to control wines, were also showed by the values of a⁄ and b⁄. Consequently, the wines treated with bentonite were the brightest while control wines were the darkest (Table 1). Others works reported similar effects of bentonite on the colour of red wines (Patil et al., 2012; Stankovic et al., 2012). In all fined wines, the brightness (L⁄) were increased (significantly by using bentonite and egg albumin) and redness (a⁄) decreased (Table 1). These results are in agreement with others previously reported (Cosme, Ricardo-Da Silva, & Laureano, 2007; Gil-Muñoz, Gómez-Plaza, Martínez, & López-Roca, 1997). Egg albumin also diminished colour intensity in all wines (9.3–14.6%) whereas gelatin affected this attribute in most of wines. Fining with plant proteins determined the lowest decline in the colour intensity and small differences in CIELAB attributes in relation to control wines. In some cases, fining with proteic agents had no effect on colour intensity, as the application of vegetable proteins in 3–2 and 4–14 m wines, and gelatin in 3–2 m wine (Table 1). This different behaviour could be related to the differences in pigment composition among wines. The colour of

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Table 1 Colour of wines. Wines (m)

TreatmentsA

CIB

L⁄C

Hue a

ab

d

C⁄D

a⁄E

b⁄F

1–2

C B VP EA G

12.04 ± 0.08 10.71 ± 0.11d 11.67 ± 0.15b 10.34 ± 0.17e 11.14 ± 0.04c

0.65 ± 0.01 0.66 ± 0.01a 0.65 ± 0.01ab 0.63 ± 0.01c 0.64 ± 0.00b

46.65 ± 0.07 50.33 ± 0.35b 47.48 ± 0.44d 51.33 ± 0.56ª 49.00 ± 0.08c

48.56 ± 0.64ª 46.56 ± 0.32d 47.74 ± 0.25ab 46.83 ± 0.33cd 47.46 ± 0.21bc

48.22 ± 0.62ª 46.26 ± 0.34d 47.45 ± 0.25ab 46.69 ± 0.33cd 47.22 ± 0.20bc

5.74 ± 0.25ª 5.27 ± 0.12b 5.24 ± 0.12b 3.62 ± 0.08c 4.70 ± 0.19c

2–2

C B VP EA G

11.52 ± 0.06a 9.80 ± 0.03d 11.11 ± 0.11b 10.44 ± 0.07c 10.46 ± 0.11c

0.70 ± 0.01b 0.74 ± 0.01ª 0.71 ± 0.01ab 0.71 ± 0.01ab 0.71 ± 0.01ab

47.40 ± 0.14c 52.65 ± 0.21ª 48.75 ± 0.49c 50.60 ± 0.14b 50.35 ± 0.49b

43.70 ± 0.96ª 39.29 ± 0.49b 43.28 ± 0.58a 41.58 ± 0.13ª 41.93 ± 0.31ª

43.28 ± 0.98ª 39.00 ± 0.52b 42.84 ± 0.69ª 41.29 ± 0.16ab 41.73 ± 0.33ª

6.05 ± 0.04ª 4.82 ± 0.18ab 6.11 ± 0.73ª 4.89 ± 0.22ab 4.15 ± 0.23b

3–2

C B VP EA G

8.44 ± 0.01ª 7.24 ± 0.02c 8.45 ± 0.06ª 7.60 ± 0.03b 8.27 ± 0.08ª

0.75 ± 0.01b 0.79 ± 0.01ª 0.76 ± 0.01ab 0.76 ± 0.01ab 0.75 ± 0.01b

58.10 ± 0.00c 62.95 ± 0.07ª 58.00 ± 0.57c 61.05 ± 0.21b 58.60 ± 0.14c

36.92 ± 0.52ª 32.72 ± 0.43b 36.05 ± 0.06ª 33.55 ± 0.42b 35.95 ± 0.43ª

36.27 ± 0.56ª 31.96 ± 0.35b 35.54 ± 0.08ª 33.22 ± 0.43b 35.44 ± 0.35ª

6.91 ± 0.13ª 6.98 ± 0.40ª 6.02 ± 0.85ab 4.76 ± 0.01b 5.98 ± 0.51ab

4–14

C B VP EA G

10.69 ± 0.30ª 8.83 ± 0.27c 10.24 ± 0.18ab 9.13 ± 0.16c 9.57 ± 0.16bc

0.66 ± 0.01b 0.69 ± 0.01a 0.66 ± 0.01b 0.65 ± 0.01b 0.65 ± 0.01b

50.55 ± 0.78c 55.60 ± 0.42ª 51.50 ± 0.28c 55.15 ± 0.07ab 53.55 ± 0.21b

46.26 ± 0.61ab 42.99 ± 0.05d 46.51 ± 0.13ª 44.05 ± 0.09cd 45.26 ± 0.24bc

45.86 ± 0.54ª 42.56 ± 0.12c 46.16 ± 0.27ª 43.88 ± 0.14b 44.99 ± 0.28ab

6.04 ± 0.61ns 6.02 ± 0.50ns 5.56 ± 1.12ns 3.81 ± 0.57ns 4.95 ± 0.36ns

Mean values ± standard deviations. Values with the same letters for each wine have no statistical significant differences, according to a Tukey test (p < 0.05). ns = not significant. A Treatment abbreviations: C = control, B = bentonite, VP = vegetable proteins, EA = egg albumin, G = gelatin. B CI = colour intensity. C L⁄ = brightness. D C⁄ = chromaticity. E a⁄ = redness. F b⁄ = yellowness.

young red wines depend on their contents in anthocyanin and derivative pigments from anthocyanins but also depend on copigmentation phenomena and pH of wine (Boulton, 2001; Fulcrand et al., 2006; Glories, 1984). Chemical structure, degree of ionisation and levels of anthocyanins primordially define the chromatic properties of young red wines and affect the formation of derivative pigments (Cheynier et al., 2006; Fulcrand et al., 2006; Glories, 1984). The condensation between anthocyanins and tannins mediated by ethanal and the cycle-addition between anthocyanins and some yeast metabolites result in the formation of anthocyanin-derived pigments that are more stable than the original anthocyanins, with consequent colour stabilization (Cheynier et al., 2006; Fulcrand et al., 2006). Fining with bentonite significantly diminished the anthocyanin contents of wines (Tables 2 and 3). At the same time, the levels of derivative pigments were diminished by most of fining and particularly by bentonite (Table 2). Decreases of the levels of anthocyanins by bentonite were comprises among 9.8% and 35.0% in relation to their concentration in control wines. These results agree with those reported in previous works realised with the same grape variety (González-Neves & Gil, 1998) and with other grape varieties (Stankovic et al., 2012). Decreases of the levels of anthocyanin-derived pigments by bentonite were comprises among 7.3% and 34.7%. The highest decreases in pigments contents by bentonite were verified in the older wine (Table 2). It is expected that polymeric pigments have a more important effect on the colour in the oldest wines (Cheynier et al., 2006) so that the impact of bentonite on the colloidal coloured matter could explain the results. On the other hand, Gómez-Plaza, Gil-Muñoz, López-Roca, De la Hera-Orts, and Martínez-Cutillas (2000) reported an increase in anthocyanin content of wines by pre-fermentation addition of bentonite, suggesting that the moment of addition influences strongly the action of fining agents. These authors explain their results

indicating that bentonite prevents anthocyanin from fixation on solids and yeasts that are remove following fermentation. Table 2 shows the values of the copigmentation indexes. Bentonite diminished the ‘‘colour due to anthocyanins’’ (CA), and the ‘‘colour due to copigmentation’’ (CC). Bentonite, egg albumin and gelatin decreased the ‘‘colour due to polymers’’ (CP) in all wines. Copigmentation is a solution phenomenon in which pigments and other non-coloured organic components form molecular associations or complexes. It generally results in an enhancement in the absorbance and in some cases, a shift in the wavelength of the maximum absorbance of the pigment (Boulton, 2001; Cheynier et al., 2006). The effects of bentonite on pigment composition and copigmentation can explain its great impact on the colour of wines. Boulton (2001) indicates that the presence of copigmented forms of the anthocyanins has great importance in the effects of fining on the colour of young red wines. This author assert that, because the copigmented form is so much more colourful than the free form, any treatments that cause some dissociation of the copigmentation stack can have more effect on colour than that due to anthocyanin depletion. Anthocyanins contents were slightly decreased by using egg albumin and gelatin, and were not affected by the utilisation of vegetable proteins in half of wines (Table 2). These results agree with the effect of those agents on the colour. Several works previously reported these effects of egg albumin and gelatin on colour and anthocyanin contents of wines (Castillo-Sánchez et al., 2006; Cosme et al., 2007; Karamanidou et al., 2011; Oberholster et al., 2013). Differences in the behaviour of the wines clarified with every agent are probably due to the different phenolic profile of each one. Anthocyanins (and tannins) are molecules containing benzene rings with adjacent hydroxyl groups that are proposed as a major source of hydrogen bonds at the basis of complex formation between gelatin and anthocyanins (or tannins) in wines. Gelatin is held to be particularly suited to hydrogen bonding because

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G. González-Neves et al. / Food Chemistry 157 (2014) 385–392 Table 2 Copigmentation indexes and pigment contents of wines. TreatmentsA

Wines (m)

CCB

CAC a

a

CPD

AnthocyaninsE

Derivative pigmentsF

1–2

C B VP EA G

0.49 ± 0.02 0.40 ± 0.01b 0.35 ± 0.03c 0.37 ± 0.01c 0.47 ± 0.01a

2.88 ± 0.01 2.56 ± 0.01c 2.97 ± 0.02a 2.76 ± 0.01b 2.80 ± 0.01ab

1.84 ± 0.01ª 1.50 ± 0.01d 1.82 ± 0,01ª 1.78 ± 0,01b 1.69 ± 0,01c

179.73 ± 1.86ª 159.85 ± 1.46c 172.92 ± 0.85b 174.78 ± 1.04b 173.89 ± 0.66b

3.34 ± 0.02ª 3.13 ± 0.01d 3.27 ± 0.01b 3.21 ± 0.02c 3.22 ± 0.01c

2–2

C B VP EA G

0.45 ± 0.01c 0.66 ± 0.02a 0.40 ± 0.01c 0.44 ± 0.02c 0.53 ± 0.01b

3.38 ± 0.01ª 2.97 ± 0.02c 3.36 ± 0.01ª 3.17 ± 0.02b 3.24 ± 0.03b

2.18 ± 0.01ns 1.81 ± 0.01ns 2.16 ± 0.01ns 1.97 ± 0.01ns 1.99 ± 0.01ns

173.68 ± 1.64ª 156.60 ± 1.29b 175.30 ± 0.81ª 172.85 ± 0.22ª 173.39 ± 0.31ª

5.55 ± 0.01ª 4.31 ± 0.01c 5.60 ± 0.01ª 5.28 ± 0.01b 5.52 ± 0.01ª

3–2

C B VP EA G

0.50 ± 0.01a 0.22 ± 0.01d 0.25 ± 0.01d 0.30 ± 0.01c 0.42 ± 0.01b

2.39 ± 0.09ab 2.15 ± 0.03c 2.58 ± 0.09ª 2.34 ± 0.01bc 2.36 ± 0.01abc

1.51 ± 0.01ª 1.19 ± 0.01b 1.48 ± 0.01ª 1.26 ± 0.01b 1.40 ± 0.01ª

120.63 ± 0.03ª 94.85 ± 0.60d 120.07 ± 0.18ab 116.88 ± 0.06c 118.18 ± 0.01b

2.96 ± 0.02ª 2.06 ± 0.01d 2.88 ± 0.02b 2.68 ± 0.01c 2.79 ± 0.03b

4–14

C B VP EA G

0.32 ± 0.01b 0.26 ± 0.01c 0.37 ± 0.01a 0.32 ± 0.01b 0.28 ± 0.01c

2.46 ± 0.04ª 1.99 ± 0.01c 2.30 ± 0.01b 2.12 ± 0.01c 2.32 ± 0.04b

2.76 ± 0.01ns 2.29 ± 0.01ns 2.70 ± 0.01ns 2.28 ± 0.01ns 2.46 ± 0.01ns

33.20 ± 0.37b 21.59 ± 0.09c 32.82 ± 0.11b 32.81 ± 0.04b 34.89 ± 0.18ª

6.56 ± 0.04ª 4.28 ± 0.02d 6.58 ± 0.01ª 5.86 ± 0.01c 6.28 ± 0.03b

Mean values ± standard deviations. Values with the same letters for each wine have no statistical significant differences, according to a Tukey test (p < 0.05). ns = not significant. A Treatment abbreviations: C = control, B = bentonite, VP = vegetable proteins, EA = egg albumin, G = gelatin. B CC = ‘‘colour due to copigmentation’’. C CA = ‘‘colour due to anthocyanins’’. D CP = ‘‘colour due to polymers’’. E Anthocyanins. F Derivative pigments from anthocyanins are expressed in mg/L of malvidin glucoside.

Table 3 Anthocyanin composition of wines. Wines (m)

TreatmentsA

DpB

PtC

PnD d

MvE

NAF

AcG

CmH

a

13.40 ± 0.15ª 10.61 ± 0.22d 11.78 ± 0.14c 12.15 ± 0.15b 12.29 ± 0.06b

1–2

C B VP EA G

8.08 ± 0.01ª 6.45 ± 0.10c 7.20 ± 0.12b 7.18 ± 0.11b 7.19 ± 0.03b

19.78 ± 0.16 17.32 ± 0.18d 19.08 ± 0.04b 18.99 ± 0.17bc 18.77 ± 0.09c

8.11 ± 0.01ª 7.94 ± 0.09ª 7.89 ± 0.02ab 7.93 ± 0.15ab 7.76 ± 0.06b

143.71 ± 1.67 128.11 ± 1.29c 138.71 ± 0.81b 140.63 ± 0.85b 140.13 ± 0.66b

133.35 ± 1.60ª 120.73 ± 0.79c 129.80 ± 0.69b 130.57 ± 0.82b 130.06 ± 0.73b

32.77 ± 0.11ª 28.32 ± 0.45d 31.10 ± 0.07c 31.95 ± 0.17b 31.36 ± 0.17+

2–2

C B VP EA G

5.67 ± 0.03ª 4.41 ± 0.02c 5.66 ± 0.04a 5.42 ± 0.01b 5.33 ± 0.01b

17.08 ± 0.08ª 14.48 ± 0.10e 16.48 ± 0.07b 16.00 ± 0.03c 15.21 ± 0.08d

8.22 ± 0.08b 7.47 ± 0.05c 9.98 ± 0.01ª 8.12 ± 0.04b 8.29 ± 0.03b

142.70 ± 1.50ª 130.23 ± 1.22b 143.19 ± 0.70ª 143.30 ± 0.16ª 144.56 ± 0.24a

131.07 ± 1.51ª 119.16 ± 0.90b 132.19 ± 0.83ª 130.79 ± 0.14ª 130.04 ± 0.44ª

32.98 ± 0.00ba 29.69 ± 0.31c 33.30 ± 0.02ba 32.67 ± 0.17b 33.40 ± 0.08a

9.40 ± 0.13bc 7.64 ± 0.07d 9.56 ± 0.05ba 9.20 ± 0.08c 9.73 ± 0.05a

3–2

C B VP EA G

3.37 ± 0.03ª 2.48 ± 0.02d 3.20 ± 0.01bc 3.14 ± 0.03c 3.27 ± 0.01b

10.08 ± 0.02ª 7.60 ± 0.05c 9.92 ± 0.09ª 9.43 ± 0.03b 9.93 ± 0.02ª

5.25 ± 0.01b 3.87 ± 0.02d 4.88 ± 0.01c 4.86 ± 0.01c 5.44 ± 0.04ª

101.92 ± 0.09ª 80.90 ± 0.55d 102.07 ± 0.08ª 99.45 ± 0.01c 100.79 ± 0.06b

92.36 ± 0.02ª 74.38 ± 0.53c 91.90 ± 0.21ª 89.82 ± 0.04b 91.55 ± 0.01ª

21.52 ± 0.07ª 16.42 ± 0.08c 21.61 ± 0.05ª 20.99 ± 0.05b 21.39 ± 0.02ª

6.74 ± 0.02ª 4.04 ± 0.01e 6.56 ± 0.02b 6.07 ± 0.03d 6.49 ± 0.02c

4–14

C B VP EA G

1.19 ± 0.02ª 0.67 ± 0.01c 1.21 ± 0.01ª 1.12 ± 0.00b 1.21 ± 0.01a

3.37 ± 0.01b 2.16 ± 0.02c 3.41 ± 0.01b 3.40 ± 0.02b 3.71 ± 0.01a

0.37 ± 0.01b 0.21 ± 0.01d 0.33 ± 0.01c 0.39 ± 0.01b 0.46 ± 0.01a

28.26 ± 0.34b 18.53 ± 0.10c 27.88 ± 0.10b 27.90 ± 0.06b 29.51 ± 0.17a

27.94 ± 0.34b 18.60 ± 0.06c 27.73 ± 0.12b 27.71 ± 0.02b 29.33 ± 0.16a

4.04 ± 0.02b 2.53 ± 0.02d 3.94 ± 0.01c 4.10 ± 0.02b 4.26 ± 0.01a

1.21 ± 0.00b 0.46 ± 0.00e 1.15 ± 0.00c 0.99 ± 0.00d 1.30 ± 0.00a

Mean values ± standard deviations. Values with the same letters for each wine have no statistical significant differences, according to a Tukey test (p < 0.05). ns = not significant. A Treatment abbreviations: C = control, B = bentonite, VP = vegetable proteins, EA = egg albumin, G = gelatin. Concentrations of each type of molecule in mg/L of malvidin glucoside. B Dp: delphinidin. C Pt: petunidin. D Pn: peonidin. E Mv: malvidin. F NA: non-acylated glucosides. G Ac: acetyl glucosides. H Cm: coumaroyl glucosides.

one third of the amino acids are glycine, where R = H, and hence steric hindrance to hydrogen bonding would be far less than with proteins containing less glycine (Marchal et al., 2002). Anyway, the

different composition of proteins used as fining agents determines a wide diversity of molecular masses, isoelectric points and surface charge densities that modify strongly their interactions with

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polyphenols and their effect on the colour of wines (Marchal et al., 2002; Maury, Sarni-Manchado, Lefebvre, Cheynier, & Moutounet, 2001; Maury et al., 2003; Sarni-Manchado, Deleris, Avallone, Cheynier, & Moutounet, 1999). Decreases in ‘‘colour due to polymers’’ verified by bentonite, egg albumin and gelatin can significantly affect the future colour of wines because colour stability depends on polymeric pigments. Boulton (2001) suggests that the level of copigmentation may influence the rates of polymer formation and oxidation, and wines that are higher in copigmentation would react more slowly than the wines lower in copigmentation, under the same conditions. The links between the copigments could be the first step in the reactions that produce new and more stable pigments (Boulton, 2001; Cheynier et al., 2006). 3.3. Anthocyanin composition of wines The additions of bentonite affected more significantly the amounts of anthocyanins based on delphinidin, petunidin, acetylglucosides and coumaryl-glucosides, although the effect was different in each wine (Table 3, Fig. 2). To our knowledge, only few studies have considered this differential effect of bentonite on the anthocyanin composition of red wines. The results reported by Stankovic et al. (2004) agreed with those found in our work. These authors highlighted that the amounts of delphinidin glucoside and acylated glucosides were the anthocyanins the most diminished in all wines clarified by bentonite. The differential effect on anthocyanin composition can modify the chromatic properties of the wines and their colour stability, changing wine quality (Cheynier et al., 2006). Bentonite, a cation exchanger clay, is an inorganic fining agent that removes wine proteins by electrostatic adsorption (Pocock, Salazar, & Waters, 2011). Additional bentonite effects include electrostatic interaction, theoretically, with all compounds bearing/ carrying a positive net charge at wine pH. Therefore, in addition to adsorbing proteins, bentonite is described to remove other positively charged molecules as anthocyanins (Chagas et al., 2012). Our results suggest that the adsorption of anthocyanins by bentonite could be compared to the adsorption of these compounds by yeasts. In our study, the anthocyanidins more adsorbed were the more polar molecules (Table 3). In agree, the relationship between the polarity of anthocyanins and their adsorption by yeasts was signalled by many reports (Medina, Boido, Dellacassa, & Carrau,

2005; Vasserot, Caillet, & Maujean, 1997), where also delphinidin and petunidin derivatives were the most adsorbed, supporting our hypothesis. This loss of polar anthocyanins could be explained by the affinity of these compounds to the cell wall of yeasts (Vasserot et al., 1997). Furthermore, Morata et al. (2003) indicate that the differences in the adsorption of anthocyanins derivatives are owing to the different polarity of each derivative according to the hydroxylation/methoxylation grade in B ring and the acyl moiety. In this context, these authors signaled that the acyl derivatives of anthocyanins (acetyl and p-coumaryl compounds) were more strongly adsorbed by the yeasts than non-acyl derivatives. Morata, Gómez-Cordovés, Colomo, and Suárez (2005) signalled that cinnamoyl derivatives (coumaroyl and caffeoyl-glucosides) were the most strongly anthocyanins molecules adsorbed while vitisins (adducts of pyruvic acid and acetaldehyde) were weakly adsorbed. This suggests that adsorption by yeasts also involves a hydrophobic interaction. In conclusion, our results could confirm that the different polarities and the hydrophilic or hydrophobic nature of the fining agents define their capacity to retain or adsorb different anthocyanins and others phenols. Therefore, wine polyphenolic composition should be evaluate for a better choice of the fining agent to employ, according to the style of wine sought in each case. The anthocyanin profiles of the wines showed differences between them (Table 3), but the proportions of each type of molecule verified in each one are similar to the typical values reported for this variety (González-Neves et al., 2007; González-Neves et al., 2010). The percentages of the different type of anthocyanins are related to the age of the wines (Cheynier et al., 2006; Fulcrand et al., 2006), with very significant differences between the wines of two months and the wine of 14 months (data not showed). In turn, the impact of each clarifier on anthocyanin profile was different in the two months aged wines in relation to the 14 months aged wine. For example, Fig. 2 shows the variations in delphinidin contents of the clarified wines in relation to their content in the respective control wines after fining. The most important changes on the anthocyanin profile and the contents of the different monomeric pigments were verified in the 4–14 m wine fined with bentonite (Table 3, Fig. 2). The impact of the applied dose of bentonite can be related to the contents of anthocyanins, because they were very lower in this wine. In turn, the matrix of the wine can influence the action of bentonite. The older wine had more acidity than the others wines (pH 3.41 for wine 4–14 m, 3.83 for 1–2 m, 3.79 for 2–2 m, and 3.82 for wine 3–2 m). These differences were based in the different acidic composition of the grapes of origin and the differential impact of malolactic fermentation in each wine. This process had less influence on the wine 4–14 m, because its initial content of malic acid was significantly lower than those of the other wines (data not showed). Because of the lower pH, the oldest wine had more proportion of ionised anthocyanins, which are more adsorbed by bentonite. Moreover, decreases in anthocyanin contents of wines along ageing are differential, because the more unstable molecules react more quickly (Cheynier et al., 2006; Fulcrand et al., 2006). 3.4. Effect on tannin contents of wines

Fig. 2. Variation (%) in delphinidin contents of the clarified wines in relation to their content in control wines after fining. B = wine treated with bentonite; VP = wine treated with vegetable proteins; EA = wine treated with egg albumin; G = wine treated with gelatin.

Table 4 shows that total polyphenol content of wines was generally diminished by fining. Egg albumin had the most important effect, with decreases of total polyphenols levels between 6.0% and 8.5% (Table 4). These results are due principally to the effect of the different fining agents on tannin contents of wines. Gelatin removed more tannins of low molecular weight (catechins) than removed the other agents in most of the wines (Table 4). In some cases, it was found that the contents of catechins of the respective control wines was inferior to those of the clarified

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TreatmentsA

Total polyphenolsB

CatechinsC

ProanthocyanidinsD

42.7 ± 0.5b 43.1 ± 0.4b 44.9 ± 1.2b 44.3 ± 0.7b 46.1 ± 0.6a

805 ± 3c 874 ± 6bc 982 ± 35ª 949 ± 31ab 829 ± 6c

2020 ± 68ª 2068 ± 27ª 1769 ± 14b 2030 ± 27ª 1788 ± 14b

44.5 ± 1.7bc 43.9 ± 0.1c 49.0 ± 0.5ab 41.2 ± 0.6c 48.0 ± 0.3a

1196 ± 6ab 1142 ± 32bc 1172 ± 2b 1094 ± 10c 1239 ± 10a

854 ± 16ab 911 ± 22a 867 ± 3ab 851 ± 31ab 794 ± 25b

1701 ± 27a 1701 ± 2ª 1759 ± 26ª 1450 ± 27c 1556 ± 14b

41.0 ± 0.3c 42.2 ± 0.2c 40.7 ± 0.1c 47.4 ± 0.4ª 44.2 ± 0.7b

1181 ± 40ns 1158 ± 29ns 1143 ± 21ns 1110 ± 13ns 1092 ± 6ns

764 ± 2ns 708 ± 8ns 785 ± 25ns 782 ± 3ns 736 ± 6ns

1759 ± 55ª 1585 ± 55ab 1759 ± 27ª 1546 ± 27ab 1392 ± 55b

39.5 ± 1.7b 42.0 ± 1.7b 38.7 ± 0.5b 40.9 ± 1.8b 46.9 ± 2.7a

C B VP EA G

1467 ± 12ª 1439 ± 22ª 1412 ± 19ª 1357 ± 26b 1415 ± 17ª

2–2

C B VP EA G

1407 ± 19ab 1433 ± 21ª 1420 ± 40ab 1313 ± 8b 1348 ± 42ab

3–2

C B VP EA G

4–14

C B VP EA G

1060 ± 17 1109 ± 20ª 1119 ± 14ª 1035 ± 48b 999 ± 20b

ab

DMAC/LAE

2097 ± 68 2194 ± 119ª 2097 ± 81ab 1904 ± 51b 1890 ± 33b

1–2

ab

Mean values ± standard deviations. Values with the same letters for each wine have no statistical significant differences, according to a Tukey test (p < 0.05). ns = not significant. A Treatment abbreviations: C = control, B = bentonite, VP = vegetable proteins, EA = egg albumin, G = gelatin. B Total polyphenols in mg/L of gallic acid. C Catechins in m/L of D-catechin. D Proanthocyanidins in mg/L of cyanidin chlorure. F DMAC/LA = tannin polymerisation index.

wines. These results were verified for all treatments in the wine 2–2 m and for all the wines fined with vegetable proteins and all wines of two months fined with bentonite. The different behaviour observed with a given fining agent in the different wines could be related to differences in the composition of these wines. Several authors indicate that the interactions between tannins and proteins are influenced by the concentrations and the characteristics of both compounds, such as molecular size and structure (Karamanidou et al., 2011; Maury et al., 2001; Sarni-Manchado et al., 1999; Versari et al., 1998). Gelatin determined significant decreases in proanthocyanidin contents in all wines (Table 4), in agreement with numerous previous works (Cosme et al., 2009; Oberholster et al., 2013; Sarni-Manchado et al., 1999; Versari et al., 1998). In all cases, the values of the polymerisation index of tannins (DMAC/LA) increased in the wines fined with gelatin (Table 4), indicating that gelatin selectively removed more the tannins molecules of high size. This effect was particularly notable in the older wine, which had also the greatest decrease in tannin content. In average, tannins of this wine had higher molecular size than those present in the wines of two months, according to the values of DMAC/LA. Several authors found that the degree of polymerisation of the proanthocyanidins, besides their composition, affect the interaction between proteins and tannins since the more polymerized molecules and those esterified with gallic acid react more than the less polymerized proanthocyanidins (Cosme et al., 2009; Maury et al., 2001; Sarni-Manchado et al., 1999). Anyway, given the great diversity of oenological gelatines available in the market, the effect of the application of this clarifying agent must refer to the characteristics of each product (Karamanidou et al., 2011; Maury et al., 2001; Versari et al., 1998). Several papers found that the interaction between proanthocyanidins and gelatin depends on the degree of hydrolysis of gelatins. Many authors suggest that the shorter protein molecules, which in the case of the gelatin correspond to a higher degree of hydrolysis, precipitate larger amounts of tannin because hydrolysis ameliorates binding site accessibility and favours protein-tannin interactions leading to precipitation (Maury et al.,

2001 and 2003; Sarni-Manchado et al., 1999). However, in other studies it was found that the largest decreases in the concentration of tannins with gelatin were obtained with lower degree of hydrolysis, attributing these results to the effect of the electrical charge of the molecules (Karamanidou et al., 2011). Egg albumin significantly decreased tannin content on three of the tested wines (Table 4), but had no effect on one of two months wines (2–2 m). The ability of tannins to complex with proteins is one of their most important properties. Indeed, the astringency is believed to result from interaction of tannins with the salivary proline-rich proteins (Cheynier et al., 2006; Maury et al., 2001; Sarni-Manchado et al., 1999). However, the fining with vegetable proteins had not significant effect on proanthocyanidin contents in three of the four wines (Table 4). Because in this case there is also a wide variety of commercial preparations, the evaluation of its use must refer to the characteristics of each particular product (Marchal et al., 2002; Maury et al., 2003; Tschiersch et al., 2010). In our case, the lack of information about the product features do not allow a better understanding of its effects. Nevertheless, the results confirm that there is a specific action of each class of fining agent on the different type of pigments, so the fining process depending of wine composition will affect the colour of wine differently. Furthermore, a more comprehensive study of the influence of each fining agent on tannic properties of wine can be achieved by analysing the proanthocyanidin composition of wines. 4. Conclusions Each fining agent had different influence on the polyphenolic composition, determining a different impact on the colour of the wine. In turn, the composition of the wine modified the effect of the fining agents. The most remarkable effects were those obtained by bentonite, which had negative impact on the anthocyanin contents and wine colour, and gelatin, which significantly decreased tannins concentrations.

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The different fining products modified in different way the tannin composition of wines. The proanthocyanidins contents were decreased significantly in all wines clarified by gelatin. Non-traditional fining agents, as vegetable proteins, may have less impact on the colour and anthocyanin content of red wines. Nevertheless, the great diversity of commercial preparations available in the market becomes necessary to know the characteristics of each product for a better comprehension of its effects. In addition, the polyphenolic composition of wine should be evaluated for a better choice of the fining agent to use, according to the style of wine sought in each case. In any case, the results of this work confirm that fining is a process that require previous assays, for a better prevision of its results and a better choice of the fining agents and their doses.

Acknowledgements This work was support by the Project CSIC-Udelar I + D 2010 ‘‘Tannat wines’’. Abastecimientos S.A. (Uruguay) sponsored gelatin and vegetable protein. The authors thanks C. Baldi, N. Hernández, S. Traverso, D. Piccardo and P. Bieito for their participation in the assays.

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Effect of fining on the colour and pigment composition of young red wines.

This work aimed to evaluate the effect of four fining agents on the colour and pigment composition of red wines of Tannat. The wines were analysed 15 ...
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