Influence Of Age On Red Wine Colour During Fining With Bentonite And Gelatin

Abstract

Two fining agents (bentonite and gelatin) have contributed to the reduction of colour of Pinot Noir wine of different ages. Bentonite caused more significant changes in the colour of young wines. Colour intensity, coloured anthocyanin forms, and polymers were reduced to a greater extent, while colourless anthocyanins and total phenolic substances were reduced to a lower extent. Smaller changes in coloured substances resulted from wine treatment with gelatin. The intensity of the share of yellow colour decreased and the intensity of the share of red colour, together with dA% value, increased. The influence of bentonite on the colour of the wine decreased while the influence of gelatin increased with wine ageing.

Keywords:

• Age of red wine
• Fining
• Bentonite
• Gelatin
• Anthocyanins
• Colour intensity

INTRODUCTION

When producing red wine, it is important to obtain good colour and clarity of wine and, if possible, preserve it for a long period of time. Colour stability and clarity of red wine are provided primarily by stabilization. Wine should be prepared for market relatively quickly, for example, in a shorter time than that necessary for obtaining nearly the same effect by spontaneous fining.[1] Fining is not employed on cloudy wines only, but also on wines showing tendency towards turbidity and settling. Fining agents of various origin are used, provided they do not cause change in the taste or aroma of wine nor provoke changes of chemical composition resulting in adverse influence on wine quality. They are intended to achieve clarity and improve colour, flavor, and physical stability of wine, and they should serve to remove only the ingredients that make wine unstable.

Ribereau-Gayon et al.[2] recommended the following fining agents of red wines: bentonite, gelatin, albumin, and casein, together with their dosage and effects that could be expected. Elimination of some phenolic compounds of colloidal nature, involved in oxidation phenomena and excess astringency of wine, has contributed to the improvement of some organoleptic characteristics of wines.[3] Bentonite and gelatin are most frequently used substances of those mentioned above. Bentonite is mainly negatively-charged clay of volcanic origin with complex hydrated aluminium silicate components. In principle, it is used to remove proteins, thus providing better clarity and stability during long-term storage. However, it also attracts other positively charged compounds, such as anthocyanins, other phenolics, and nitrogen. It is not reactive towards small phenolic compounds. In fact, it binds large phenolic compounds, such as anthocyanins, and may also bind phenolic compounds complexed with proteins.[4] Gelatins, which are also positively charged, are used for removal of excess tannins from wine. They are most commonly used to reduce the level of astringency and bitterness in the press fraction of red wines, with reference to soften red wines. Although some enochemical characteristics of these agents are known, their effects on phenolic substances in wine are not fully studied.[5] These fining agents do not have the same effect on phenolic substances in wines of different ages. In order to clarify the effect of the use of bentonite and gelatin on anthocyanins and colour intensity in red wine, the research on wine fining of red Pinot Noir variety of different ages was carried out.

MATERIALS AND METHODS

Reagents and Standards

Deionized water, purified with a Milli-Q water system (Millipore, Bedford, MA). HPLC-grade methanol (Merck, Darmstadt, Germany), was used after filtration through 0.45-lm pore-size membranes. Standards of delphinidin-3-glucoside (Dp 3-G), cyanidin-3-glucoside (Cn 3-G), petunidin-3-glucoside (Pt 3-G), peonidin-3-glucoside (Pn 3-G), and malvidin-3-glucoside (Mv 3-G) were obtained from Extrasynthese (Lyon, Genay, France) and dissolved in methanol–water–formic acid, 40:55:5 (v/v). Other chemicals and solvents used were of the highest analytical grade.

Fining Treatment of Wine

The treatment design contained two factors: the age of the Pinot Noir wines including 6 (A), 18 (B), and 42 (C) month old wines, and the level of the fining agent. The following experiment has been set: T ₀ – control group wines, which received no fining agents; T ₁—bentonite (0.3 g/L); T ₂—bentonite (0.6 g/L) and T ₃—bentonite (1.0 g/L) treated wines; and T ₄—gelatin (0.05 g/L) and T ₅—gelatin (0.1 g/L) treated wines. Fining of wines was carried out with bentonite 'Clarol,' produced by 'Combine Aleksinac' from Aleksinac, Serbia, and gelatin 80 B1 produced by 'Italo-gelatin' from Italy. For each variant, 200 ml of wine were used. Variants T ₁, T ₂, and T ₃ have been treated with bentonite suspension, variants T ₄ and T ₅ with gelatin solution. After eight days, wines were separated from sediment and analysed.

pH, Anthocyanins, Colour Intensity, Tint, and Spectrum Form

pH was measured by means of a digital pH-meter (Hanna Instruments, Germany). The iodometric determination of free sulphur dioxide was done by titration in acid solution according to Recueil OIV.[6] Content of free, coloured (flavilium cation and quinodal base), and colourless anthocyanins (carbinol pseudo-base and bisulphite compound) and polymers was determined after the adidition of excess SO_2, at pH = 1, by measuring absorbance of the wine at 520 nm, in accordance with Somers and Evans method.[7] According to the official OIV method, colour intensity (I), and tint (T) were:

Colour intensity was determined by measuring the absorbance at 420, 520, and 620 nm in a 1 mm cell using a Unicam 5625 UV-Vis spectrophotometer (Unicam Ltd., Cambridge, UK) with a 1 mm glass cell.[8] Acording to Glories,[8] intensity of yellow (A ₄₂₀%), red (A ₅₂₀%), and blue (A ₆₂₀%) colours, as well as proportion of red colour produced by flavylium cations of the free and bound anthocyanins spectrum form (dA%), were:

Total Phenols

Total phenols (IFC) were determined by the spectroscopic method using Folin-Ciocalteu reagent and sodium carbonate.[6] Total phenolics index was determinated by measuring the absorbance at 280 nm (A ₂₈₀) of the wine sample.[9]

Extraction of Anthocyanins from Wine

Sep-Pak C18 cartridges (Waters, Milford, MA, USA) were prewashed with methanol diluted with water (1/1:v/v). Then, wine samples were passed through the cartridges, where anthocyanins (and other phenolic compounds) were absorbed. The column was washed with 4 ml of distilled water and dried under enhanced vacuum for several seconds. Finally, anthocyanins were recovered with methanol containing 0.01% (v/v) of HCl. The methanolic extract was concentrated under vacuum at 35°C by rotary evaporation and the pigments were dissolved in methanol–water–formic acid, 40:55:5 (v/v).

HPLC Analyses

The HPLC analyses were carried out using Hewlett Packard HPLC equipment, type HP 1100, equipped with Pye Unicam UV-Detector Model PU 4025.[10 11 12] Chromatographic separation of anthocyanins was carried out by means of a 250 × 4 mm i.d., 5 μm phase column using LiChrospher type 100 RP 18, protected by LiChroCART Guard Columns, Merck (Darmstadt, Germany), both thermostated at 40°C. All samples were analyzed in duplicate.

The mobile phase was a linear gradient of methanol containing 0.1% of HCl (solvent B) in 5 mM phosphate buffer, pH = 1.8 (solvent A), at a flow rate of 0.8 mL/min. The following gradient was used: 0 min, 25%B; 0–20 min, 25–30%B; 20–47 min, 30–65%B; 47–49 min, 65–35%B; 49–55 min, 35%B; 55–58 min 35–25%B; 58–60 min, 25%B. Ultraviolet–visible spectra ranging from 200 to 600 nm were recorded for all peaks. The anthocyanins were identified by comparison of retention times with those of authentic standards and by comparison of ultraviolet-visible spectra with those in the literature.[13 ^– 15] Quantification of the anthocyanins was based on peak areas at 520 nm. Calibration curves were plotted after chromatography of solutions containing different concentrations of the standards. Unknown concentrations were determined by means of linear regression equations.

Statistical Analysis

The data obtained were statistically analyzed by SAS (2001), 8.02 version, through PROC ANOVA procedure. Tukey's Honestly Significant Difference was adopted as a multiple comparison procedure. The results are presented as mean values ± S.D.

RESULTS AND DISCUSSION

Some relevant analytical parameters in control wines of the red Pinot Noir of different ages are given in Tables 1 and 2 (variant T ₀). The data obtained showed that the wines differed in many parameters of analysis, such as colour intensity and tint, total phenolic substances, the content of free, coloured and colourless anthocyanin forms, in terms of chemical factors, such as colour, total, and volatile acidity, alcohol content, reducing sugar content, etc. It is possible to differentiate wines made by different grape cultivars.[16]

Table 1 Some relevant analytical parameters in the control wines

		Variants
Parameter	Wine A (6 months old)	Wine B (18 months old)	Wine C (42 months old)
pH	3.22 ± 0.03	3.32 ± 0.02	3.43 ± 0.02
Free SO2 (mg/L)	14.3 ± 0.58	17.3 ± 0.61	8.0 ± 0.42
Total phenols (A280)	24.2 ± 0.87	22.7 ± 0.77	18.4 ± 0.67

Table 2 Influence of fining agents for fining of red wines on changes of certain colour parameters

			Treatments
Colour parameters	T0	T1	T2	T3	T4	T5
Wine A
Colour intensity	0.465 ± 0.0062^a *	0.413 ± 0.006 ^c	0.378 ± 0.0036 ^d	0.338 ± 0.0026 ^e	0.431 ±0.0265 ^b	0.425 ±0.0026 ^b
Nuance	0.628 ± 0.0057 ^b	0.666 ± 0.0056 ^a	0.684 ± 0.0050 ^a	0.678 ± 0.0139 ^a	0.618 ± 0.0047 ^c	0.606 ± 0.0042 ^d
Free antocyanins	166.2 ± 1.201 ^a	142.6 ± 0.346 ^d	133.1 ± 0.854 ^e	128.0 ± 0.265 ^f	154.6 ± 0.557 ^b	149.6 ± 0.458 ^c
Coloured antocyanins	27.77 ± 0.208 ^a	22.30 ± 0.1000 ^d	20.70 ± 0.173 ^e	18.60 ± 0.100 ^f	26.40 ± 0.173 ^b	25.60 ± 0.173 ^c
Colourless anthocyanins	138.43 ± 1.002 ^a	120.30 ± 0.361 ^d	112.40 ± 0.700 ^f	109.40 ± 0.361 ^e	128.20 ± 0.721 ^b	124.00 ± 0.600 ^c
Polymers	22.40 ± 0.173 ^a	19.10 ±0.100 ^d	16.80 ± 0.100 ^e	14.70 ± 0.100 ^f	21.10 ±0.100 ^b	20.17 ± 0.058 ^c
IFC	25.50 ±0.100 ^a	23.20 ± 0.100 ^d	22.90 ± 0.100 ^e	22.57 ± 0.058 ^f	24.00 ± 0.100 ^b	23.70 ± 0.100 ^c
Wine B
Colour intensity	0.347 ± 0.0046 ^a	0.319 ± 0.0026 ^b	0.282 ± 0.0026 ^c	0.272 ± 0.0040 ^e	0.318 ± 0.0036 ^b	0.306 ± 0.0030 ^d
Nuance	0.705 ± 0.0044 ^e	0.736 ± 0.0035 ^c	0.759 ± 0.0044 ^b	0.780 ± 0.0036 ^a	0.694 ± 0.0046 ^de	0.689 ± 0.0040 ^d
Free antocyanins	98.50 ± 0.173 ^a	85.70 ± 0.100 ^c	81.00 ±0.265 ^d	73.10 ±0.265 ^e	89.90 ±0.200 ^b	85.90 ± 0.173 ^c
Coloured antocyanins	15.80 ±0.100 ^a	13.40 ± 0.100 ^c	12.50 ± 0.100 ^d	11.40 ±0.100 ^e	14.33 ± 0.115 ^b	13.65 ± 0.050 ^c
Colourless anthocyanins	82.70 ± 0.100 ^a	72.30 ±0.100 ^c	68.50 ±0.173 ^d	61.70 ±0.200 ^e	75.57 ± 0.231 ^b	72.25 ± 0.218 ^c
Polymers	21.20 ±0.100 ^a	18.40 ±0.100 ^c	16.30 ±0.100 ^d	14.37±0.058 ^e	19.30 ±0.173 ^b	18.50 ±0.100 ^c
IFC	23.60 ±0.100 ^a	21.60 ± 0.100 ^b	21,27 ±0,058 ^e	21.00 ±0.100 ^c	21,07 ±0,058 ^ce	20.53 ±0,058 ^d
Wine C
Colour intensity	0.241 ± 0.0046 ^a	0.227 ± 0.0026 ^b	0.220 ± 0.0015 ^b	0.211 ± 0.0023 ^c	0.211 ± 0.00265 ^c	0.202 ± 0.0015 ^d
Nuance	0.902 ± 0.0044 ^d	0.920 ± 0.0040 ^c	0.932 ± 0.0045 ^b	0.948 ± 0.0053 ^a	0.885 ± 0.0010 ^e	0.875 ± 0.0026 ^e
Free antocyanins	40.20 ± 0.173 ^a	35.77 ± 0.153 ^d	33.77 ± 0.0577 ^e	32.60 ± 0.100 ^f	36.97 ± 0.115 ^b	36.53 ± 0.0577 ^c
Coloured antocyanins	8.97 ± 0.0577 ^a	7.73 ± 0.058 ^d	7.40 ± 0.100 ^e	7.03 ± 0.0577 ^f	8.43 ± 0.0577 ^b	8.07 ± 0.058 ^c
Colourless anthocyanins	31.23 ± 0.153 ^a	28.07 ± 0.0577 ^c	26.37 ± 0.058	25.57 ± 0.058	28.57 ± 0.058 ^b	28.43 ± 0.0577 ^b
Polymers	13.60 ± 0.100 ^a	12.37 ± 0.0577 ^b	12.03 ± 0.058 ^c	11.63 ± 0.0577 ^d	11.73 ± 0.0577 ^d	11.20 ±0.100 ^e
IFC	18.40 ± 0.100 ^a	17.17 ± 0.0578 ^b	17.03 ±0.0578	16.67 ±0.0577 ^c	17.20 ±0.100 ^b	16.87 ± 0.058 ^bc
*Mean values of 3 determinations ± standard deviation.
^a–fMeans within a row followed by different superscript letter are significantly different (P < 0.05).

Red wine phenolic compounds consisted of both grape polyphenols, including essentially anthocyanins, proanthocyanidins, and phenolic acids, and new phenolic compounds derived from them during winemaking and the ageing process. Phenolic compounds are important constituents of red wine, due to their contribution to sensory properties, such as colour, flavor, astringency, and bitterness.[17] Table 2 shows that treatments with bentonite led to a more significant decrease in colour intensity, which is particulary emphasized with a dose of 1.0 g/L (T ₃). The decrease of colour intensity was greatest in young wine (from 11.2 to 27.4%) than in 18 month old wine (from 8.1 to 21.6%), while the smallest decrease included 42 month old wine (from 5.8 to 12.5%). The participation of yellow colour increases in intensity, while red and blue colour decreases (Table 3).

Table 3 Intensity of yellow (A ₄₂₀%), red (A ₅₂₀%), blue (A ₆₂₀%) colours, and spectrum form (dA%) of examinated wine

			Treatments
Colour parameters	T 0	T 1	T 2	T 3	T 4	T 5
Wine A
A 420%	35.5 ± 1.33	36.8 ± 1.16	37.5 ± 1..31	38.0 ± 0.41	35.2 ± 0.36	34.9 ± 0.44
A 520%	56.5 ± 0.65	55.2 ± 1.06	54.7 ± 0.97	54.4 ± 0.63	57.0 ± 0.57	57.5 ± 0.44
A 620%	8.0 ± 0.25	8.0 ± 0.33	7.8 ± 0.37	7.6 ± 0.37	7.8 ± 0.39	7.6 ± 0.41
dA%	61.51 ± 0.98	59.45 ± 0.87	58.61 ± 0.79	58.04 ± 0.96	62.22 ± 1.04	63.10 ± 1.31
Wine B
A 420%	37.67 ± 0.54	38.62 ± 0.61	39.44 ± 0.73	40.14 ± 0.81	37.33 ± 0.94	37.22 ± 0.65
A 520%	53.39 ± 1.19	52.50 ± 0.28	51.94 ± 0.36	51.45 ± 1.51	53.80 ± 0.58	54.02 ± 0.74
A 620%	8.96 ± 0.33	8.88 ± 0.52	8.62 ± 0.61	8.46 ± 0.55	8.87 ± 0.69	8.76 ± 0.46
dA%	56.33 ± 1.01	54.76 ± 1.25	53.74 ± 1.44	52.77 ± 0.75	57.06 ± 0.87	57.44 ± 0.96
Wine C
A 420%	42.31 ± 0.34	42.86 ± 0.41	43.14 ± 0.53	43.59 ± 0.66	41.97 ± 0.94	41.75 ± 0.68
A 520%	46.90 ± 0.55	46.58 ± 0.76	46.29 ± 0.69	45.98 ± 0.77	47.43 ± 0.89	47.69 ± 1.15
A 620%	10.79 ± 0.34	10.56 ± 0.51	10.57 ± 0.44	10.43 ± 0.51	10.60 ± 0.62	10.56 ± 0.71
dA%	43.40 ± 0.51	42.66 ± 0.63	42.00 ± 0.46	41.26 ± 0.49	44.58 ± 0.78	45.16 ± 0.69

Tint increased (from 5 to over 10%) and values for determining the form of the spectrum decreased, pointing out to slight browning of wine, which is more emphasized with a higher dose of bentonite. These results indicated that bentonite decreased participation of red and blue colour and increased the share of the intensity of yellow colour in wines ageing for a longer period of time. Treatments with gelatin caused less significant influence on colour intensity, so in young wine this parameter decreased from 7.3 to 8.6%, while in 18 month old wine it decreased from 8.4 to 11.8%. Gelatin produced more significant influence on reduction of colour intensity of 42 month old wine (up to 16.1%). It has an affinity for phenols and reacts by forming hydrogen bonds between the phenolic hydroxyl and the peptide bonds of the protein component. Thus, gelatin produced a lower effect on colour in younger wines than in older products.[18] Large phenols, such as condensed tannins and polymerized anthocyanins, are preferentially removed (the latter generally have a greater percentage of larger polymeric phenolics).

Unlike bentonite, gelatin removes yellow colour more intensively, and thus increases the share of red colour in intensity, which affected the reduction of colour tint from 2 to 5% in variant with a higher dose of gelatin, while the values for the form of the spectrum increased. Results of the statistical analysis showed that there was a significant difference (P < 0.05) between almost all applied treatments of wine, regarding examined parameters. Colour intensity, coloured and colourless anthocyanins, polymers, and IFC are highest in the control group in all examined wines (6, 18, and 42 months old), (Table 2). Also, in all cases they showed a significant difference (P < 0.05) within particular treatment between examined wine of different ages.

Figure 1 shows the impact of the fining agent and applied dose on changes of phenol composition of young red Pinot Noir wine. Bentonite caused significant reduction of colour intensity, ionized anthocyanins, and polymers, and a low reduction of colouress anthocyanins and total phenol substances, such as tannin substances. This is especially emphasized with a dose of 1.0 g/hl. Reduction of total anthocyanins was 23%, ionized up to 33.1%, and polymers up to 34.4%. Bentonite influenced tannin substances (tannin substances index up to 9%) to a lower extent, probably as a result of negative charge of bentonite and tannin colloid particles. Treatments with gelatin produced a lower influence on these parameters than bentonite (ionized anthocyanins 7.9%, and polymers 9.8%) with the exception of the index of tannin substances (up to 13%).

Figure 1 Impact of fining agents on the changes phenolic composition in Pinot Noir (6 months old) (color figure available online).

Figures 2 and 3 show the influence of the same fining agent on changes of phenolic composition of 18 and 42 month old wine. The older the wine is, the less the influence of bentonite on coloured substances content, such as on colour intensity. In 42 month old wine, reduction of coloured anthocyanins ranges from 14.5% to 22.2% and polymers from 8.8% to 14.7%. Gelatin reduces significantly the content of polymers in old wine from 14% to 17.7%, while reduction of coloured anthocyanins is between 6.7% and 10%. The influence of bentonite decreased with the oldness of the wine. This could be explained by the fact that young wines contain anthocyanins in colloid form, which is unstable.

Figure 3 Impact of fining agents on the changes phenolic composition in Pinot Noir (42 months old) (color figure available online).

Figure 2 Impact of fining agents on the changes phenolic composition in Pinot Noir (18 months old) (color figure available online).

According to Ribereau-Gayon et al.,[2] bentonite has proven to be a significant agent for removal of colloid coloured substances, which consist of flavilium cations (ionized anthocyanins), tannins, polysaccharides, and proteins. Adding bentonite to eliminate unstable complexes and ensure stabilization of wine to prevent its loss of colour is important and enables wines to remain stable at low temperatures and not change during several months.

Proportional representation of certain anthocyanins in wine is given in Table 4. Treatment of wine with bentonite and gelatin influenced reduction of the share of Dp 3-G, Cn 3-G, and Pt 3-G and increase of the share of Pn 3-G and Mv 3-G, at the same time. The 18 month old wine did not have Cn 3-G, while the trend of decrease and increase of certain anthocyanins remained the same. In 42 month old wine, only Pn 3-G and Mv 3-G are present. The share of Mv 3-G increased by using fining agents.

Table 4 Influence of red wine fining on proportional representation of certain anthocyanins

		Anthocyanins (%)
Variants	Treatments	Dp 3-G	Cn 3-G	Pt 3-G	Pn 3-G	Mv 3-G
Wine A	T 0	7.1	2.3	9.1	20.0	61.5
	T 1	65	1.7	8.4	20.3	63.1
	T 2	5.8	1.2	7.9	20.9	64.2
	T 3	5.1	0.8	7.2	21.4	65.5
	T 4	6.9	2.2	8.9	20.1	61.9
	T 5	6.8	2.0	8.6	20.4	62.2
Wine B	T 0	3.6	0	5.2	19.2	72.0
	T 1	3.4	0	4.8	19.3	72.7
	T 2	3.0	0	4.5	19.5	73.0
	T 3	2.6	0	4.0	19.8	73.6
	T 4	3.2	0	4.9	19.4	72.5
	T 5	3.0	0	4.6	19.5	72.9
Wine C	T 0	0	0	0	14.0	86.0
	T 1	0	0	0	13.7	86.3
	T 2	0	0	0	13.2	86.8
	T 3	0	0	0	12.7	87.3
	T 4	0	0	0	13.6	86.4
	T 5	0	0	0	13.0	87.0

CONCLUSION

The use of the agents for red wine fining caused reduction of the content of phenol substances, but the quantitative effect varied depending on the types of agents, the dose applied, and the age of the wine. Major changes in phenol structure of the young wine resulted from the treatment of wine with bentonite. Ionized anthocyanins, polymers, and colour intensity were reduced most with colourless anthocyanins and total phenolic substances reduced to a lower degree. The share of yellow colour increased, while the share of red and blue colour decreased in intensity with simulataneous increase in the tint of colour and reduction of the value of the spectrum form. The influence of bentonite on wine colour reduced with the ageing of the wine. Minor changes in phenolic compounds of the young wine, except in the case of index A ₂₈₀, were the result of wine treatment with gelatin. Unlike bentonite, gelatin reduced the share of yellow colour in intensity and increased the share of red colour, thus decreasing the tint and increasing the value of the spectrum form. With the ageing of wine, the impact of gelatin on phenolic composition of wine increases, particulary as far as reduction of colour intensity, as well as the content of polymers, were concerned. Results of statistical analysis showed that regarding examined parameters there was a significant difference between almost all applied treatments of wine. Application of bentonite and gelatin reduced the participation of Dp 3-G, Cn 3-G, and Pn 3-G in the total anthocyanins and increased the share of Pn 3-G and Mv 3-G. In 42 month old wine, the share of Mv 3-G increased, while the share of Pn 3-G decreased.