Influence of addition of insoluble solids, different ... - Wiley Online Library

Research article Received: 8 December 2012

Institute of Brewing & Distilling

Revised: 3 May 2013

Accepted: 8 June 2013

Published online in Wiley Online Library: 24 August 2013

(wileyonlinelibrary.com) DOI 10.1002/jib.79

Influence of addition of insoluble solids, different yeast strains and pectinesterase enzyme on the quality of apple wine Vinod Kumar Joshi,1* Dhanwant Kaur Sandhu2 and Vikas Kumar1 Rate of fermentation and physico-chemical characteristics of apple wines, owing to the addition of different yeast strains, insoluble solids and pectinesterase enzyme, were examined. The highest rates of fermentation and ethanol production were found in the wine fermented by yeast strain UCD 505, while strain UCD 595 gave the smallest amount of methanol. The addition of insoluble solids to the apple juice significantly increased the levels of methanol, vitamin C, amyl alcohol, total volatiles, rate of fermentation, tannins, colour units, Mn and Zn. Addition of insoluble solids decreased pH, titrable acidity, ethanol, total sugars, total esters, and K, Mg, Ca, Cu and Fe content of the wine. Addition of pectinesterase enzyme significantly increased all the parameters examined except for pH, vitamin C, total esters, Mn and Mg content. Application of cluster analysis to the results of rate of fermentation, reducing sugars, volatile acidity and ethanol showed that the influence of the yeast strains was more than the influence of insoluble solids or pectinesterase enzyme addition. Consideration of more parameters showed that there was a clear interaction between the yeast strain, insoluble solids and the pectinesterase enzyme. Addition of insoluble solids to the must led to the production of some undesirable quality characteristics. In contrast, specific yeast strains and enzyme addition improved various physico-chemical characteristics of the wine. Pre-settled or clarified juice was preferred to produce a quality apple wine. Copyright © 2013 The Institute of Brewing & Distilling Keywords: apple; insoluble solids; pectinesterase; wine; Saccharomyces cerevisiae; cluster analysis

Introduction

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* Correspondence to: Vinod Kumar Joshi, Department of Food Science and Technology, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan (HP)-173230, India. E-mail: [email protected] 1

Department of Food Science and Technology, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan (HP)-173230, India

2

Department of Microbiology, Guru Nanak Dev University, Amritsar (PB), India

Copyright © 2013 The Institute of Brewing & Distilling

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The production of wine is an ancient practice. Wine has been prepared and consumed by man since antiquity (1). Apple wine and apple cider are alcoholic beverages prepared and consumed worldwide. Fresh or concentrated apple juice is used to make apple wine, which has a long tradition in Europe and has acquired an important place in the global wine industry (2). To produce red wine from grapes, fermentation is carried out with the skin, while for white wine it is the juice without the skin that is fermented. Unlike grape wine, apple wine and cider are produced from juice. To prepare these from concentrate, it is diluted to an appropriate level (3–6). The fermentation behaviour of apple wine is influenced by a number of factors. Addition of pectinesterase enzyme has led to a considerable improvement in the quality of apple wine (7). Wang et al. (8) have studied the fermentation kinetics of different sugars by a Saccharomyces cerevisiae apple wine yeast. Although glucose and fructose are both utilized by this yeast, glucose was used more rapidly than fructose (9). In the production of grape wines, several practices are employed (10). Some of the practices that influence the quality of product include rapid separation of pomace and clarification or fermentation with pomace similar to white and red wine production. Quality-wise, the white wine from turbid juice was rated much lower than that made from clarified juice. The former wine was also rated harsher and more bitter than the latter (11). Pomace contact increased the content of total phenols and consequently increased astringency. An improvement in the quality of white wine, by removal of the sediment from the musts prior to

fermentation, has been reported (11,12). However, clarification of the free run musts and separation of the deposit by decantation, centrifugation or filtration can result in the production of large amounts of acetic acid by the yeast, thus adversely affecting the quality of the wine (13,14). On the other hand, the addition of pressed or macerated must to the clarified and racked free run must was reported to decrease acetic acid production by the yeast, if this occurred before the onset of alcoholic fermentation. However, if such addition was made after 4–5 days, then it did not affect such characteristics. It has also been demonstrated in the case of grape must that the addition of bentonite or other solids to the clarified must increased the fermentation rate and assured a complete fermentation, similar to what occurred in the turbid must when treated with 0.5% naturally suspended solids (15). The addition of clarification deposit had the strongest and most significant effect both in the clarified grape must and in a synthetic nutrient medium. It decreased the pyruvic acid, acetic acid formation and the latent phase, and increased the fermentation rate. Production of esters in must with insoluble solids ISS is also controversial. Gelatin and silica addition caused

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a strong release of these acids. Such effects, if any in apple wine preparation, are totally lacking in literature reports. To produce white wine from grapes a free run juice is fermented; in apple such a practice is not possible, as the grated fruits must be pressed to obtain the juice. However, the quality of solids present in the turbid juice must could affect the quality of the apple wine. At the same time, how the different wine yeast strains (Saccharomyces cerevisiae var. ellipsoideus) influence the quality of wine, in the presence of different levels of insoluble solids as such, or with enzyme, is interesting from both a scientific and a practical point of view. Lack of information on these aspects led to the investigations reported in this paper.

Materials and methods Materials The apple juice used in this study was manufactured by Fruit Canning unit of the Department of Horticulture, Navbahar Shimla-2 (HP) India. The pectinesterase used was manufactured by M/S Triton Chemical, Mysore (India) and was used at a concentration of 0.5%. The strains of yeast cultures of Saccharomyces cerevisiae var. ellipsoideus used were procured from the Department of Enology and Viticulture, Davis, California, USA and coded UCD 595, UCD 522 and UCD 505. The ‘W’ strain was a local isolate of Saccharomyces cerevisiae var. ellipsoideus tested for wine making. Insoluble solids used were the sediment in the form of slurry from apple juice deposited in the bottles after processing and storage.

Experimental To study the effect of insoluble solids on the fermentation behaviour, physico-chemical characteristics of apple wine, different combinations of yeast strains and insoluble solids were tested. The level of insoluble solids (ISS), viz. 2.5, 5.0 and 10.0% along with the control (no addition of ISS), and four yeast strains (strain W, UCD 505, UCD 522 and UCD 595), with and without enzyme, were employed and the fermentations were conducted with the respective yeast strains as described earlier. Sediment in bottles of processed apple juice was used as a natural source of insoluble solids. All treatments were replicated twice. The final total soluble solids (TSS) of the musts were adjusted to 24°B with canesugar syrup. Other details of fermentations were the same as outlined earlier (7). After the completion of fermentation, the wines were racked, filtered and filled into 200 mL bottles, keeping 2.5 cm head-space, followed by crown corking and mild pasteurization. After maturation for a year, the wines were again filtered and analysed.

Analysis During the fermentation, the fall in TSS (°B), and the pH, titrable acidity and ethanol were monitored at the appropriate time intervals. The wines were analysed for different physico-chemical characteristics, viz. total sugar, ethanol, total soluble solids, pH, titrable acidity, volatile acidity, tannins/total phenols, total esters, methyl alcohol and amyl alcohol as per the standard methods (16–18). Total soluble solids (°B) were measured using a hand refractometer and pH by digital pH meter; ethanol was measured colourimetrically by the potassium dichromate method, while colour was measured using a Lovibond tintometer and expressed as units of red and yellow colour (16). Total phenols were measured colourimetrically using tannic acid as a standard. All the analyses were carried out in triplicate. The methyl and amyl alcohol content in the products was measured by GLC (Shimadzu, gas chromatograph GC-9A). For methanol analysis, a nitrogen flow of 30 mL/min as a carrier, and column and injection temperature of 130 and 220°C, respectively, in a ‘Porapack’ column, were used. Amyl alcohol was measured using a SE-30 column, with an injection temperature of 40 and 110°C and a nitrogen flow of 18 mL/min. A flame ionization detector was used for both the estimations. Standards were analysed similarly and used to calculate the methyl alcohol and amyl alcohol content in the samples. All other operational conditions were the same as recommended by the manufacturer. Both methyl and amyl alcohol were expressed as μL/L. Analyses of data The data obtained from the physico-chemical analyses of the apple wine were subjected to analysis of variance using a completely randomized design (CRD) and the means with critical differences are reported. The data were also analysed by cluster analysis. The statistical computer program was run on a personal computer in the Computer Centre of Dr YSPUHF, Nauni, Solan using a CLUST.BAS computer package (19). The output was obtained in the form of cluster number, distance, etc. These results were plotted as a dendrogram and the interpretation of data was made, accordingly.

Results and discussion Effect of insoluble solids The increase in the ISS enhanced the rate of fermentation (RF) value significantly, but only in the treatment where more than 5% ISS was added (Table 1). However, in an earlier study, within

Table 1. Changes in physico-chemical characteristics apple wine as affected by insoluble solids addition Insoluble solids (%)

Rate of fermentation (oB)/24 h

Ethanol (% v/v)

Titrable acidity (%)

pH

0.98 1.00 1.03 1.09 0.02

11.63 11.33 10.43 10.84 0.250

0.38 0.38 0.37 0.35 0.017

4.02 4.01 3.59 4.02 0.036

0 2.5 5.0 10.0 Critical difference, p ≥ 0.05

Total Reducing Red colour Yellow soluble sugar (%) (units) colour solids (oB) (units) 6.98 6.81 7.42 7.11 0.279

0.38 0.33 0.32 0.37 0.03

1.30 1.35 1.38 1.57 0.11

3.82 4.74 3.86 4.95 0.26

Vitamin C (mg/L)

Volatile acidity (% acetic acid)

9.02 9.74 10.95 9.54 0.59

0.031 0.036 0.038 0.029 0.003

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NS, Not significant.

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Effect of insoluble solids, yeast strains and pectinesterase on apple wine a given level of insoluble solids, the fermentation rates were found to be identical (15). The increase in the levels of ISS in the must decreased the ethanol content in the wine, but nonsignificantly. Addition of insoluble solids might have increased the turbidity of the must, thus decreasing the fermentability with a reduction in ethanol content as reported earlier in grape wines (20). Significant differences in the methanol content of wines fermented with variable ISS levels were evident and the relationship was found to be concentration dependent (Table 1). The titrable acidity, TSS and residual sugar levels were also affected significantly by the addition of insoluble solids in the apple must but only up to 5% ISS (Table 1). The reduction in titrable acidity could probably be attributed to adsorption of some acids by the insoluble solids (21). The increase or decrease in titrable acidity were too small to influence the taste of the wines to any significant extent, as was the trend with the pH values of the wines fermented with different levels of ISS. Increasing the quantity of ISS increased the red colour units of the wines but non-significantly. Addition of the insoluble levels significantly affected the vitamin C content of the wines and increasing levels up to 5% were recorded. Addition of ISS to the apple must significantly increased the methyl alcohol, amyl alcohol and the total volatiles of the wine (Table 1), but the reverse was the trend for the total ester content compared with the control. Fermentation with insoluble solids significantly altered the volatile acidity of the apple wine, again without any distinct pattern, which was probably related more to fermentation conditions than to the amount of insoluble solids. Generally, with an increase in the ISS level, the tannin content also increased, which was desirable. The potassium content of the wines was significantly influenced by the addition of ISS (Table 1). In general, with an increase in the insoluble solids, K decreased and the Na content increased. Sodium did not exhibit a clear trend with respect to the ISS level. Unlike K and Na, the Mg content of the wines was not significantly influenced by level of the ISS. The Ca content of wines was significantly decreased by the addition of ISS (Table 1). Addition of insoluble solids reduced the Cu content. With the addition of ISS, Zn content increased significantly. The addition of insoluble solids to the must generally decreased Fe content. Effect of yeast strain The type of yeast significantly influenced the RF (Table 2) and it can be seen from the data that the highest RF was seen with UCD 505 and UCD 595, and there was more dependence on the type of yeast used to conduct the fermentation than


on the insoluble solids. Similar results have been previously reported where the yeast strain, agitation and non-soluble solids levels were found to be the key factors affecting the rate of grape juice fermentation (15). The ethanol content of the apple wines was significantly affected by the yeast strain. Differences in the ethanol content owing to the yeast strain were observed. The results corroborated those for the RF described earlier. There were also significant differences in the wines fermented by different yeast strains for methanol content. Both the yeast types (strain) and the addition of enzyme invariably significantly reduced the total soluble solids. Different yeasts strains significantly affected the titrable acidity, and hence the pH of the finished wine (Table 2). The colour of the wines was influenced to a greater extent by the use of different yeast strains and fermentation with yeast strains UCD 505 and W gave significantly higher red and yellow colour units, imparting an attractive appearance to the wine. Addition of different yeast strains to the apple must enhanced significantly both the amyl alcohol content and the total volatiles of the wine. The effect of yeast type reflected a more pronounced effect than the insoluble solids. Significant differences were obtained in the total ester content of the wines from the must fermented by different yeasts (Table 2). Generally, the yeast types (Table 2) influenced the tannin content as well as the total ester content of the wines. Wines fermented by different yeasts strains had a different K content, probably owing to differences in the uptake of K and finally its release from the autolysed yeast cell into the wine. The use of different yeast strains significantly influenced the Na content of the wine. Unlike K and Na, the Mg content of the wines was not significantly influenced by the yeast strains (Table 2). Compared with the grape wine, the Mg content was quite low (16). Wines fermented by different yeasts differed significantly for Ca, Fe and Cu content. Mn and Zn levels of the wines remained intact (Table 2). Pectinesterase enzyme addition The rate of fermentation and ethanol content of the apple wines were significantly enhanced by the addition of a pectinolytic enzyme (Table 3), but in absolute values the differences were too marginal to have any drastic impact on the quality of wine. With the addition of a pectinolytic enzyme, an increase in ethanol content took place, probably owing to release of fermentable sugar from the pulpy material of the insoluble solids as result of enzyme action. It significantly increased the methanol content in the wines and reduced the total soluble solids and reducing sugar. It affected neither the titrable acidity nor the pH of the finished

Table 1. (Continued) Methanol (μL\L)

Amyl alcohol (μL\L)

Total volatiles (μL\L)

Total esters (mg/L)

Tannins (mg/L)

K (mg/L)

Na (mg/L)

Mg (mg/L)

Ca Cu Fe Zn Mn (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)

60 97 84 94 1.26

347 412 393 440 7.88

610 759 750 866 13.65

94 88 71 69 3.08

143 151 136 153 3.2

1513 1478 1433 1400 14.4

14 15 18 14 0.13

15 18 16 14 5.90

98 92 92 85 9.94

0.12 0.12 0.10 0.12 NS

1.9 1.8 1.7 1.9 0.05

0.32 0.35 0.39 0.39 0.02

0.33 0.33 0.33 0.34 NS

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Table 2. Changes in physico-chemical characteristics of apple wine as affected by different yeast strains Yeast type

Isolate W UCD 505 UCD 522 UCD 595 Critical difference p ≥ 0.05)

Rate of fermentation (oB)/24 h

Ethanol (% v/v)

Titrable acidity (MA) (%)

pH

1.03 1.04 1.00 1.04 0.02

10.2 11.9 11.6 10.04 0.25

0.37 0.39 0.37 0.37 0.005

3.97 4.00 4.05 4.01 0.036

Total Reducing soluble sugar (%) solids (oB) 7.08 6.82 7.18 7.17 0.028

Red colour (units)

0.38 0.34 0.32 0.36 0.03

1.53 1.58 1.20 1.12 0.11

Yellow Vitamin Volatile colour C acidity (units) (mg/L) (% acetic acid) 4.68 4.28 3.97 3.45 0.26

9.52 10.20 9.85 9.68 0.59

0.031 0.036 0.032 0.034 0.003

MA, Malic acid.

Table 3. Changes in physico-chemical characteristics of apple wine as affected by the enzyme addition Parameters o

Rate of fermentation ( B)/24 h Ethanol (%v\v) Methanol (μL\L) pH Total soluble solids (oB) Titrable acidity (% malic acid)

With enzyme

Without enzyme

Critical difference, p ≥ 0.05

1.20 11.15 81.1 4.02 6.78 0.38

0.87 10.91 77.9 4.00 7.40 0.37

0.02 0.17 1.1 NS 0.19 NS

1.56 4.83 9.87 422 0.035 134 78.6

1.19 3.86 9.76 373 0.032 160 81.0

0.08 0.19 NS 5.00 0.002 2.3 NS

Colour (tintometer colour units) Red Yellow Vitamin C (mg\L) Amyl alcohol (μL\L) Volatile acidity (% acetic acid) Tannins (mg/L) Total esters (mg/L) Minerals (mg/L) K Na Mg Ca Cu Fe Zn Mn

1518 18.3 16.9 83.7 0.11 2.17 0.51 0.40

1399 12.1 15.1 100.3 0.12 1.50 0.22 0.27

10.2 0.10 NS 3.1 NS 0.04 0.02 0.02

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wines (Table 3). As expected, the colour of the wines was significantly improved by the addition of enzyme, along with better retention of vitamin C. Addition of enzyme to the apple must significantly increased the quantity of amyl alcohol in the wine but did not influence the ester content. Unlike K and Na, the Mg content of the wine was significantly influenced by the addition of enzyme (Table 3) and significantly reduced the Ca levels. The addition of enzyme increased the Mn content of the wines, which might be due to the liberation of Mn owing to the activity of pectinesterase enzyme on the sediment (Table 3).

and alcohol content, cluster analysis distinctly grouped the different yeasts, irrespective of insoluble solids content, showing that the influence of yeast type was more than the influence of ISS on these parameters. It can be seen that yeast W, UCD 522 and UCD 595 formed a separate cluster from UCD 505, showing better closeness in fermentation behaviour. Consideration of other characteristics such as total esters, amyl alcohol, tannins, methanol and total volatiles (Fig. 2) however, showed a clear interaction of the insoluble solids levels with yeasts types and the enzyme addition, as different insoluble solids were grouped separately.

Cluster analysis

Discussion

Cluster analysis output of the data on the effect of insoluble solids in the apple wine, using flexible strategy with Euclidean distance as an index was plotted as a dendrogram (Fig. 1). Based upon the rate of fermentation, reducing sugar, volatile acidity

One of the three factors was the addition of insoluble solids prior to the fermentation. The clear advantage of addition of insoluble solids to the must is the enhanced rate of fermentation, an aspect documented earlier in the grape fermentation (22). Considering



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Effect of insoluble solids, yeast strains and pectinesterase on apple wine


Table 2. (Continued) Methanol (μL\L)

Amyl alcohol (μL\L)

Total volatiles (μL\L)

Total esters (mg/L)

Tannins (mg/L)

95 84 86 77 1.62

427 325 393 448 7.88

769 705 605 907 13.65

55 91 99 69 3.08

165 162 154 137 3.2

K Na (mg/L) (mg/L) 14.57 1462 1369 1425 14.44

13.7 15.2 15.0 16.8 0.137

Mg (mg/L) 14.6 14.8 17.3 17.3 NS

Ca Cu Fe Zn Mn (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 89.8 88.0 99.8 99.3 4.44

0.14 0.11 0.12 0.08 NS

1.68 1.92 2.03 1.71 0.05

0.33 0.37 0.42 0.34 0.02

0.33 0.33 0.34 0.40 NS

Figure 1. Dendrogram of physico-chemical analysis data of apple wine as affected by addition of insoluble solids and yeast types, based on rate of fermentation, reducing sugar, volatile acidity and ethanol (ISS = Insoluble solids, W = Isolate W, 595 = UCD 595, 505 = UCD 505, 522 = UCD 522).

J. Inst. Brew. 2013; 119: 191–197

sediment in apple juice (24), its addition as insoluble solids enhances the methanol content. The presence of methanol in wine is therefore not unexpected and is attributed to the activity of pectinolytic enzymes added or present in the fruit juice used in wine preparation. Consumption of the alcoholic beverages with higher concentration of methyl alcohol has been associated with acute toxicity, although not supported by toxicological considerations at the level of methanol found in this study (25). Thus, the danger of methanol toxicity from wine consumption would appear to be remote. At the same time, the methanol levels should be as low as possible. The lowering of the titrable acidity of the finished wines by the addition of the insoluble solids could probably be attributed to adsorption of some of the acid by the insoluble solids (21). Changes in titrable acidity were too small to influence the taste



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both the absolute values and the legal standards, the volatile acidities were in the range normally found in wines, so this was not a factor of concern. Since ethanol is an important parameter for wine-making, its reduction, owing to addition of ISS, is not desirable. Differences in the residual sugars, total esters and fusel oil contents were noted in relation to the ISS (15). The changes which took place in apple must as a result of the addition of ISS were predictable when a grape fermentation was taken as a model. The RF value is one of the attributes of yeast suitable for wine production. A slight increase in the methanol content of the grape wines by the addition of pectolytic enzyme was documented earlier (23). Methanol is not generated by the action of yeast per se during production of wine, and most of it is formed from hydrolysis of pectin by pectinolytic enzymes present in the crushed grapes. Since the pectin is the major fraction of

V. K. Joshi et al.


Figure 2. Dendrogram of physico-chemical analysis data of apple wine as effected by addition of insoluble solids and yeast types, based on ester, amyl alcohol, tannin, methanol and total volatiles (ISS = Insoluble solids, W = Isolate W, 595 = UCD 595, 505 = UCD 505, 522 = UCD 522).

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of the wines to any significant extent. The addition of pectinolytic enzyme increased the acidity of wines, but only to a small extent, probably owing to the hydrolysis of pectic substances into methyl alcohol and galactouronic acid (26). Irrespective of addition of enzyme or insoluble solids, in terms of absolute values the sugar content was comparable in wines fermented with different yeasts. The changes in the colour of the wine by the addition of insoluble solids were undesirable. The sediment of apple juice (source of ISS) is known to contain tannins (24) that upon oxidation by polyphenol oxidase impart a brown colour, thus increasing colour units. It has been reported that higher insoluble solid levels during grape juice fermentation increase the levels of both the isobutyl and isoamyl alcohol (15,21). Fusel oil formation in wine fermentation is a consequence of amino acid utilization by the yeast as a source of nitrogen. Our findings are in conformity with those obtained earlier (27) in that the wines produced from extremely turbid grape must invariably have fusel oils far in excess of wines from clarified juice of the same fruit. It was postulated that addition of insoluble solids might have provided aerobic conditions of fermentation owing to the availability of more active interfaces for oxygen absorption, thus creating favourable conditions for the formation of higher alcohols and vice-versa (27). It is also possible that ISS might have enhanced the enzymatic activity (28) or ISS might have added precursor compound(s) of higher alcohols, that


is, amino acids and other carbon sources (29), thus increasing the formation of higher alcohols. Vinification practices, such as must clarification prior to alcoholic fermentation are also known to affect the formation of volatile compounds (15,20,23,27). Our results on volatile production are consistent with those reported earlier for grapes, as volatile esters generally increase with increased concentration of ISS or from the must with sediment, than from must without it (15,30). Even inert solids have been found to enhance the amount of fusel oils, especially under vigorous aerobic conditions (27). No doubt, volatile fractions are important in the flavour development of wines (16); abnormally high concentrations would not be desirable. Use of enzyme might have released K, thus increasing its content. The K content of apple wine was comparable with that of grape wine (22). In terms of absolute values, the Na content was quite low, which is desirable (16). The sediment of the apple juice, in addition to other constituents, is known to contain tannins and pectin, and its addition in the form of ISS might have increased the tannin content (24). Addition of enzyme is known to reduce the quantity of tannins owing to the precipitation, hydrolysis and degradation of phenolic compounds by the enzymatic reactions (31). Since an optimum quantity of tannins is important in imparting astringency to wines, an increase in its concentration in wine might make the wine excessively


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Effect of insoluble solids, yeast strains and pectinesterase on apple wine astringent. Therefore, an increase in the levels owing to ISS addition is undesirable, while reduction owing to enzymatic degradation is a satisfactory development. The reported results are similar to the earlier findings that addition of insoluble solids reduced the ester content in grape wine (15,21). Esters contribute to the aroma of the wine (16) and therefore their reduction by addition of ISS is undesirable. The Mg content was quite low and may be the inherited characteristic of the fruit (16). Utilization of Ca by yeast for its metabolic activities during fermentation may have decreased Ca quantities in the wines. The reduction in Ca with the increase in ISS could be the consequence of its precipitation as calcium pectate, normally found in the sediment. Since Ca plays an important role in nutrition (32), its reduction by the addition of ISS content is undesirable. The values of Zn found in this study are far below the level required for any toxicity or to impart any metallic taste to the beverages (16). The element Mn is not involved in the stabilization of wines and has no toxic effect (16). The Fe content in a beverage is important as this element is essential in human nutrition (32), in addition to its role in the stabilization of wines (16), and the values obtained were below the 5 mg/L required for appearance of iron casse. Similarly, the copper content of the wines was