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Production Wine Different Strains Yeast Comparison Sensory Attributes Biology Essay

At present there is a demand for highly refined wine with special and fresh quality attributes. The use of yeasts with improved and desired properties is becoming a very important part of Enology. The influence of yeast on the phenolic content and colour of wine is considered as an important criterian for yeast selection. The experiment was conducted to determine the effects of different types of yeast on grape must from a single source. All the parameters were kept the same by varying the type of yeast used for fermentation. The level of total polyphenols was measured using Folin-Ciocalteau method and the total amount of antioxidant present was analyzed using TEAC assay. Even though the result showed slight variation in the Phenol concentration, statistical analysis on the samples showed that there existed no significant difference between the samples (P>0.05). The same was observed in the case of antioxidant capacity of the samples. the samples were also analyzed for the phenol concentration and antioxidant capacity at the 4 stages of wine making. the highest concentration was observed after fermenting the grape must. It was see that the phenolic concentration and the antioxidant capacity showed a very strong correlation. The analysis for colour and flavour variation showed no significant difference between the samples.

Introduction

Mankind has been drinking wine for thousands of years. Wine is made from grapes. But it is not a grape juice. The presence of alcohol in wine makes it very different from grape juice. Wine contains spirit alcohol, which is formed as a result of fermentation reaction. Fermentation is the process of interaction of yeast (mostly Saccharomyces Cerevisiae – True Wine Yeast), which is a single celled living organism and with the sugar in the grape juice. Alcohol and carbon dioxide are the by-products of this reaction. Fermentation is a vitally important stage in winemaking. The yeast not only converts sugar to alcohol but also produce esters and other compounds, which contribute to the wine’s fruit aromas. Extraction of flavor and color from the grape skins (for red wines) also occurs during fermentation.

The production of wine occurs best in the absence of oxygen. This reaction continues until the alcohol content becomes so high that the yeast dies off due to the alcohol it created. The yeast sinks to the bottom and is removed. What began as grape juice and yeast has now become wine. This wine must be filtered and stored before it can be consumed.

Apart from the principal wine yeast, saccharomyces cerevisiae, the immediate fermentation of the wine must to alcohol is a complex process carried out by the step by step activity of different species of yeast (Heard and fleet, 1988), found on grapes and in the must and these help contribute flavour to the wine (Lambrechts and Pretorius, 2000). For the first one to three days of a alcoholic fermentation, the wild yeast (non-Saccharomyces yeast – low fermentative power) predominates. Their growth is significant and can influence the chemical composition of wine. As alcohol levels reaches 3-4%, the wild yeast give way to the increasing numbers of alcohol tolerant Saccharomyces so that by two to five days this yeast predominates (Margalith, 1981; Kunkee, 1984). The wild yeasts are capable of growth under aerobic and anaerobic conditions and may persist during the fermentation. They compete with Saccharomyces for nutrients, and may also produce secondary metabolites as a result of their growth, which affects the characteristic smell of wine. At the end of a natural fermentation usually only Saccharomyces cerevisiae is present. Whereas in inoculated fermentations, Saccharomyces cerevisiae predominates from beginning to end because of the high level of inoculum.

It has also been revealed from studies n S. cerevisia that in addition to the production of alcohol they generate many secondary metabolites that play major role in determining the wine quality (Fleet, 1990; Lema et al., 1996; Lambrechts and Pretorius, 2000; Fleet and Heard, 1993). The uses of modern instruments have shown that flavour of wine is as a result of large number of compounds. More than 1000 volatile compounds have been identified in wine and of that yeasts produce 400 during fermentation (Nykanen, 1986). Thus the nature and concentration of the volatiles in wine depends on the yeast species that participate in fermentation.

Grapes contain large amount of different phenolic compounds in skin, pulp and seeds and these phenolic compounds are responsible for some of the major organoleptic properties of wines, specially colour and astringency. Wine phenolic composition is mainly dependent on the kind of grapes used and the fermentation conditions (Cheynier, Hidalgo Arellano, Souquet, & Moutounet, 1997). Flavanoids are one of the major classes of polyphenols. Flavanoids include flavanol (quercetin, myricetin), flavan-3-ols (catechin and epicatechin), as well as polymers of flavan-3-ols, which are called procyanidins, and anthocyanins. These are the pigments responsible for the colour of red wine (Soleas & Goldberg, 1999). The initial colour of wine is due mainly to anthocyanins extracted from the grape skin during crushing, pressing, and fermentation. Various experimental evidences prove that condensed pigments are formed in the gradual manner between free anthocyanins and colourless phenols present in grapes – particularly monomeric and polymeric flavanols (catechin and procyanidins), flavanols and phenolic acids – during storage and aging of red wines. These compounds may result in the presence or absence of oxygen, so polymerization reaction occurring during wine storage in bottles are essentially anaerobic and more strongly influenced by temperature than by the dissolved oxygen concentration (Gomez Plaza, Gil Munoz, Lopez Roca, Martinez Cutllas, & Fernandez Fernandez, 2002). Numerious mechanisms have been put forward to explain the formation of these pigments that involve copigmentation (Baranac, Petranovic, & Dimitric-Markovic, 1996; Baranac, Petranovic, & Dimitric-Markovic, 1997; Darias Martin et al., 2002), direct condensation between anthocyanins and flavanols, and acetaldehyde-mediated reactions between them (Dallas, Ricardo da Silva, & Laureano, 1996; Francia-Aricha, Guerra, Rivas-Gonzalo, & Santos-Buelga, 1997; Vivar-Quintana, Santos-Buelga, & Rivas-Gonzalo, 2002). These chemical changes cause colour changes from full red to orange-brown in wine during storage or ageing (Somers, 1971; Zamora, 2003).

It was found that the French people had low incidence of coronary heart disease despite having a diet high in fat and being heavy smokers. The reason suggested was that French people drank wine as part of their daily diet. Wine is rich in flavanoid content (Aruoma, 1996). Flavanoids are polyphenolic compounds, and they have the ability to act as antioxidants by a free radical-scavenging mechanism (with the formation of less reactive flavanoid phenoxyl radical) and metal ion chlation (Arora, Nair, & Strasburg, 1998; Lodovici, Guglielmi, Casalini, Meoni, Cheynier, & Dolara, 2001). Wide ranges of studies have shown that the antioxidative properties of these compounds may protect against arteriosclerosis and coronary heart disease (Struch, 2000; Sun et al. 2002).

The objective of this investigation was to determine if there existed a difference in the various attributes of wine produces by 6 different strains of yeast. All the samples were exposed to the same conditions for the same amount of time. The various attributes of wine analysed were the phenolic concentration and antioxidant capacity of the samples. These samples were then compared to investigate if there was a significant difference between the wines produced. Folin-Ciocalteau method was chosen to analyse the phenolic concentration in the samples and TEAC (Trolox Equivalent Antioxidant Capacity) assay was used to measure the antioxidant capacity of the wine. As flavour and colour are important factor in the acceptance of wine, the effect of different yeast strains on the flavour and colour were also investigated.

Materials and methods

Winemaking Instruction for Beaverdale Red Wine Kit:

The given grape must was diluted with water to 6gallons in a larger canister, mixed thoroughly and distributed equally to sterilized 2 Lit conical flasks. Six different strains of yeast were selected for the analysis. Each of these samples was taken in triplicates to ascertain the values. Therefore the diluted grape must was distributed equally (1400ml) to 18 conical flasks. To analyze the change in specific gravity before and after fermentation process a hydrometer reading is taken. The hydrometer gave an expected reading of 1.086.

Table 1: Name and amount of yeast used

Yeast

Quantity

Code

Vintner’s Harvest - CR51

0.448 g

A

Gervin Wine Yeast – Varietal A, Strain GV8

0.304 g

B

Gervin Wine Yeast – Varietal D, German Strain

0.304 g

C

Gervin Wine Yeast – Varietal D, Strain GV2

0.304 g

D

Lalvin – Bourgovin RC 212 (S. Cerevisiae, B1VB)

0.304 g

E

Lalvin – 1122 (S. Cerevisiae, INRA – Narbonne)

0.304 g

F

Table showing the yeast used and the amount added to produce wine. Each wine was coded as shown in the third column.

All the 6 types of yeasts were equally divided 3 times and added to the diluted wine must. Each set was marked as trial 1, trial 2, and trial 3. After adding the yeast to the samples it was shaken vigorously to ensure even distribution. All the conical flasks were closed to ensure no further contamination and for the fermentation process to take place effectively. The samples were left undisturbed to ferment at room temperature (20°C - 25°C) for a period of 20 days.

Ones the fermentation is complete recheck the specific gravity with hydrometer. The values were found to be in between 990 and 994 as required. Ones its sure that fermentation is completed the next step is to add the Stabiliser (0.705ml each) and shake thoroughly for 3-4mins. The addition of stabilizer to wine is to remove the CO2 content. The wine should be left overnight for 3 days along with thorough mixing. The more this is done the easier it becomes to clear the wine.

The wine should have the finings (kieselsol and chitosan) added. 1ml of kieselsol is added to each conical flask and shake for 10secs to mix. Leave it undisturbed for 24hrs. Now add chitosan (1ml) to each container. Shake for 10secs to mix thoroughly. Leave the mix for 7days. The wine should be clear at the end of 7days.

The next step is to transfer each of the samples to separate bottles. The processing is called syphoning. The syphoning is carefully conducted using a U-bend and plastic tubing directly into wine bottles. Syphoning is done to clear the sediments (yeast sediments) from the wine. The wine is ready for drinking immediately, but will improve if matured for several weeks or months.

Sample preparation:

Direct dilution: Each wine samples was diluted 15times (0.67ml wine in 10ml distilled water) in 10ml volumetric flasks. 1ml from these volumetric flasks are used to the further experimentation.

Free polyphenol determination (15times dilution): 2 ml wine is diluted with 1ml of water in a plastic tubes that can be tightened with screw caps. All the samples are then subjected to hydrolysis at 95°C for 2 hrs (in hot water bath). Weight of all the samples is measured before and after the hydrolysis to ensure any loss of water in the form of gas through the gaps in between the screw cap. This is then cooled to room temperature and 1 ml is pipetted out from each into 10 ml volumetric flasks and each made upto 10 ml with distilled water. 1 ml from these volumetric flasks are used to the further experimentation.

Total Polyphenol determination (15times dilution): 2 ml wine sample is mixed with 0.8 ml of 1.2M HCl and 0.2 ml of distilled water in a plastic tubes that can be tightened with screw caps. All the samples are then subjected to hydrolysis at 95°C for 2 hrs (in hot water bath). Weight of all the samples is measured before and after the hydrolysis to ensure any loss of water in the form of gas through the gaps in between the screw cap. This is then cooled to room temperature and 1 ml is pipetted out from each into 10 ml volumetric flasks and each made up to 10 ml with distilled water. 1 ml from these volumetric flasks are used to the further experimentation.

Methods of the compositional analysis of the red wine

Folin-Ciocalteau method: The total phenolic content was calculated by the Folin-Ciocalteau method using gallic acid as a standard (Singleton and Rossi, 1965). The absorbance was calculated at a wavelength of 760nm.Folin-Ciocalteau method is based on the method of Singleton where metal oxides are reduced by polyphenols resulting in a blue solution (Roura et al, 2006). The polyphenol content is expressed as Gallic Acid Equivalent (GAE) Polyphenols are reducing agents, however there are other reducing molecules present in the sample, which will also react with FC reagent. As a result the phenolic value maybe higher than that actually present in the samples.

The phenolic content in wine was analyzed at 4 stages:

Pre-fermentation stage (initial diluted grape must)

Fermented Wine

Wine after yeast proteins are removed (before bottling)

Bottled Wine (1 weeks after bottling)

Standard solution of Gallic acid is used. 0.1 g Gallic acid is dissolved in few drops of 50% methanol (1:1 ratio dilution of methanol). The solution is then made up to 100ml with distilled water in a 100 ml volumetric flask. From this a series of dilutions are made covering a range from 0 μg/ml to 100 μg/ml (0, 20, 40, 60, 80, 100 μg/ml), into 10 ml standard flasks and made up to 10 ml with distilled water. From each 1ml is taken into test tubes. To this add 5 ml of Folin-Ciocalteau Reagent (100 ml FC Reagent in 1000 ml volumetric flask) along with thorough mixing using a Whirlimix and its left to stand for 3 mins. Followed by the addition of 7.5% Na2CO3 (75g Na2CO3 in 1000 ml water), mixed on a Whirlimix and allow the mix to stand for 2 hrs at room temperature. Blank is prepared by using 1 ml of distilled water instead of Gallic acid.

1ml from the prepared wine samples are taken into test tubes. To this add 5 ml of Folin-Ciocalteau Reagent (100 ml FC Reagent in 1000 ml volumetric flask) along with thorough mixing using a Whirlimix and its left to stand for 3 mins. Followed by the addition of 7.5% Na2CO3 (75g Na2CO3 in 1000 ml water), mixed on a Whirlimix and allow the mix to stand for 2 hrs at room temperature.

The absorbances for the tubes are then read at 760 nm ones the spectrophotometer was set to zero using blank. Each sample was read in duplicates.

Trolox Equivalent Antioxidant Capacity (TEAC) Assay: TEAC is to estimate the antioxidant capacity and the free radical scavenging property of polyphenols. When ABTS is mixed with potassium persulphate free radicals are created, resulting in blue colouration. Trolox is used as a standard in the experiment because it is known for free redical scavenging. In the prsence of trolox the free radicals produced by mixing ABTS and potassium persulphate is reduced resulting in de-coloration. The ability of the trolox to react with the reaction products gives a slightly higher anitoxidant value than that is actually present in the sample (Klening.T, 2007; Arts, 2004).

The antioxidant nature of wine was analyzed at 4 stages:

Pre-fermentation stage (initial diluted grape must)

Fermented Wine

Wine after yeast proteins are removed (before bottling)

Bottled Wine (1 weeks after bottling)

Trolox is used as standard for the assay. 0.1 g of Trolox is mixed in 10 ml of 95% ethanol and this was made up to 100 ml with PBS (Phosphate buffer saline solution). PBS is prepared by mixing 810 ml of Na2HPO4 (5 mM, 0.709 g in 1000 ml water), 190 ml of NaH2PO4 (5 mM, 0.709 g in 1000 ml water) and 9g of NaCl. The pH of this solution is made to 7.4 by adding NaH2PO4 to make it more acidic or by adding Na2HPO4 to make it more alkaline. From the prepared trolox standard, series of dilutions are made covering a range from 0 μg/ml to 100 μg/ml (0, 20, 40, 60, 80, 100 μg/ml), into 10 ml volumetric flasks and made up to 10 ml with distilled water.

ABTS stock solution is prepared by mixing 0.0962 g ABTS and 0.0165 g of potassium persulphate and made up to 100 ml with distilled water in a 100ml volumetric flask. The solution is light blue initially but left over night turn deep blue due to the formation of free radicals in the presence of potassium persulphate. In the presence of light the free radicals are absorbed and the colour reduces, hence the ABTS stock solution is kept wrapped in aluminum foil and stored in dark place. ABTS stock solution is diluted with PBS to give an absorbance of 0.7 or 0.8, and this solution should be used for further steps of the experiment. PBS is used as a blank. In another cuvette 80 μl of PBS is mixed with 4 ml of ABTS and the absorbance is read each time a standard is read. This is the control to which the standard is compared.

From the series of trolox dilutions prepared in 10 ml volumetric flasks, 80 μl is taken into test tubes. To this add 4 ml of ABTS and shake well and let it stand for a minute for the reaction to occur. The absorbance is read in a spectrophotometer at 734 nm with PBS as the Blank.

80 μl from the prepared wine samples are taken into test tubes. To this add 4 ml of ABTS, shake well and allow it to stand for 1 min and read the absorbance at 734 nm. ABTS continued to loose its colour in the presence of a free radical scavenger for a period of 20 mins after which the reaction was assumed to stop. But during investigation it was found that the reaction proceeded beyond 20 mins till the complete colour of the solution deteriorated and the solution turned colourless. The sample were read in duplicates.

Analysis for Difference in flavour: To analyze the differences in the flavour produced by using different yeast a Sensory panel was conducted. Six characteristics of wine were analyzed during the session.

Sweetness

Dryness

Body

Acidic nature

Berry fruit/ Red fruit

Red and black fruit mix

18 untrained assessors were selected for the panel. The panel was asked to rate each characteristics of wine, given a 9point hedonic scale based on the intensity of the each characteristic.

Fig. 1. The 9point hedonic scale used for the flavour analysis (Sensory Panel). Assessors were asked to put a cross in the box according to the intensity of the attributes perceived by them.

Analysis for difference in colour: To analyse the difference in colour produced by the different yeasts an instrument called Lovibond RT 100 Colour device is used. Samples were taken in a 5 ml beaker, placed on a white background and the tip of the instrument was placed at the opening of the beaker. The instrument was calibrated first as mentioned in the manual and the reading was taken with the first sample as reference and comparing the remaining 5 samples with the reference. The software shows the difference between colour intensities of each sample.

Results

It was observed that hydrolysis helped to increase the phenol content and the antioxidant activity of the wine. Thus direct dilution was insignificant in calculating the actual phenol content and the antioxidant capacity of the samples. And also the two methods of hydrolysis (to calculate free and total phenols) gave similar results.

Total phenol content: The samples that were analyzed after hydrolysis gave a higher phenol content compared to the wine samples that were analysed after direct dilution. Large amounts of phenols remain attached to the proteins and are not detected by the Folic-Ciocalteau method. Thus for analysis through out the experiment wine samples that were analysed were hydrolyzed at first to free the phenols from the proteins.

Wine samples were analyzed for phenol concentration at 3 stages of wine production. Fig. 2. shows the phenol quantity calculated at different stages of by the wine samples produced by 6 different yeasts. Although from the graph it is evident that there is a small variation in the phenolic concentration of wine produced by the different strains of yeasts, statistical analysis, i.e., Paired T-test and ANOVA proved the variation among the different samples to be insignificant (P>0.05). The small variation in the phenolic concentration among samples maybe due to the various metabolites produced by the different yeast.

The first stage that was analysed for phenol concentration was the grape must that was diluted initially before fermentation (Pre-fermentation stage). The sample gave the almost similar phenolic concentration as wine soon after fermentation. The second set of samples were analysed soon after fermentation, showed the highest concentration of polyphenols as expected. All the polyphenols that are attached to the wine must are released after fermentation and thus the phenol content in wine is high. Results show a decrease in the phenolic concentration after clearing and following after 1 week of wine bottle aging. But at each stage all the 6 samples showed similar decrease in the phenolic concentration.

The samples were analysed in duplicates to guarantee the validity of the observed results. The concentrations are expressed in µg/ml.

Fig. 2. Graph showing the phenol concentration in the 6 wine samples at the 3 stages of wine making. The first bar stands for wine after fermentation, second bar for wine after clearing and third bar for wine analyzed after 1 week of bottle ageing.

Antioxidant Capacity: The total antioxidant property of the wine samples analysed was determined by the bleaching of pre-fromed ABTS radical cations. The addition of free radicals-scavangers to a solution containing ABTS-derived radical cations leads to a decrease in the absorbance of the samples at 734 nm that is proportional to the size of the wine aliquot (Campos, Escobar, & Lissi, 1996).

Samples were prepared the same way as it was prepared for Folin-Ciocalteau method. Wine sample by direct dilution gave showed a lower antioxidant content compared to the samples that were hydrolyzed. Thus samples were first hydrolyzed before analysis.

Wine samples were analyzed at 4 stages for the concentration of antioxidant capacity. The first analysis was done on the grape must diluted to before the fermentation. An unexpected result was obtained at this stage. The solution turned colourless in the span of few seconds. The next analysis was conducted on the samples soon after fermentation. The results showed the highest concentration of antioxidants capacity as expected, because as the grape must is being fermented there would be more flavanoids produced. These flavanoids exhibit free radical scavenging activity. More over phenols also exhibit antioxidant activity, i.e., more phenols are released from the must due to fermentation and this increases the concentration of antioxidant capacity.

Fig. 3. shows a slight variation in concentration among the 6 samples. But statistical analysis proved that the variation among the samples were insignificant (P>0.05). The small variation in the antioxidant capacity concentration among samples maybe due to the various metabolites produced by the different yeast. Concentrations are expressed as μg/ml.

Fig. 3. Graph showing the Antioxidant capacity in the 6 wine samples at the 3 stages of wine making. The first bar stands for wine after fermentation, second bar for wine after clearing and third bar for wine analyzed after 1 week of bottle ageing.

Analysis for the variation in Flavour: ANOVA was conducted to analyse the variation between the samples. The data was entered into anexcel sheet and fed into R-console. The data showed an interaction between the assessors and the samples. Thus Friedman single-factor with ANOVA was used for the analysis of the data obtained from the taste panel. The results obtained from the R-console showed that there existed no significant difference between the samples (P>0.05).

Analysis for variation in colour: Lovibond RT Colour is a reliable instrument in measuring the colour intensity of coloured samples. The instrument plots the result in a colour intensity graph. From the investigation results it was evident that the samples exhibited similar colour with no significant difference.

Fig. 4. Comparison between the Phenolic Concentration and Antioxidant capacity of the 6 six samples and 3 stages of Winemaking.

Relationship Between Phenolic concentration and Antioxidant capacityof bottled wine: Fig. 4. showing a relationship between the antioxidant content and the phenolic concentration of the different wine samples. From the graph it is evident that as the phenolic concentration reduces there is a reduction in the antioxidant content of the wine. The same was observed for the 3 stages analysed. Sample A (Vintner’s Harvest - CR51) showed relatively a higher value of antioxidant concentration.

Statistical Analysis: The same samples were analysed in 3 replications and the results were expressed as mean values ± standard error. The direction and the magnitude of the relation between the variable were calculated using ANOVA (Analysis of Variance) test. The criterion for statistical significance was p≤0.05.

Discussion

Phenol Concentration: The results of investigations proved that the wine produced from the different yeast was statistically proved to be almost the same in term of the phenolic concentration. The small variation in the phenolic concentration could be as a result of different secondary metabolites produced by yeasts during their growth phase. These metabolites might have higher reducing properties as Folin-Ciocalteau method in the measurement of any reducing compounds. Thus the values observed might be slightly higher than that is actually present in the samples.

The first sample analysed was the Pre-fermented grape must, which was diluted to the required amount. The phenolic concentration observed at this stage was slightly lower than that was observed after the fermented stage. Most of phenols are observed in the grape skin and pulp, during fermentation the yeast helps in breaking down these and releasing the phenols. This can be suggested as a reason for the increase in phenol concentration in the fermented wine. The fermented grape must showed the highest phenolic concentration among all the four stages as expected. During the third stage of wine making, Clearing, finings were added to the samples. The role of these finings is to adsorb the proteins in the wine and precipitate at the bottom. Therefore at this stage the phenols are adsorbed to the yeast cell wall and they are precipitated clearing some of the phenols from the solution (Plaza-Gomez et al., 2002).

The main structural component of the cell wall of Saccaromyces Cerevisiae are glucans and mannams with a minor portion of chitin (Walker, 1998). Mannoproteins are located on the outer surface of the of the yeast cell wall and they determine almost all the surface properties of the cell wall, which includes it’s ability to bind to wine molecules such as anthocyanins and phenols (Morata, Gómez-Cordovés, Colomo, and Suàrez, 2005; Morata, Gómez-Cordovés, Suberviola et al., 2003; Vasserot, Caillet, and Maujean, 1997).

The last stage analysed was the bottled wine after aging it for 1 week. The result showed unexpected reduction in phenol content. The reason for this was not well documented. It can be assumed that the phenols were broken down by the presence of fraction of yeast in the bottled wine or maybe the metabolites produced by yeast that might take part in other interactions resulting in new compounds.

Li, H., et al. (2009), analysed the phenolic content in red wine. The content of total polyphenols varied from 1402 to 3130 mg/L, giving an average value of 2068mg/L. The values obtained were found to be similar to the phenolic concentration values calculated by this investigation.

Antioxidant capacity: ABTS radicals are most widely used as stable chromogen compound to measure antioxidant activity of a biological material. High TEAC values indicate that the elevated mechanism of antioxidant action of extracts. This is because they act as a hydrogen donor and it could terminate the oxidation process by converting all the free radicals into a stable form (Hua Li, et al., 2008).

The initially diluted wine must prior to fermentation (Pre-fermented grape must) was analysed for the concentration of antioxidant content. It was observed the TEAC test for the sample gave an error in result. The observed colour was fading and the solution turned colourless in few seconds. This might have occurred due to the interference of sugar molecules but the mechanism for this to occur was not clearly understood.

It has been proved through this investigation that different strains of yeast produce wine with slight variation in antioxidant capacity, colour and flavour. But statistical analysis proved that these small variations do not make the wines significantly different from each other. The small variation that was noted might be due to the different metabolites produced by the different yeast during fermentation or due to the interaction between yeast and polyphenols. Among the 4 stages analysed, the samples analysed after fermentation showed the highest concentration as expected. More flavanoids and phenols are liberated from the wine must as a result of fermentation.

The third stage analysed was after the clearing process. At this stage finings were added to precipitate the yeast proteins. The investigation showed a reduction concentration of antioxidant capacity. During this stage finings were added to the wine. The reason for the reduction in antioxidant capacity of wine sample might be same as that was stated in the case of phenols. The addition of finings was to help in adsorb proteins present in the wine and precipitate at the bottom (Plaza-Gomez et al., 2002). Therefore at this stage adsorption of polyphenols to the yeast cell wall might take place (Morata, Gómez-Cordovés, Colomo, and Suàrez, 2005; Morata, Gómez-Cordovés, Suberviola et al., 2003; Vasserot, Caillet, and Maujean, 1997). When the finings are added to the fermented wine the yeast proteins precipitate along with the adsorbed molecules.

The forth stage analysed was the bottled wine after 1 week aging. This stage showed a further unexpected reduction in concentration. This might be because of the presence of certain yeast metabolites that results in the degradation of polyphenols and also participate in certain interactions with pigments thereby reducing the colour or can also be due to the formation of new compounds that have lower antioxidant capacity.

During wine maturation and aging, anthocyanins participate in frequent condensation reactions that result in the formation of oligomeric and polymeric pigments. These pigments present more stable structures and modify the initial colour (bright red) of young wine to more brick-orange hues. The wine yeast has a two-fold implication on the red wine colour. The wine yeast enhances the extraction of grape anthocyanins during maceration and fermentation, depending on the alcohol production capacity of yeasts. They also influence the formation of more stable forms of anthocyanins during maturation and ageing. On the other hand they promote the degradation of anthocyanins and participate in certain interactions with pigments that result in colour loss (Maria Monagas, et al., 2006).

Flavour and Colour of the wine samples: The taste panel rated the samples based on a 9point hedonic scale for the 6 attributes of wine. R-Gui was conducted to analyse if there existed any difference between the samples. Using Friedman single factor with anova it was concluded that there existed no significant difference between the samples. (i.e. P>0.05). Similarly the analysis for colour difference between samples produced by the different yeasts also showed a slight difference in colour but using statistical tools proved that these differences were insignificant.

Relationship between Phenol Concentration and the Colour Spaces: Fig. 5. shows that all the samples showed similar L*, a* and b* values. The wine samples showed similar phenolic concentration and thus the Colour spaces were similar for all the samples. Thus we can assume that the phenolic concentration plays a very important role in determining the colour of the wine samples.

Fig. 6. Graph showing correlation between phenol content and the Colour spaces in the 6 samples.

Fig. 5. The correlation between the antioxidant capacity and the phenolic concentration of the different. The graphs were found to show a linear correlation with r2 values ranging between 0.927 and 0.999. This was found to me similar to that mentioned in many literature.

Relation ship between Phenol concentration and the Concentration of antioxidant capacity of wine: Fig. 5. Shows the correlation between the antioxidant capacity and the phenolic concentration of the different wine sample. A very close relationship was observed between the total phenolic content and the total antioxidan potential, for all the wines observe (Campos And Lissi, 1996). The r2 value was in between).988 and 0.992. It is evident from the graph that there exists a linear correlation between the two factors (r2 in between 0.927 & 0.999). These results were in agreement with other reports in the literature (Campodonico et al., 1998; Fogliano, Verde, Randazzo, & Ririeni, 1999; Henn & Stehle, 1998; Sánchez-Moreno, Larrauri, & Saura-Calixto, 1999a; Sato et al., 1996; Simonetti et al., 1997)

In literature, contrasting and confusing reports exists about the relation between the phenolic content of wine and the antioxidant capacity. The results obtained by this investigation are in agreement with few authors who suggested a linear correlation exists between the antioxidant capacity and the total phenol content (Ghiselli, Nardini, Baldi, and Sacaccini, 1998). Also López-Vélez et al. (2003) concluded in his study that the total antioxidant activity of wine investigated was well correlated with the phenol content. And thus he stated that red wine polyphenols are, in-vitro, significant antioxidants. Many other claim that there exists a statistical correlation between the penolic content and just the flavanoid fraction and that it is not so when considering the non-flavanoid fraction (Katalinić, Milos, Modun, Musić, & Boban, 2004).

It was found that the total flavanols are the class of polyphenols that accounts for radical scavenging efficiency of wine and to a lesser extent to a reducing ability. But there was a link between antioxidant capacity of wine and it total phenolics and only a weak relation to the amount of anthocyanins present (Anous A, Makris DP, and Kefalas P, 2001).


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