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Evaluation of Antioxidant and Antimicrobial Activity of Wine from Various Sources

Wine rich in antioxidant property an alcoholic beverage also is considered as an excellent source for many bioactive compounds like antioxidants, polyophenolic and flavonoids. Reports suggest that wine contains antioxidant power that reduces the oxidative stress and prevents risk for many coronary diseases and cancer (Danila Di Majo et al., 2008). The respirative cycle of oxidative phosphorylation produces free radicals which may attack biological macromolecules like DNA, and cause cell ageing, cardiovascular diseases, and even cancerous tumor growth due to denaturation of single and double strand DNA. When immune system of an organism fails to rectify the problem then the situation of ‘oxidative stress’ occurs, which damages the macromolecules present in the body. Consumption of food containing natural antioxidant activity is one of the effective way to overcome the problem (Reşat Apak et al., 2007; Just J.R. et al., 2003; Daroch et al., 2001). Besides these properties, wine also shows significant antimicrobial activity. Recently, the antimicrobial effects on various plant extracts against certain pathogens like Staphylococcus aureus, Candida albicans and E.coli have been reported by a number of researchers. The International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 3(2016) pp. 26-35 Journal homepage: http://www.ijcmas.com


Introduction
Wine rich in antioxidant property an alcoholic beverage also is considered as an excellent source for many bioactive compounds like antioxidants, polyophenolic and flavonoids. Reports suggest that wine contains antioxidant power that reduces the oxidative stress and prevents risk for many coronary diseases and cancer (Danila Di Majo et al., 2008). The respirative cycle of oxidative phosphorylation produces free radicals which may attack biological macromolecules like DNA, and cause cell ageing, cardiovascular diseases, and even cancerous tumor growth due to denaturation of single and double strand DNA.
When immune system of an organism fails to rectify the problem then the situation of 'oxidative stress' occurs, which damages the macromolecules present in the body. Consumption of food containing natural antioxidant activity is one of the effective way to overcome the problem (Reşat Apak et al., 2007;Just J.R. et al., 2003;Daroch et al., 2001). Besides these properties, wine also shows significant antimicrobial activity. polyphenols like resveratrol, hydroxytyrosol, quercetin and a number of phenolic acids present in the wine shows anti-oxidant, anti-carcinogenic, antiinflammatory and anti-microbial activity (Chrissanthy Papadopoulou et al., 2005;Sandipan Goswami and Subhajit Ray, 2010;Bapat, 2010).
Wine is produced from wide variety of sources. For the winemaker's continuous production of wine throughout the season, its cost effectiveness, easy transportation, storage are essential factors. The quality of the fruit, amount of fermentable sugars, acid content and characteristics flavor provides suitability for wine making (Sandipan Goswami and Subhajit Ray, 2010). Most of the winemaker prefers grape for wine production. The total phenolics in red wine are higher in comparison to the white wine. Wine phenolics are categorized as flavonoid and non-flavonoids. Few fruits with balanced sugar, acid, tannin, nutritive salts as a suitable substrate for the growth of yeast with adequate amount of water are also used for wine production (Robinson Jancis, 2006). The Arunachal Pradesh Horticulture Produce Marketing & Processing Board (APHM&PM) in India have funded the project for the wine processing from kiwi fruit. This fruit has high aroma and a refreshing flavor with high acidic nature and sugar almost similar to grapes (Business Standards, 2015). Sucrose if generally added for the fruits that have excess acids can split into fermentable fructose and glucose. In order to maintain the specific gravity of the juice at the initial is too high with water or acidulated water it can be adjusted to the winemaker's target range (Bapat et al., 2010).
In this present study wine was prepared from four different sources; rice, barley, grapes and strawberry under different fermentable conditions. The characterization of both red and white wine was carried out and compared with the commercially available wine for comparative analysis. The quantification and purification of the wine was carried out by HPLC. A comparative evaluation was performed for the estimation of total antioxidant compounds, phenolics concentration and its antimicrobial activity.

Production of Wine from Black Grapes (Vitis vinifera)
250g of grapes was weighed and washed by removing the stems with 1% HgCl 2 then with sterile distilled water for 10 minutes. The grapes was soaked in 5% sodium chloride solution for 15minutes. After the completion of surface sterilization finally it was washed with sterile sterile distilled water. The inoculum was prepared for batch fermentation by homogenizing 250g of grapes with 25g of sugar and 200ml of sterile water into an pre-autoclaved wine jar fitted with rubber cork and bent glass tube for the release of CO 2. The yeast cell was cultured and maintained for 48 hours in yeast extract, peptone dextrose medium at 27 0 C. The inoculum size of 3 x 10 4 cells /mL was transferred into the fermentation wine jar containing grape juice. The inoculum bottle was incubated for 48 hours at room temperature under slow shaking condition. Later 750g of grapes was homogenised along with 50g of sugar and 300ml of water. A pinch of potassium metabisulphite was added. The inoculum was transferred completely to this fermentation bottle and allowed for production of wine. The contents are mixed well and incubated for 28 days at room temperature under stationary condition during the incubation period. After the completion of the incubation period of 28 days quantification of antioxidants, total phenolic compounds and other characterization of wine was performed for every week until 4 weeks.

Production of Wine from Strawberries (Fragaria ananassa)
The strawberries was cleaned by removing the stems and washed under tap water. 250g strawberries are then firstly washed with 1 % HgCl 2 then with sterile distilled water. The cleaned fruits was soaked in 5% sodium chloride solution for 15 minutes washed with sterile distilled water. The strawberries was then weighed and homogenised into pulp in a blender along with 25g of sugar and 200ml of water and transferred into a brown bottle. The same concentration of yeast culture was added as a starter culture to the fruit extract and incubated at 27 0 C for 48 hours under slow shaking condition. For the batch fermentation process same set up was made for the wine production as in the case of grapes. The remaining 750g of strawberries are then homogenized into pulp along with 50g of sugar and 300ml of water. A pinch of potassium metabisulphite is added to it and the inoculum is transferred. The contents are mixed well and incubated for 28 days (Sandipan Goswami and Subhajit Ray, 2010) under stationary condition.

Production of Wine from Rice (Oryza sativa) and Barley ( Hordeum vulgarae)
The wine production from rice and barley was performed under two conditions: With sugar and without sugar as the sugar content in both are comparatively lesser than the fruits.

Production of Wine from Rice (Half Boiled Rice)
The rice grains was cleaned and washed with water properly. It was soaked in clean sterilized water for an overnight and was grind to small particles. In the first setup: 500g of partially grinded rice in a 1liter wine jar with single holed rubber cork with a bent glass fitted to release the CO 2 during the fermentation and 200ml of water is taken. To this 17g of sugar and same inoculums size of precultured yeast added to the jar. In the second setup: Another 500g of grinded rice was taken similar to the previous set without adding sugar. A pinch of potassium metabisulphite was added to both the jars and the contents were mixed well and incubated for 48 hours under slow shaking condition then transferred to stationary condition for 26 days of incubation.

Production of Wine from Barley
The barley was cleaned and washed under tap water. It was soaked in clean sterilized water for an overnight and was grind to small particles. Two experimental set up was established-with sugar and without sugar. In the first set 500g of partially grinded barley was taken in a 1liter wine jar with 22g of sugar and 200ml of water. The rest 500g of grinded barley was set for wine production without sugar. An yeast inoculum of 3 x10 4 cells/mL was added to both the wine jars and incubated for slow shaking condition for initial 48 hours and later maintained the fermentation process under stationary condition for 02 days. A pinch of potassium metabisulphite is added to both the jars and the contents were mixed well and incubated for 28 days (Sandipan Goswami and Subhajit Ray, 2010).

Estimation of Percentage of Alcohol, Total Acid and Volatile Acid
Estimation of alcohol The concentration of alcohol was estimated by alcoholmetry method using AOAC chart (Table 1).

Estimation of Total Acidity and Volatile Acidity
10ml of the standard wine as well as sample wines was taken in a 100ml conical flask. Diluted to 1:1 ratio by adding 10ml of distilled water and a few drops of phenolphthalein indicator. This solution was titrated against 0.1N NaOH for the colour appearance from pink to green. The titration was repeated to get concordant values (Ronald et al., 2010). The total acidity and volatile acidity was estimated from the titrated value (Table 1).

Purification and Quantification of Bioactives Compounds of Wine Samples by HPLC
The column used for HPLC system consisted of a C18 RS column (250, 4.6mm ID, S-5μm. 8nm) separation was performed at room temperature. Injection volume was 10μL, flow rate set at 1 mL/min with UV detector was carried out at 290 nm. Solvent used were 2% acetic acid and 0.5% acetic acid / 50% acetonitrile. 1 mL of wine sample was diluted with 5mL of methanol and filtered through 0.45μm filter before injection into the HPLC 4 ( Table 2).

Estimation of Antioxidants Present in Different Wine Samples Phosphomolybdenum Assay Protocol
An aliquot of 0.3 ml wine sample (5 mM in MeOH) was combined in a 4-ml vial with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The vials was capped and incubated in a water bath at 95ºC for 90mins. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 695nm against a blank. The antioxidant activity was (Kashif Ghafoor et al., 2011) expressed relative to that of ascorbic acid (Kashif Ghafoor et al., 2011) Table 3.

Antimicrobial Assay (Disc Diffusion Method)
The pure bacterial and fungal culture was inoculated in a freshly prepared nutrient broth and potato dextrose broth respectively. The inoculated broth was incubated until the OD at 620nm reaches 0.7. The cultures from the broth was swabbed on a Mueller Hinton agar plate for bacteria and potato dextrose agar plate for fungal culture. The discs with wine samples of various concentrations was placed on the petri plates with swabbed cultures using sterile forceps. The plates was incubated for 24hrs for bacterial culture and 3 days for fungal culture. After the completion of incubation period the zone of inhibition was measured and compared with standards (Zahid Hussain, 2011) Kirby Bauer Chart . The antimicrobial activity shown by different wine samples for microbial cultures with the zone of inhibition was compared for different wine samples (Table 4).

Estimation of Total Phenolic Compounds
The total phenolic compounds was analyzed using Folin Ciocalteau method. A 200 μL properly diluted wine sample or standard solution of varying concentrations mixed with 400 μL Folin Ciocalteau reagent. The deionized water was used for dilution and control. The solution was diluted to a total volume of 4.6 mL using deionized water followed by thorough mixing. After incubation for 10 min at room temperature, 1 mL of 10% Na 2 CO 3 solution was added followed by immediate thorough mixing and incubated for 2hrs at room temperature. The absorbance was read at 765nm on a spectrophotometer (Kashif Ghafoor et al., 2011). Gallic acid of 1 mg/mL was used as the standard and the total phenolic compounds of the samples was expressed in milligram gallic acid equivalent per mL of wine sample (mg GA/1 mL) ( Table 5).

Characterization of Prepared Wine Samples
The prepared wine samples was characterized by the colour, aroma, flavor, astringency, pH, alcohol percentage, total acid and volatile acid (Table1). The observations after the 3 rd till 9 th week fermented wines was found as grape wine was light reddish purple color, strawberry wine was pinkish, rice and barley wine (with and without sugar) was creamish white. The pH of grape and strawberry wine was found to be 4.7 after fermentation whereas pH was 3 before fermentation. Rice and barley wine (with and without sugar) the pH was found to be 4.0 after fermentation and pH 6 before fermentation (Table1).
The alcohol concentration ethyl alcohol, propanol, butanol, etc) was estimated by specific gravity method using specific gravity bottle. The specific gravity of all the wine samples was calculated and percentage of alcohol present was estimated using AOAC chart (Table 1).
Total acid in this study was expressed in terms of percentage of Tartaric acid (Table  1) and was calculated by the formula: Total acidity (TA) =Volume of NaOH added x Normality of NaOH x 7.5/10. Volatile acid is expressed in terms of percentage of Acetic acid (Table 1) and is calculated by the formula:Volatile acidity (VA) = Volume of NaOH added x Normality of NaOH x 6/10. Total acid and volatile acid directly effects the color and flavor of wine. Tartaric acid is one of the major acidic material present predominantly in compare to other acids like malic acid, acetic acid etc. and helps in the ageing of the wine. At the same time among the various volatile acids present in the wine acetic acid plays the major role (Wahab et al., 2005).
The tartaric acid in Roselle wine was found to be 0.65% which is almost close to grape wine in the present study (Arubi et al., 2009). The VA of the Roselle wine showed 0.04% of acetic acid, where the rice wine prepared with sugar in the present study showed 0.035% of acetic acid (Table 1). It is observed that grape wine has highest percentage of TA and VA, followed by strawberry, rice (with sugar), rice (without sugar), barley (with sugar) and lastly barley (without sugar). Reddy et al, in 2005(Reddy et al., 2005 optimised wine production from six varieties of Mangifer indica L and it was found that ethanol produced was 7-8.5%, TA (tartaric acid) 0.6-0.8 % and VA (acetic acid) was 0.01-0.2% v/v.
In 2009 Soni et al 21 reported during the batch process alcohol produced was 12% with supplementation of ammonium sulphate, potassium dihydrogen phosphate, proline and biotin to hot water extract of Emblica officinalis (Indian gooseberry), but later in a fed batch process the concentration of alcohol was increased to 16.1 %.

Identification of Bioactives and Purification of Prepared Wine Samples by High Performance Liquid Chromatography (HPLC)
The HPLC was performed for all the prepared wine samples in the 5th week of fermentation and the amount of antioxidant present in each sample is calculated, and is expressed in μg/mL. Gallic acid was used as a standard ( Table 2).
The result represents that the concentration of antioxidants presents in strawberry was highest 20μg/mL. and for grapes 12μg/mL. and barley with sugar 10μg/mL.. The other samples contained lower concentration of antioxidant.

Estimation of Antioxidants Present in Different
Wine Samples by Phosphomolybdenum Assay The assay was conducted for four consecutive weeks for comparative analysis ( Table 3).The amount of antioxidants present in each wine sample was estimated and expressed in terms of μg/mL. Ascorbic acid was used as standard. It was observed that the amount of antioxidants in all the wine samples increases as the weeks for fermentation increase. Strawberry wine showed constant increase in the concentration from 60 μg/mL to 100 μg/mL. Grape wine, rice with sugar and barley with sugar were also showed presence of antioxidant very comparatively close to each other. Hence, in the upcoming days huge interest could be shown in the cultivation of strawberries and the production of strawberry wine.  Krachai-Dum wines showed increase in the total phenolic content, flavonoids and total antioxidants as ageing increases but the study also suggests that the antioxidant activity depends on number of factors including the phenolic compounds present in the wine 9 . The research conducted by Korsak Towantakavanit, Yong Seo Par, Shela Gorinstein, 2010, proves that the antioxidant activity of the wine depends on the quality and ripening stages of the fruit choosen and usually increases with increase in fermentation 10 .

Antimicrobial Assay (Disc Diffusion Method)
Assay was performed against three bacterial strains of E.coli, Pseudomonas aeroginosa, and Streptococcus pyogenes and the zone of inhibition produced by all the wine samples were measured (Table 4). The recent study conducted by Aleksandra N Radovanovićet et al supports the antimirobial activity of the serbian red wine against various strains of bacteria by disc diffusion method 11 .

Estimation of Total Phenolic Compounds
The total phenolic compounds was estimated by Folin Ciocalteu method. It was observed that similar results like antioxidant concentration, strawberry wine shows the highest even for phenolic content as 1.14mg/mL. Wine from dried apricot, raisin using S. cerevisae were prepared with an fermentation period of 28 days, were found to be rich in Vitamin A, lycopene, carotenes, polyphenols and antioxidants compounds 13 .