Antimicrobial Activity and Health Benefits of Red Wine from Grape (Vitis vinifera Linn.)

Universities Research Journal 2011, Vol. 4, No. 3 Antimicrobial Activity and Health Benefits of Red Wine from Grape (Vitis vinifera Linn.) Nu NuYi Abstract The phytoconstituents in pulp, skins and seeds of grape (Vitis vinifera Linn.) and in red wine made from grape juice of such variety by fermentation using Saccharomyceae cerevisiae were investigated. Phenolic compounds, anthocyanins and tannins in red wine were also checked by general methods. The bioactivities of red wine against some pathogenic microorganisms: gram positive bacteria: S.aureus, B.subtilis and B.pumalis; gram negative bactreia: P.aeruginosa, E.coli and fungi, C.albicans were described in this paper. Key words : Vitis vinifera, Saccharomyceae cerevisiae, pathogenic microorganisms, S.aureus, B.subtilis, B.pumalis, P.aeruginosa,.. Introduction In this research, the fruit being focused for its phytoconstituents of biological importance is grape (Vitis vinifera Linn.), family Vitaceae (Figure 1). It is one of the major economic importance in wine making because of their right levels of acid, sugar, flavour and their nutritional values. Investigation of phytoconstituents in red wine and its antimicrobial activity on pathogenic microorganisms are the aims of the present study. Health Benefits of Grape ( Vitis vinifera Linn.) and Red Wine Recent studies have reported that the red wine and grape juice have the same health benefits: reducing the risk of blood clots, reducing low density lipoprotein(bad cholesterol ), preventing damage of blood vessels in the heart, and maintaining a healthy blood pressure. Both red wine (Figure 2) and grape juice contain antioxidants, some flavonoids which can increase HDL (High Density Lipoprotein or " good cholesterol") and lowers the risk of clogged arteries (atherosclerosis) and thus may help lower blood pressure. Phenolic compounds in red wine inhibit oxidation of human LDL (Low Density Lipoprotein) levels (Stephanie et al., 2009). Lecturer, Department of Chemistry, Meiktila University

154 Universities Research Journal 2011, Vol. 4, No. 3 Figure 1. Plant of Vitis vinifera Figure 2. Red wine from Grape Antioxidants in Grape and Red Wine Oligomeric proanthocyanidin complexes (OPCs), powerful antioxidants found in grape juice, skin, seeds and red wine from grape are useful to treat a range of health problems related to free radical damage including heart diseases, diabetes and cancers. OPCs can lower cholesterol, help to treat hypertension by protecting blood vessels from damage and can reduce the risk of developing cancer by preventing the growth of breast, stomach, colon, prostate and lung cancer cells in vitro. Moreover, OPCs have potential benefits against the platelet aggregation and other risk factors of atherosclerosis, loss of physical performance and mental activity during ageing and hypertension in humans. Proanthocyanidins found in red wine have antioxidant activity and these compounds play a role in the stabilization of collagen and maintenance of elastic, the two critical proteins in connective tissue that support organs, joints, blood vessels and muscle. Proanthocyanidin's antioxidant capabilities are 20-times more powerful than vitamin C and 50- times more potent than vitamin E. It causes strengthen all blood vessels and improve the delivery of oxygen to the cells. Antioxidants in red wine are in two main forms, flavonoids and nonflavonoids. Cardioprotective effect of red wine are due to the presence of polyphenolic compounds such as flavonoids and resveratrol (nonflavonoid). Isoflavonoids and polyphenols in red wine possess the function of antioxidant, lipid lowering, immunomodulator, antiosteoporotic and anticancer properties (Stephanie et al., 2009).

Universities Research Journal 2011, Vol. 4, No. 3 155 Some flavones have antiestrogenic effects. This may benefit people at risk of breast and prostate cancers. Thus, it possesses anticarcinogenic effects. Flavonoid intake inhibit the aggregation and adhesion of platelets in blood, which lower the risk of heart diseases. Flavonoid such as quercetin, kaempferol in grape and red wine could interfere sulfaction-induced cancer growth. Quercetin is also vasorelaxant and it showed a marked cytoprotective capacity in vitro test. Various Medicinal Uses of Different Parts of Grape and Red Wine Red wine enriched with resveratrol, flavonoids and other phenolic compounds possesses the beneficial effects of calories restriction in heart, skeletal muscles and brain which inhibit gene expression associated with heart and skeletal muscle aging and thus prevent age-related heart failure (Corder et al., 2006). Various medicinal uses of different parts of grape and red wine are described in Table 1. Table 1. Medicinal uses of different parts of grape and red wine Parts used Sap of grape vines Leaves Unripe grape Ripe grape Dried grape (Raisin) Grape skin to treat skin, eye diseases. Medicinal values to stop bleeding, anti-inflammation, to relief pain, hermorrhoids. to treat sore throats. to treat cancer, cholera, small pox, nausea, eye infections, skin, kidney and liver diseases. to treat constipation and thirst. to prevent cancer, to protect heart and brain damage, to inhibit the degenerative nerve diseases.

156 Universities Research Journal 2011, Vol. 4, No. 3 Table 1. Medicinal uses of different parts of grape and red wine (Cont'd) Parts used Grape seed extract Red wine Medicinal values to treat chronic venous insufficiency, blood pools in the legs, pain, swelling, fatique, visible veins, diabetes and edema, to protect collagen and elastin in skin (i.e anti-ageing) to treat hemorrhoids, to prevent damage to human liver cells caused by chemotherapy medication. to reduce the risk of leukemia, breast, skin and prostate cancers, to protect heart and brain damage. Materials and Methods Sample Collection Ripe and undamaged fruits of the grape plant (Vitis vinifera), used as raw materials for wine fermentation were collected from Tawma Village, Meiktila Township, Mandalay Region, Myanmar. Botanical Description of Grape Family - Vitaceae Genus - Vitis Species - vinifera Botanical Name - Vitis vinifera Common Name - Grape Preparation of Red Wine from Grape (Vitis vinifera) Ingredients for Making Red Wine Red grape (fruits) 1 viss (680 ml grape juice) Sugar 35 g Yeast (Saccharomyces cerevisiae) 8 g Sodium metabisulphite 0.3 g Ammonium phosphate 0.1 g Distilled Water 750 ml

Universities Research Journal 2011, Vol. 4, No. 3 157 Process of Wine Making Wines are produced from grape by multistep process. The basic wine making steps are (1) grape processing (2) fermentation (3) clarification (4) stabilization (5) bulk aging and (6) bottling. Red Wine was prepared by the following procedure. Before fermentation, clean, undamaged and fresh fruits of grape (Vitis vinifera) were pressed manually with little pressure (free run) to obtain the clearest liquid from middle of the pulp. After this, pressing was continued with heavier pressure to get the grape juice and volume of the juice was measured. Grape juice with grape seeds and grape skins were placed in the fermenter. Brix of the juice was checked to get 22 24 degree. ph and temperature of the filtrate was also measured. Sodium metabisulphite solution, prepared by dissolving 0.3 g of Na 2 S 2 O 5 crystal in 350 ml of distilled warm-water was added to the combined grape juice with stirring to inhibit the wild yeast (Wang, 2004). After being added sulphite, sugar was added to this grape juice. Then, yeast solution (8 g yeast (Saccharomyces cerevisiae) was added to 200 ml of warm-water and set aside for 10 min before adding to the juice) was added to the grape juice in the fermenter. At the same time, 0.1 g of ammonium phosphate and 100 ml of distilled water were also added to the juice. The fermenter was covered with air tight stopper. Fermentation was allowed for two weeks at the dark place at room temperature in anaerobic condition. After two weeks, decantation of turbid red wine was rapidly carried out to remove sediment including death yeast cells, grape seeds, grape skins and other impurities. Fermentation was allowed to continue. After three weeks, decantation of turbid red wine was rapidly filtered through cotton filter. Decantation was repeated at every one week to get desired clarity of red wine. After fermentation, red wine was stabilized and aging of wine was allowed for at least three months. Then, a little sodium metabisulphite solution (0.1 g in 100 ml) was added to wine just before bottling to ensure the absence of microorganisms. Finally, clear, stable and properly aged wine with good aroma and flavor was bottled in pasteurized glass bottles of brown colour with air tight

158 Universities Research Journal 2011, Vol. 4, No. 3 stopper. Thus, red wine was stored in glass bottle for many years before drinking. Process of wine making from mature and undamaged grapes (Vitis vinifera)of good quality with grape seeds and skins by biochemical processing with careful handling, pre-treatment of juice, clarification, chemical composition of the juice, temperature and ph of the fermentation and the condition to inhibit other micro-organisms were carried out to achieve the red wine with distinctive flavor and aroma. In this processing, the original microbial population on the grape skin was eliminated by treatment with SO 2 produced from sodium metabisulphite by the following reactions. Na 2 S 2 O 5 2SO 2 + Na 2 O sodium metabisulphite sulphurdioxide sodium oxide Temperature between (20-28 C) and ph of grape juice (4-4.5) are optimum condition for yeast population. So, in this research, ph of grape juice was 4 and the temperature was kept at 20-28 C. This temperature is enough to extract the pigment, anthocyanin compounds from the grape skin. Aging of wine in the dark for years is the crucial step of wine making to obtain wine of good flavor and aroma. The chemistry of the process of wine aging are phenols + phenols------------->more complex phenols Alcohol + Acids -------------->Aldehydes------------>Esters Esters + Esters ------------->Delicate and more complex Esters Results and Discussion The flavor, aroma and quality of wine merely depend on the value of fruit juice and subsequent processing of fermentation. In this research, freshly prepared grape juice collected from ripe and undamaged fruits of grape (Vitis vinifera) cultivated in Tawma village, Meiktila Township, Mandalay Region were fermented by dry yeast (S. cerevisiae) strain for wine making. From about 1 viss of fruits, 680 ml of grape juice were obtained. Yield percentage of red wine from grape is 85%. Some physical parameters of grape juice are control factors for wine fermentation. The optimum ph for microorganisms is near to neutral point (ph 7.0). Yeasts are usually acid tolerant and it can grow well in ph range

Universities Research Journal 2011, Vol. 4, No. 3 159 of 4-4.5. To inhibit other microorganisms except the population of S. cerevisiae, ph of grape juice is one of the important factors. Specific gravity and brix (the total solid content, especially sugar) of grape juice should also be measured because grape juice with 22-24 brix is common for wine fermentation. So, some physical parameters of grape juice were measured and data obtained were given in Table 2. Table 2. Results of physicochemical investigation on grape juice of Vitis vinifera L. No. Parameter Grape Juice 1 Acidity 0.65 2 ph 4 3 Specific gravity 1.08 4 Brix 23º Phytoconstituents in the various parts of fruits (grape) and red wine made from grapes were described in Table 3. Table 3. Results of phytochemical tests on various parts of fruits and red wine of grape No Tests Extract Observations Pulp Skin Seed Red Wine 1 Alkaloids 1% HCl - - - - 2 Flavonoids 95% EtOH + + + + 3 4 Phenolic Compounds Terpenes & Terpenoids 5 Glycosides Distilled H 2 O + + + + 95% EtOH - - - - (1) Distilled H 2 O + + - + (2) 95%EtOH + + - +

160 Universities Research Journal 2011, Vol. 4, No. 3 No Tests Extract Observations Pulp Skin Seed Red Wine 6 Tannins Distilled H 2 O + + + + 7 Steroids 95% EtOH + + + - 8 Carbohydrate Distilled H 2 O + + + - 9 Saponins + = present - = absent (1) Distilled H 2 O + + - - (2) 95% EtOH + + - - The major constituents necessary for fermentation of grape juice to red wine are soluble sugars, monosaccharides mainly glucose, fructose and mannose. These sugars were determined qualitatively in the grape juice of Vitis vinifera and results obtained were stated in Tables 4 and 5. Table 4. General tests for detection of carbohydrates in grape juice No. Test Reagents Observations Inferences 1 Molisch Molisch's reagent & conc: H 2 SO 4 Purple colour ring at junction of two liquids Carbohydrates are present. 2 Fehling Fehling's A and B solution Brick red ppt Reducing sugars are present. 3 Benedict Benedict's reagent Reddish ppt Reducing sugars are present. 4 Barfoed Barfoed's reagent Orange to Red ppt Monosaccharide is present. 5 Moor 2% NaOH solution Yellow to Reddish brown colour Glucose is present.

Universities Research Journal 2011, Vol. 4, No. 3 161 No. Test Reagents Observations Inferences 6. Seliwanoff Seliwanoff's reagent Red to Orange colour & Faint orange colour (>7min) Fructose is present. 7. rapid furfural 1% α - naphthol & conc: HCl Purple colour Glucose is present. Table 5. Microchemical tests for types of monosaccharides (sugars) in grape juice (qualitative test) Test Observation Inference Osazone Test Needle shaped Yellow Osazone Crystals (within 5 min) Glucose, fructose and mannose are present Physical parameters measured at Qality Cntrol Department of Development Centre of Pharmaceutical Technology (DCPT) for red wine were described in Table 6. Table 6. Some physical parameters in red wine from grape Parameters Colour Description Solubility Wine Red (Red Wine) Miscible with water Refractive Index 1.347 Specific Gravity 0.9951 Clearity of solution satisfy From the results of phytoconstituents in red wine mentioned above in Table 3, it can be noticed that flavonoids, phenolic compounds, glycosides and tannins are all present in red wine.

162 Universities Research Journal 2011, Vol. 4, No. 3 Table 7. Results from detection of phenolic compounds in red wine No Types of Phenols 1. All Phenols Group tests 2. Flavin Derivatives (Anthocyanins) 3. Polyphenols (Tannin) Tests Observations Inferences H 2 SO 4 tests FeCl 3 test dil HCl test NaOH test 2% gelatin Saturated KIO 3 red Red Greenish blue colour Red colour Blue colour Curdy white ppt brown colour Phenol is present Anthocyanins are present Cyanidin & Malvidin are present Pelargonidin is present Cyanidin is present Tannin is present Tannin is present HNO 2 blue colour Ellagic tannin is present The results obtained from the detection of phenolic compounds in red wine were given in Table 7. From this results, it can be seen that flavin derivatives, anthocyanins and polyphenol,tannin are present in red wine fermented from grape (Vitis vinifera). Phenolic compounds, flavonoids, anthocyanin and anthocyanidins are all possess potential effects on human health which have already described in introduction of this research paper. So, these compounds are especially detected in red wine of grape for many medicinal values to prevent and treat such diseases as hypertension, diabetes, obesity, damage of blood vessel, damage of brain cells, leukemia, cardiovascular diseases, various types of cancers at breast, skin, stomach, colon, prostate, lung, age-related diseases. Bioactive constituents of grape possess bioactivities which can inhibit or against the population of six microorganisms inoculated in agarwell on plate. Results given by plate diffusion tests on red wine upon such microorganisms and their inhibition zones were given in Figure 3. From such results, it can be seen that wine has potent action on many diseases which mostly occurs in daily life around the world. Types of

Universities Research Journal 2011, Vol. 4, No. 3 163 microorganisms and their related diseases which can be affected by red wine were given in Table 8. 1 2 3 18mm(++) 17mm(++) 14mm(+) 4 15mm(++) 5 6 16mm(++) 15mm(++) Figure 3. Inhibitory zone of six microorganisms : (1) B. subtilis (2) S. aureus (3) P.aeruginosa (4) B. pumalis (5) C.albicans (6) E. coli on Agar-well Plate affected by red wine of grape Table 8. Bioactivity of red wine according to agar well plate diffusion test (Agar well 10 mm) Types of Microorganisms Gram Positive Bacteria Gram Negative Staphylococcus aureus Inhibitory Zone 17mm (++) Infection induced by such microorganisms Skin, wound, respiratory, urinary tract, bones and joints, pneumonia, carbuncle, food poison. Bacillus subtilis 18mm(++) Non- pathogenic. Bacillus pumalis 15mm(++) Eye, soft tissue. Pseudomonas aeruginosa 14mm(++) Skin, especially at burn sites, wounds,

164 Universities Research Journal 2011, Vol. 4, No. 3 Types of Microorganisms Bacteria E.coli Inhibitory Zone 15mm(++) Infection induced by such microorganisms pressure sore and ulcers, urinary tract. Urinary tract, wound, bed sore, dysentery, diarrhea, haemorrhagic, pyelitis, cystitis, appendix abscess, septic wounds, gastroenteritis, peritoritis. Fungi Candida albicans 16mm(++) mouth and skin, bloating vaginal, candidacies, poor digestion, fingernail and toenail, inflammation, invasion, vaginal mucosa. Conclusion Grape ( Vitis vinifera ) and 85% yield of red wine fermented from grape, cultivated in Tawma Village, Meiktila Township, Mandalay Region have good quality for commercial wine production. Flavonoids, phenolic compounds and tannins are present in the grape pulp, skins, seeds and red wine of grape juice. The occurrence of phytoconstituents and phenolic compounds including anthocyanin and resveratrol has great health benefits to prevent many diseases being encountered around the world. Especially, red wine possesses the bioactivities against some bacteria and fungi and their related diseases. So, grape and red wine made from grape cultivated in Myanmar should be consumed as medicinal foods as well as beverage of moderate drink daily.

Universities Research Journal 2011, Vol. 4, No. 3 165 Acknowledgements The author would like to express sincere and profound gratitude to Rector Dr. Maung Thinn and responsible personnel of Meiktila University for giving the opportunity to do this research programme. The author wishes to convey heartfelt gratitude to Professor Dr Chaw Khin, Head of Department, Department of Chemistry, Meiktila University for kindly allowing to make use of the research facilities and for her guidance and advice in this project. Special thanks are also due to the officials from DCPT, Yangon, especially Daw Myint Myint Lwin for their help to measure bioactivity and some physical parameters of grape-wine sample for this project. References Corder, R., Mullen, W., Khan, N.Q., Masks, S.C., Wood, E.G., Carrier, M.J, and Crozier, A. (2006), "Red Wine Procyanidins and Vascular Health", J-nature, 444, 566, Nov. Stephanie, D., Wollian, H., Peter, J. (2009), "Alcohol, Red Wine and Cardiovascular Disease", School of Dietetics and Human Nutrition, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada. Wang, N.S. (2004) "Wine Fermentation", Research Article, Dept. of Chemical Engineering, University of Maryland, College Park, MD 2074-211, EN CH 485.