OPTIMUM PARAMETERS FOR WINE PRODUCTION FROM POMEGRANATE FRUIT JUICE

OPTIMUM PARAMETERS FOR WINE PRODUCTION FROM POMEGRANATE FRUIT JUICE

Samson, Ashok Kumar Singh and Gauri Singh^*

Department of Microbiology, Dolphin (PG) Institute of Biomedical & Natural Sciences, Dehradun- 248007, Uttarakhand, India.

ABSTRACT: Pomegranate fruits are a very rich source of antioxidants, Vitamin B5 (pantothenic acid), potassium, flavonoids and have numerous health benefits. It may help decrease the risk of having a heart disease, heart attacks, and strokes. These characteristics of pomegranate have great impact on the quality of wines. So there might be a scope for large scale manufacturing of the pomegranate wine as it has many health benefits than grape wine Thus, the present study is aimed at investigating the fermentation parameters of pomegranate wine.  Fermentation of pomegranate was carried out by a yeast strain. The present study provides information on the parameters like inoculum size, pH, incubation temperature, substrate concentration and incubation period involved during wine production. These parameters are the part and parcel of wine production, without the knowledge of these parameters one cannot make a tasty and good quality wine. We see a significant variation between residual sugar % (w/v) & alcohol % by applying ANOVA for all parameters. Analysis reveals that best wine from pomegranate can be produced at an inoculum size of 8% v/v and pH 4.0 at the incubation temperature of 37 °C. Further, it can be defined better if we incubated neither less nor more than 7 days. Under these conditions, alcohol content of the wine reached to 10.1%.  This study will provide a good reference for future industrial production of pomegranate fruit wine.

Alcohol, Fermentation, Residual Sugar, Pomegranate wine

INTRODUCTION: Wine drinking has been a part of our lives for many centuries. Wine brings pleasure to the person who would drink it. It is incontestable that wine has an innate taste that can lift one´s spirit. Now a days, wine is not consumed only for pleasure. Many people make wine drinking a part of their routine because of its health benefits. Wine is packed with anti-oxidants that help fight different types of diseases and delay the signs of aging.

Wine is an alcoholic drink made from fermented fruit juice. Generally, fruits contain quantities of sugar that can be used by yeast during the fermentation process. In addition to the inherent characteristics of fruit (pH values, sugar contents and nitrogen contents), other factors must be taken into account during fruit wine production. The initial sugar concentrations, fermentation temperatures, SO₂ concentrations and specific yeast strains are key factors in determining successful fermentative processes of fruit wine ¹.

India is the second largest producing country of pomegranates. In the current study, pomegranate fruit was used to develop wine by fermentation. Pomegranate has long been regarded as a promising diet source of phytochemicals, including ellagitannin, flavonoids, anthocyanins ² which possess several health promoting characters, like maintaining redox balance of internal environment, prevention and treatment of cardiovascular disease, diabetes, cancers, Alzheimer`s ³. It is an abundant source of various Vitamins. Pomegranate is a fine, natural source of Vitamins A, C and E. It also contains folic acid. Pomegranate wine as an emerging beverage not only offers an alternative to employ underused pomegranate fruits ⁴, but also provide multiple beneficial effects on health management ⁵.

The soluble polyphenolic content of pomegranate juice (0.2 to 1.0%) includes anthocyanins, catechins, tannins, and gallic and ellagic acids ⁶. The pomegranate wine has up to 3 times more antioxidants than red wine! Pomegranate wine has greater protection capacity than red wine on low-density lipoprotein oxidation ⁷.

An  efficient  ethanol  production  requires  four  components: fermentable carbohydrates, an efficient   yeast   strain,   a   few  nutrients  and  simple  culture  conditions.  The yeast cell contains enzyme catalysts that provide an energetically   favourable pathway for the reaction. At the moment, most of the wine production processes are relying on Saccharomyces cerevisiae strains that allow rapid and reliable fermentations, reduce the risk of sluggish or stuck fermentations and prevent microbial contaminations ⁸. Yeast starter cultures that are specifically selected for the winemaking process on the basis of scientifically verified characteristics typically complement and optimize the raw material quality and individual characteristics of the wine, producing a more desirable product ⁹.

We defined factors namely inoculum size, temperature, pH, substrate concentration and incubation period to achieve the optimum fermentation condition for pomegranate wine.

MATERIALS AND METHODS:

Culturing of yeast: The yeast strain Saccharomyces cerevisiae (MTCC-36) was obtained from IMTECH Chandigarh and taken as model strain. The cultures were kept in refrigerator until used.

Preparation of inoculums: The yeast strain was inoculated in the glucose yeast extract broth .The inoculated broth was incubated at 37 °C in orbital shaker for 48hr. After 48hr the count was done using haemocytometer to check the optimum number of yeast cells. The broth with spore count 3 x 10⁸ was taken.

Preparation of pomegranate juice: The fresh pomegranate fruits were collected from the fruit vendor shop Premnagar, Dehradun. The juicy arils were separated from fresh fruits with the help of stainless steel knife. The arils were crushed to juice in fruit mixer. 700 ml of juice was collected from 2 kg of pomegranate fruit.

The juices were transferred into five 250ml conical flask with each conical flask containing 100ml of juice. The inoculum was added to juice and allowed for fermentation.

Optimization of culture parameters:

Inoculum size: Keeping all the parameters constant the inoculums ((Saccharomyces cerevisiae) of different size i.e. 2%, 4%, 6%, 8% and 10% (v/v) were added into the juice and incubated for 7 days.

pH: The pomegranate juices were incubated with taking optimized inoculum size but at different pH viz; 3.0, 3.5, 4.0, 4.5, 5.0 for 7 days.

Temperature: The pomegranate juices were incubated with taking optimized inoculum size and pH but at different temperature viz; 25°C, 27°C, 30°C and 37°C for 7 days.

Substrate concentration: The pomegranate juices were incubated with taking optimized inoculum size, pH and temperature but with different substrate concentration viz; 20%, 40%, 60%, 80%, 100% (v/v). After 7 days the substrate concentration was optimized.

Incubation period: The pomegranate juices were incubated with taking optimized inoculum size, pH, temperature and substrate concentration but for different incubation period (5 days, 10 days and 15 days). After 15 days the incubation period was optimized. Glucose yeast extract broth was taken as control for optimization of above parameters.

Estimation of residual sugar content: The residual sugar content of the fermented juice was estimated by the DNSA method ¹⁰.

Estimation of Alcohol content: The Alcohol content of the fermented juice was estimated by Dichromate Titration method ¹¹.

Statistical Analysis: Data analysis was done using ANOVA.

RESULTS AND DISCUSSION: The quality of wine can be characterized by its ethanol content which is the chief component found in all types of wine ¹². It is worthy to note that final sugar concentration has inverse relationship with ethanol concentration. Therefore, in the current study pomegranate juice were optimized for wine production for above factors. The different parameters were studied after 7 days of Incubation. We let a null hypothesis between residual sugar % & alcohol % for statistical analysis

Inoculum size: The optimization of inoculum size is important as sugar consumption is a balance between biomass development and ethanol production and a high inoculum size will thus be a compromise on amount of ethanol produced. The pomegranate juice were incubated with different inoculums size viz; 2, 4, 6, 8 and 10%. We observed that ethanol production increase with increase in inoculums size up to 8% and decreases beyond this level while reverse were found in reducing sugar concentration (Table 1). It was, therefore, concluded that 8% inoculum is the optimum and selected for further studies.

TABLE 1: OPTIMIZATION OF INOCULUM SIZE FOR ALCOHOL PRODUCTION AND RESIDUAL SUGAR

When we take Inoculum % as a single factor and apply ANOVA to find out whether there is any variation, we see a significant variation between  residual sugar  % (w/v) & alcohol  % (p-value=0.022386<0.05) and (C_alculated =7.969947 > C_ritical=5.317655), which rejects the null hypothesis. The outcome of this study was almost similar to that of other studies v/v. Similar trends have also been reported by Singh and Kaur (2009) ¹³. Where they observed 10% (v/v) as optimized inoculum level for litchi wine production. An optimized inoculum level of 10% v/v for alcoholic fermentation of jamun, plum, apple, pear juice, guava and 7.5% inoculum size for kinnow wine production has been observed in other research reports.¹⁴

Effect of pH: The pH of juice is important parameter for the successful progress of fermentation. Control of pH during wine fermentation is important for two reasons. The growth of bacteria is retarded by acidic solution and yeast grows well in acidic conditions ¹⁵. So, different pH ranges from 3.0 to 5.0 was tested It has been observed in pomegranate wine, alcohol production significantly increase with increase in pH up to 4.0, and decrease beyond that pH level 4.0. (Table 2).

TABLE 2: OPTIMIZATION OF PH FOR MAXIMUM ALCOHOL PRODUCTION AND RESIDUAL SUGAR

There is a significant variation between residual sugar % (w/v) & alcohol % (p-value= 0.000121 <0.05) and (C_alculated =47.972357 > C_ritical= 5.317 655) in case of pH. With increase in pH the ethanol content was reduced gradually, because yeast produce acid rather than alcohol with increase in pH ¹⁶. The maximum alcohol production was found to be at pH 4.0 with alcohol content 9.8% v/v. However prior study on screening of pomegranate for wine production. Matapathi et al., 2004 ¹⁷. was found to be at pH 2.9-3.4 with maximum alcohol content 12.9% v/v.

Effect of Temperature: Temperature can affect the sensitivity of yeasts to alcohol concentration, growth rate, rate of fermentation, viability, length of lag phase, enzyme and membrane function, etc. It has been observed that alcohol production was high at higher temperature.(Table 3) When we take Temperature as a single factor and apply ANOVA to find out whether there is any variation, there is a significant variation between  residual sugar  % (w/v) & alcohol %   (p-value=0.334254>0.05) and (C_alculated  = 1.102027 < C_ritical=5.987378),

TABLE 3: OPTIMIZATION OF TEMPERATURE FOR MAXIMUM ALCOHOL PRODUCTION AND RESIDUAL SUGAR

As the temperature increases initial fermentation rate are increased due to the enzyme activity of the metabolic pathway. The current study showed the same optimum incubation temperature (37 °C) for maximum wine production as made by Torija, et al., 2001¹⁸.

Effect of substrate concentration: Optimum substrate concentration is important for maximum alcohol production during fermentation. Substrate concentration is always related to enzyme activity of yeast which ferments the sugar content of substrate to alcohol. The pomegranate juice was adjust to different substrate concentration i.e. 20%, 40%, 60%, 80% and 100%. It was observed highest production of alcohol takes place in a 100% substrate (Table 4).  Jadhav and Darikpar (2010) ¹⁹ found that maximum ethanol was produced at 40% concentration Ficus glomerata fruit.

When we take substrate concentration as a single factor, we see a significant variation between residual sugar % (w/v) & alcohol % (p-value = 0.21245 > 0.05) and (C_alculated= 1.835902 < C_ritical = 5.317655.

Effect of Incubation Period: Incubation period is very important in wine production as it determines the ability of yeast sample to degrade the sugar content in the juice. The pomegranate juice was incubated for different periods of time viz: 3, 5, 7, 10, and 14 days respectively (Table 5).

TABLE 4: DIFFERENT SUBSTRATE CONCENTRATION AND THEIR RESPECTIVE SUGAR AND ALCOHOL CONTENT

TABLE 5: DIFFERENT INCUBATION PERIOD AND THEIR RESPECTIVE SUGAR AND ALCOHOL CONTENT

There is a significant variation between residual sugar % (w/v) & alcohol % (p-value = 0.000199 <0.05) and (C_alculated=41.54344 > C_ritical=5.317655) with respect to incubation period. The incubation period for the maximum alcohol production was found to be 7 days in current study, however ^{20, 21} revealed that optimum incubation period was 10-14 days.

Similar work has been done by other workers by using different substrates  such as Pooja and Kocher (2017) ²² Optimized  the parameters on grape juice of Punjab MACS purple and H-144 cultivars that lead to 12.0 and 11.2 (%v/v) ethanol production, respectively. The results found in the work of Chakraborty et al., (2017) ²³ showed that the optimum conditions for production of wine from household wastes of vegetable peels having maximum yield 6.69% ethanol are temperature 32°C, pH 5.5 with incubation time of 2 days.

CONCLUSION: The results showed that the optimal condition for pomegranate fermentation was defined as inoculum size 8%, pH 4.0, temperature 37 °C, incubation period 7 days and 100% substrate concentration. Under these conditions, ethanol from the juice could be achieved reaching up to 10.1% (v/v), This study will provide a good reference for future industrial production of  pomegranate fruit wine.

ACKNOWLEDGEMENT: The authors would like to thank to Mr. Vijay Ratan Pawar (Department of Biostatsitics Dolphin (PG) Institute of Biomedical & Natural Sciences, Dehradun, India) for statistical analysis.

CONFLICT OF INTEREST: We declare that we have no conflict of interest.

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    How to cite this article:

    Samson, Singh AK and Singh G: Optimum parameters for wine production from pomegranate fruit juice. Int J Pharm Sci Res 2017; 8(11): 4826-31.doi: 10.13040/IJPSR.0975-8232.8(11).4826-31.

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