Research Paper. Preeti Yadav 1, Neelima Garg 1 * and Deepa H Diwedi 2 1

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, Vol. 8(4), 2009, pp.406-418 Research Paper Introduction Mahua also known as Mowra Butter tree (Madhuca indica J. F. Gmel.

1 , Vol. 8(4), 2009, pp Research Paper Introduction Mahua also known as Mowra Butter tree (Madhuca indica J. F. Gmel.) is found in mixed deciduous forests of Central India, Maharashtra, Andhra Pradesh and Madhya Pradesh. It is also planted in the plains of northern India and Deccan Peninsula. It is one of the most valued trees among tribal communities of central India and its every part is used for various purposes 1. The succulent cream-coloured corollas when fall on the ground during March and April are collected and dried. These are a rich source of sugars and contain appreciable amounts of vitamins and calcium. Production of liquor from mahua flower is a traditional practice for centuries Effect of location of cultivar, fermentation temperature and additives on the physico-chemical and sensory qualities on Mahua (Madhuca indica J. F. Gmel.) wine preparation Preeti Yadav 1, Neelima Garg 1 * and Deepa H Diwedi 2 1 Central Institute for Subtropical Horticulture Rehmankhera, P.O. Kakori, Lucknow , Uttar Pradesh, India 2 Baba Saheb Bhim Rao Ambedkar University, Lucknow *Correspondent author, neelimagargg@rediffmail.com Received 12 April 2009; Accepted 11 June 2009 Abstract Mahua (Madhuca indica J. F. Gmel.) has been used for liquor production, for centuries, by the tribals and local people. However, wine from mahua flower is not common. Wine made from different mahua germplasm showed no significant difference in terms of biochemical and sensory quality. Wine fermented at 16 C had higher content of alcohol (9.9%) and ascorbic acid (0.9 mg) compared to that at 20 and 25 C. Nutrients in the form of yeast extract, added to the must, improved wine quality while tannin addition lowered its sensory quality. Sweetened wines scored better than dry wines. Lemon peel addition improved aroma and acceptability of these wines. HPLC analysis reflected the presence of phenolics, viz. gallic acid, chlorogenic acid, catechin, epicatechin, caffeic acid, 4-hydroxybenzaldehyde, ascorbic acid and tannic acids in mahua wines. Keywords: Mahua, Madhuca indica, Wine, Phenolic compounds, Alcohol, Sensory quality. IPC code: Int. cl. 8 C12G 1/00 However, the drink thus, prepared is of low quality and sometimes even health hazardous. Moreover, one disadvantage with liquor over wine is that former contains mostly alcohol and other non-volatile components of the fermenting substrate are destroyed during the process of alcohol distillation which is otherwise present in wine. With the government planning to showcase Wines of India across the globe, the Agricultural and Processed Food Products Export Development Authority (APEDA) is working out the strategy 3. Mahua, being an indigenous tree of high economic value has vast potential for good quality wine which may find extensive export market. As yet, no systematic report is available on preparation of good quality mahua flower wine, preparation of indigenous mahua liquor, fermentation was carried out under ambient temperature conditions. In northern India, the temperature during summer rises as high as 40 C, hence under such conditions if the wine is made, the quality of wine will be far inferior. Tannins are excellent antioxidant that helps in giving the structure, texture and flavour to wine. Wines that are fermented with the skins Mahua flowers

2 (such as red wines) usually contain enough natural tannin but for other wines (such as flower or fruit wines), tannin addition may be desirable. Similarly, mahua wine has strong mahua flavour and may need modification to make it more appealing. Present study documents the work on optimization of conditions for mahua wine preparation. The effect of location of the cultivar, fermentation temperature and additives on the physicochemical and sensory qualities of wine has been described in this paper. Materials and Methods Mahua flower collection In the month of April, mahua flowers from Rehmankhera area were collected in morning hours on polythene sheets (20 m 20 m) laid down under the trees, filled in clean polythene bag and brought to lab under hygienic conditions. Juice extraction Since, there was no reference available on juice extraction or wine preparation from mahua flower, the entire procedure was standardized by us. Mahua flowers were washed thoroughly under tap water. Diseased and damaged flowers were sorted out, crushed in a fruit mill and pressed with hydraulic press and the juice (from all the parts of the flower) was collected in clean stainless steel utensils. Yeast culture The yeast culture Saccharomyces cerevisiae St-2, used in the present investigation, was obtained from the culture collection of microbiology laboratory of CISH, Lucknow. The culture was maintained on Yeast Extract Peptone Dextrose (YEPD) Agar slants and was re-cultured every month. Preparation of wine In the present study, fresh mahua flower juice was used for wine preparation as per our preliminary work 4. Unless specified, the total soluble solids (TSS) of mahua juice were adjusted to 20 Brix with cane sugar granules. The acidity of the juice was maintained at 0.5% using citric acid. The juice was treated with 200 ppm KMS and inoculated with yeast maintained on YEPD slant and mass multiplied in YEPD broth. The inoculated juice was transferred into glass jars. Mouth of the jar was closed with cotton plugs which were replaced with fermentation locks after five days and kept for fermentation for 15 days (till TSS became constant) at room temperature (30±5 C). The wine was siphoned, aged for one month and then, bottled in 200ml capacity glass bottles, pasteurized at 70 C for 5 min and stored at room temperature. Effect of location of cultivar on quality of wine: Germplasm growing in following area surrounding Lucknow were screened for quality of mahua wine as per standard protocol described earlier. The germplasm growing in area surrounding Lucknow was divided in four regions, viz. Malihabad ( ), Kakori (T 1 ), Rehmankhera (T 3 ) and Bakshi-ka-talab (T 4 ) and collected flowers and wine made from these was compared. Effect of post-fermentation sweetening: To make wine more palatable, sweet wine was made by raising the sugar level of the wine by 5 and 10 B above the control level. Different treatments used were: C No adjustment of final TSS (Control); T 1 -Final TSS of mahua wine adjusted to 11 B; -Final TSS of mahua wine raised to 16 B. Standardization of fermentation temperature: Fermentation at ambient temperature conditions was compared to that of controlled conditions. The treatment included: C-Room temperature (30±5 C), T 1 = 16 C, = 20 C, T 3 = 25 C. Effect of tannin addition: To check it, tannin addition is desirable in mahua wines, treatments designed were: C-No tannin addition, T 1 -Addition of 0.15% tannic acid, -Addition of 0.3% tannic acid. Effect of yeast nutrient addition: Effect of addition of nutrients in the form of diammonium phosphate, potassium hydrogen phosphate and yeast extract was studied. The treatments were: C-No addition, T 1 -Addition of 5% diammonium phosphate, -Addition of 1% yeast extract, T 3 -Addition of 1% potassium hydrogen phosphate. Effect of herbal/plant extract additives: Herbal additives were used to improve its flavour. Various treatments were: T 1 -Control (No additive), -Addition of raw mango and mint extract (1%) during the course of fermentation, T 3 -Treatment with lemon slices (1%) along with peel during the Vol 8(4) July-August

3 course of fermentation, T 4 -Addition of cinnamon after mahua wine fermentation as per standard protocol. The water extracts were prepared by soaking the herb/plant part (10 g w/v) in water overnight. Physico-chemical and sensory analyses Biochemical analysis of mahua juice and wine was carried out for TSS, acidity, volatile acidity, ascorbic acid, tannins and reducing sugars as per methods of AOAC 5. Ethanol concentration in the fermented mahua wine was determined spectrophotometrically 6. The phenolics in mahua wine were analyzed by High Performance Liquid Chromatography 7,8. Microbiological quality of mahua wine was carried out as described by Speck 9. For judging the sensory attributes of the wine, sensory evaluation was conducted by a panel of seven semi-skilled judges 8. The attributes considered in the scoring were colour, clarity, nose/aroma, taste, tannin, astringency, freedom from acetic acid, sugar and impression. The overall final rating was obtained by calculating the average of the scores. Statistical analysis For all the experiments, three replicates were kept and the experiment was laid in two factor CRD design and the data were subjected to statistical analysis using statistical package for agricultural workers developed by O.P. Sheoran of CCSHAU, Hisar. Results and Discussion The biochemical analysis of mahua flower juice showed that it has TSS ( B) 13; acidity (%) 0.11; ascorbic acid (mg/100ml) 3.15, tannins (%) 0.11 and reducing sugar (%) 1.04 and it can be utilized for production of wine. The wines made from all the screened germplasm had no significant difference in terms of biochemical (Table 1) and sensory quality (Fig. 1). Therefore, the germplasm from Rehmankhera region was selected for further study on the ground of easy accessibility. To make it more palatable, sweet wine was made by raising the sugar level of the wine 5 and 10 B above the control level resulting in the different sugar acidity ratios, viz. 5 B : 0.6%; 11 B:0.6%; 16 B:0.6%. Table 1: Screening of local mahua germplasm* for wine preparation Parameters Storage Treatments* C.D. at P= 0.05 (Months) T 1 T 3 T 4 TSS ( B) Acidity (%) N.S Volatile acidity (%) N.S N.S Ascorbic acid (mg/100ml) Tannins (%) Alcohol (%) Non-enzymatic browning N.S N.S. N.S *T 1 - Kakori; - Malihabad; T 3 - Rehmankhera; T 4 - Bakshi-ka-Talab 408

Score/100 Score/100 T1 T2 T3 T4 55.75 55.7 55.65 55.6 55.55 55.5 55.45 0 time 6 months Storage s (Months) Fig.

2 : Effect of sugar acid ratio on sensory quality of mahua wine Table 2 presenting post fermentation sweetening revealed that except sweetness there was no significant difference in various

4 Score/100 Score/100 T1 T2 T3 T time 6 months Storage s (Months) Fig.1 : Sensory evaluation of mahua wine prepared from different germplasm Control T1 T Storage s (Months) Fig. 2 : Effect of sugar acid ratio on sensory quality of mahua wine Table 2 presenting post fermentation sweetening revealed that except sweetness there was no significant difference in various parameters of wines. During storage, the total and volatile acidity, ascorbic acid and tannins decreased while the reducing sugar content increased in all the treatments. However, during sensory evaluation, T 1 (11 B: 0.6%) was liked most (Fig. 2). This suggested that sugar acid blend in T 1 was better compared to the other two treatments. Fessler 10 have suggested a residual sugar level of 10-12% and acidity 0.4% in dessert wines. Vine 11 has recommended a sugar acid blend of 12 B and 0.5% acidity. Many of the world s great wines, such as those from Sauternes (including Barsac or Tokaj) have a high level of residual sugar which is carefully balanced with additional acidity to produce a harmonious blend. Table 3 reflects that wine fermented at 16 C had highest content of alcohol (9.9%) and ascorbic acid (0.9mg%). The same treatment scored higher organoleptic scores than others (Fig. 3). Temperature is perhaps the most critical factor influencing fermentation kinetics 12. Sener et al 13 observed that kinetic and yield parameters of wine fermentation were both temperature dependent. Sensory evaluation showed wine fermented at 18 C was better than others. Temperature as low as 13 C decreased both the fermentation and the growth rates 14. Vol 8(4) July-August

5 Table 2 : Effect of post-fermentation sweetening (sugar acid ratio) on mahua wine Parameters Storage Treatments* C.D. at P=0.05 (months) C T 1 TSS ( B) Acidity (%) Volatile acidity (%AA) Vitamin-C (mg/100ml) Tannins (%) N.S Reducing Sugar (%) Alcohol (%) N.S Non-enzymatic browning C - No adjustment of final TSS (Control); T 1 - Final TSS of mahua wine adjusted to 11 B; - Final TSS of mahua wine raised to 16 B; AA= Acetic acid 410

Score/100 100 90 80 70 60 50 40 30 20 10 0 Tannin addition reflected better retention of ascorbic acid, while alcohol content was lower (Table 4).

6 B), which reflects better conversion of sugar into alcohol. Least browning was observed in the control which further improved its acceptability.

6 Score/ Tannin addition reflected better retention of ascorbic acid, while alcohol content was lower (Table 4). Latter might be due to anti-microbial property of tannins. Higher alcohol (8.45%) in the control treatment is well supported by lower TSS (6.6 B), which reflects better conversion of sugar into alcohol. Least browning was observed in the control which further improved its acceptability. Sensory evaluation studies further confirmed that tannin addition is not required in mahua wines (Fig. 4). T 1 treatment (with lower tannin content) was liked better than (with higher tannin content). Modern wine makers take great care to minimize undesirable tannins from seeds by crushing grapes gently when extracting their juice, to avoid crushing the seeds 15. The sensory acceptability of all mahua wines including Control T1 T2 T Storage s (Months) Fig. 3 : Effect of fermentation temperature on sensory quality of mahua wine those treated with tannin improved after ageing. This is in concurrence with observation in case of red wine that should Score/ be aged and improved for perhaps three or more years. As the wine ages, the tannin softens and becomes less noticeable 16. Effect of addition of nutrients, viz. diammonium phosphate, potassium hydrogen phosphate and yeast extract revealed higher alcohol, ascorbic acid (Table 5) and better sensory acceptability (Fig. 5) in yeast extract treatment. This might be due to the fact that yeast extract might have provided a complex growth medium that best supports the growth of yeast 17. Osho 18 reported that ethanoltolerant yeast responds differently to different nutrients used for growth supplementation. The wine yeast NCYC 125 had its highest biomass concentration of 13.78g/l in medium supplemented with yeast extract, while strain Y271 had 13.49g/l in medium supplemented with urea. Other yeast strains Y276 and Y279 had 10.93g/l and 9.79g/l, respectively in Control T1 T Storage s (Months) Fig. 4 : Effect of tannin addition on sensory quality of mahua wine Vol 8(4) July-August

7 Table 3: Effect of fermentation temperature* on mahua wine Parameters Storage Treatments* C.D. at P=0.05 (Months) C T 1 T 3 TSS (0 B) Acidity (%) Volatile acidity (%) Ascorbic acid (mg/100ml) Tannins (%) Reducing sugar (%) Alcohol (%) Non-enzymatic browning *T 1-16 C; - 20 C; T 3-25 C 412

8 Table 4: Effect of tannin addition* on biochemical quality of mahua wine Parameters Storage Treatments C.D. at P=0.05 (months) C T 1 TSS ( B) Acidity (%) Volatile acidity (%) Vitamin-C (mg/100ml) Tannins (%) N.S Reducing Sugar (%) N.S Alcohol (%) Non-enzymatic browning * C No tannin addition; T 1 Addition of 0.15 % tannic acid; Addition of 0.3 % tannic acid Vol 8(4) July-August

9 Table 5: Effect of yeast nutrient* addition on quality of mahua wine Parameters Storage Treatments* C.D. at P=0.05 (Months) C T 1 T 3 TSS ( B) Acidity (%) Volatile acidity (%) Ascorbic acid (mg/100ml) Tannins (%) N.S. N.S. N.S Reducing sugar (%) Alcohol (%) Non-enzymatic browning *C No addition; T 1 Addition of 5% Di-ammonium phosphate; Addition of 1% Yeast extract; T 3 Addition of 1% Potassium hydrogen phosphate 414

Score/100 Score/100 90 80 70 60 50 40 30 20 10 0 Fig.

11. These results have clearly shown improvement in aroma and acceptability of herbs (lemon, cinnamon, raw mango and mint) treated mahua flower wines Control T1 T2 T3 3 6 9 12 Storage s (Months) T1

10 Score/100 Score/ Fig. 5 : Effect of yeast nutrient addition on sensory scores of mahua wine during storage media supplemented with combination of all nutrients used for supplementation 11. These results have clearly shown improvement in aroma and acceptability of herbs (lemon, cinnamon, raw mango and mint) treated mahua flower wines Control T1 T2 T Storage s (Months) T1 T2 T3 T Storage s (Months) Fig. 6 : Effect of additives on sensory quality of mahua wine over the control (Table 6 and Fig. 6). However, lemon treated wine was found superior to cinnamon treated wine. Raw mango-mint treated wine scored least. Lemon is used for flavour improvement of burnt rice and vegetables. Citronellal, geranial, 1,8-cineole are the major flavour producing compounds of lemon 19. HPLC analysis reflected the presence of phenolics, viz. gallic acid, chlorogenic acid, catechin, epicatechin, caffeic acid, 4-hydroxybenzaldehyde, ascorbic acid and tannic acid in mahua wine. Lemon treatment of wine resulted in variation in phenolics pattern and quantity. Goldberg et al 20 have reported that the concentrations of (+)-catechin and (-)-epicatechin were highest in red Burgundy and Canadian wines Arts et al 21 determined the levels of (+)-catechin, (- )-epicatechin, (+)-gallocatechin, (-)- epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate in 8 types of black tea, 18 types of red and white wines, apple juice, grape juice, iced tea, beer, chocolate milk, and coffee using HPLC. All the wines were subjected to microbiological analysis during storage and no microbial growth could be observed up to 12 months of storage in any of the samples. Conclusion It is concluded from the study that mahua flower has potential as substrate for making good quality wine, irrespective of germplasm. Wine sweetened to a final TSS of 11 B with 0.5% acidity were found most acceptable. A fermentation temperature of 16 C was optimum for better quality wines. Tannin addition is not required for yeast extract addition is desirable for better quality mahua wine. Lemon peel addition improved the flavour of mahua wine. Fermentation enriches mahua wine with Vol 8(4) July-August

11 Table 6 : Effect of additive* on flavour improvement of mahua wine Parameters Storage Treatments C.D. at P=0.05 (Months) T 1 T 3 T 4 TSS ( B) Acidity (%) Volatile acidity (%) Ascorbic acid (mg/100ml) Tannins (%) N.S. N.S. N.S Reducing sugar (%)

12 Parameters Storage Treatments C.D. at P=0.05 (Months) T 1 T 3 T 4 Alcohol (%) Non-enzymatic browning *T 1 Control (No additive); Raw mango and mint extract (1%); T 3 Lemon slices along with peel (1%); T 4 Cinnamon extract (1%) phenolics, viz. gallic acid, chlorogenic acid, catechin, epicatechin, caffeic acid, 4-hydroxybenzaldehyde, ascorbic acid and tannic acid. References 1. The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products Raw Materials Series, Publications and Information Directorate, Council of Scientific & Industrial Research, New Delhi, Vol. VI, 1962, Mande BA, Andreasen AA, Sreenivasaya M and Kolachov P, Fermentation of Bassia Flowers, Industr Eng Chem, 1949, 41, Baisya RK, India Fast Emerging as a Major Market for Wine, Pocess Food Ind, 2006, 9(3), Yadav P, Garg N and Diwedi DH, Standardization of pre-treatment conditions for mahua wine preparation, J Ecofriendly Agric, 2009, 4(1), Association of Official Analytical Chemists (AOAC), Official Methods of Analysis, Washington, D.C., U.S.A., Caputi AJ, Ueda M and Brown T, Spectrophotometric determination of ethanol of wine, Am J Enol Viti, 1968, 19, Basha SM, Musingo M and Colova VS, Compositional differences in the phenolics compounds of muscadine and bunch grape wine, Afr J Biotechnol, 2004, 3(10), Amerine MA, Pangborn RM and Roessler EB, Principles of sensory evaluation of food, Academic press, New York, 1965, p Speck M, Compendium of methods for the microbiological examinations of foods, 2 nd edition, American Public Health Association, Inc., 1985, pp Fessler JH, Guideline to practical wine making, P.O. Box 5276, Elmwood Station, Berkelery, CA, 1971, p Vine RP, Commercial Wine making, AVI Publishing Co Inc., Westport, Connecticut, 1981, pp Coleman MC, Fish R and Block DE, Temperature-dependent kinetic model for nitrogen-limited wine fermentations, Applied Envir Microbiol, 2007, 73(18), Sener A, Canbas A and Unal MU, The effect of fermentation temperature on the growth kinetics of wine yeast species, Turkish J Agric For Sci, 2007, 31(5), Beltran G, Rozas N, Mas A and Guillamom J, Effect of low temperature fermentation on yeast nitrogen metabolism, World J Microbiol Biotechnol, 2007, 23(6), Grenier P, Pressing techniques and automation of the pressing cycle, Semana Vitivinicola, 2001, 56, Bissell P, The role of tannins in Merlot varietals Vol 8(4) July-August

13 and blends, Austr New Zeal Wine Ind J, 2002, 17(4), 24, Jackson RS, Wine Science: Principles and Applications, Academic Press, 1994, pp Osho A, Ethanol and sugar tolerance of wine yeasts isolated from fermenting cashew apple juice, Afr J Biotechnol, 2005, 4(7), Sawada M and Yamada T, The analysis of oxygenated compounds fraction of lemon oils prepared by different extraction methods and application to a beverage, Nipp Shok Kag Kog Kaishi, 1998, 45(2) Goldberg DM, Karumanchiri A, Sang T and Soleas GJ, Catechin and epicatechin concentrations of red wines: Regional and Cultivar-Related Differences, Am J Enol Vitic, 1998, 49(1), Arts ICW, Putte B and Hollman PCH, Catechin contents of food commonly consumed in the Netherlands, 2. Tea, Wine, Fruit Juices and Chocolate Milk, J Agric Food Chem, 2000, 48(5),

PRODUCTION OF ETHANOL FROM MAHUA FLOWER (MADHUCA LATIFOLIA L.) USING SACCHAROMYCES CEREVISIAE 3044 AND STUDY OF PARAMETERS WHILE FERMENTATION

PRODUCTION OF ETHANOL FROM MAHUA FLOWER (MADHUCA LATIFOLIA L.) USING SACCHAROMYCES CEREVISIAE 3044 AND STUDY OF PARAMETERS WHILE FERMENTATION Pranav Mandal 1 and Niren Kathale 2 1 Contributory Lecturer,