Indian Journal of Traditional Knowledge Vol. 15(2), April 2016, pp. 254-259 Influence of different fermentation conditions on the formulation and development of Amla (Emblica officinalis Gaertin.) wine Vaishali Punjahari Argade* & Vishal Vijay Pande Department of Pharmaceutics and Quality Assurance, Sanjivani College of Pharmaceutical Education and Research, Kopargaon, District Ahmadnagar-414001, MS E-mails: [email protected]; [email protected] Received 23 February 2015, revised 14 October 2015 Amla wine is one of the most acidic alcoholic beverages prepared by using various fermentation conditions such as fermenting agent, temperature, duration, juice concentration variation or by addition of jaggery. In this present study, Amla wine was analyzed for titrable acidity, volatile acidity, free SO2 content, total soluble solids, pH, reducing sugar, alcohol content by GC, ascorbic acid content, total phenolic content, total flavonoids content, etc. The marker constituent in Amla wine was identified by TLC, HPTLC, IR and antioxidant activity was determined using DPPH free radical scavenging assay. The influence of the yeast (Saccharomyces cerevisiae) on self generated alcohol (SGA) content as ethanol was observed in range of 1.20-2.40%. Whereas by using flowers of Woodfordia fruticosa (L.) Kurz as the source of fermenting yeast, ethanol reached up to 1.0-1.70%. Feeding of excess jaggery in the formulation increases the ethanol content up to 8.69% and 9.29%. The trace amount of SGA like n-butanol, n-propanol, iso-butanol and methanol were also determined. From this study, it can be concluded that phenolics, flavonoids and vitamin C were found abundant in the Amla wine, which may help in protecting the biological system against the free radicals and make immune system strong. Keywords: Amla wine, Fermentation conditions, Self generated alcohol, Ethanol IPC Int. Cl.8: C12G 1/00, C12C 11/00, C12Q, C12N 1/16-C12N 1/18 The Ayurvedic system of medicine comprises of the very popular dosage forms such as Asava (fermented infusion) and Arishta (fermented decoction), which contain the self generated alcohol (SGA) which is prepared from natural products (fruits, flowers, root, leaf and others)1. Amla is known as Indian gooseberry (Embilica officinalis Gaertin.; Family- Euphorbiaceae)2,3. It is reported that Amla possesses greater antioxidant, antibacterial, antimutagenic, anti-ulcer, antitumour, anti-aging, hepatoprotective activities and also useful in giving relief from cough, bronchitis haemoptysis, tuberculosis and scurvy2,3,4. Wine is recognized to be potentially beneficial in anti-aging, improving lung functions, antioxidants effect, reduction of coronary heart disease, decreasing cancer risk in women and make the bone stronger5. Moderate consumption of wine lower the risk of type-2 diabetes, heart attack, artery damage, Alzheimer’s disease and decrease in the level of cholesterol (LDL). Percent of ethanol present in wines is about 7-16%6. —————— *Corresponding author In the present study fermentation process is carried out using two types of fermenter, i.e., yeast Saccharomyces cerevisiae (Family-Saccharomycetaceae) and flowers of plant Woodfordia fruticosa (L.) Kurz (Family-Lytheraceae) without any source of nutrients. Flowers are used as source of yeast, from these six-types of yeast; cultures are isolated and identified them as a S. cerevisiae and Rhodutorula mucilaginosa 7. Methodology Collection and authentification of the drug and fermenter Fresh Amla fruits were collected from the local market of Kopargaon, District Ahmadnagar, MS, India and the flowers of plant Woodfordia fruticosa (L.) Kurz (Dhataki) were collected from Mahatma Phule Krushi Vidyapeeth, Rahuri in June 2013, District Ahmadnagar, India. Its authentification was carried out at Department of Botany, S S G M College Kopargaon, District Ahmadnagar, India. The standard culture of yeast S. cerevisiae was collected from Sanjivani sugar factory Sahajanandnagar Kopargaon, India and stored at 4 0C in refrigerator. ARGADE & PANDE: INFLUENCE OF DIFFERENT FERMENTATION CONDITIONS ON FORMULATION OF AMLA WINE Physicochemical examination of Amla fruits The physicochemical examination and preliminary phytochemical screening of Amla fruit powder was carried out as per WHO guidelines2,8,9. Preparation of Amla juice (AJ) and yeast culture Collected amla fruits were washed with water. Amla seeds were separated from the amla fruits and juice as extracted by crushing and pressing the fruits pulp with cotton cloths. The 10% w/v yeast of S. cerevisiae was used as a starter culture. Formulation of amla wine Amla wine was formulated by using two types of fermenter, i.e., yeast S. cerevisiae and flowers of Woodfordia fruticosa (L.) Kurz. Formulations were prepared as stated in Table 1, labeled them accordingly and set for the desired fermentation period. Amongst these we selected the A1, Aa formulation for evaluation. Amla wine analysis Total Soluble Solids (TSS) content was ideally estimated using Abbey’s Refractometer. It is also expressed as 0Brix 12 (0Brix = 1 gm sucrose /100 gm juice) and pH was determined using digital pH meter. Titrable acidity (TA) and Volatile acidity (VA) was resolved by using acid base titration10. Free-SO2 determination was carried out by using Acid base titration11. Vitamin C determination was carried by using Redox titration12. Reducing sugar determination (RS) was determined by using Nelson-Somogyi method13. Total Phenolic Content (TPC) was measured using Folin ciocalteu assay14,15. Total Flavonoid Content (TFC) was determined using Aluminium chloride colorimetric assay14,15. Antioxidant activity was measured using DPPH free radical scavenging assay16,17. GC analysis This study used the Thermo Scientific Chemito GC-1000 along with the Flame ionization detector and SGP BP624 GC Capillary column (30m length, 3mm thickness). Wine sample of 0.5µL was directly injected in to GC with help of syringe and the separation was carried out with the help of FID. Nitrogen was used as a carrier gas at constant flow rate of 3ml/min with 10 or 15min sample running time. 255 HPTLC analysis Qualitative analysis of Aa formulation was done by using High Performance Thin Layer Chromatography (HPTLC). Sample was spotted on thin layer of adsorbent, i.e., silica gel 60 F254 aluminium plate (10×10cm) using camag Linomat 5 applicator and developed by using reported Ethyl acetate: Glacial acetic acid: Formic acid: Water (6: 1: 1: 2v/v/v/v) mobile phase for 30 min and scanning was performed on Camag TLC scanner at 254nm wavelength18. FT-IR analysis A drop of sample A1 and Aa was squeezed in between the two sodium chloride plates (0.1-0.3mm thin), it’s infrared spectra was recorded at 4000400cm-1 using Fourier transform infra-red spectrophotometer ( Model . 8400S Shimadzu) Results and discussion The preliminary phytochemical analysis showed the presence of carbohydrates, glycosides, steroids, alkaloids, flavonoids, vitamin C, tannins or phenolics in amla fruits. Amla wine thus produced had brownish color and clear appearance with alcoholic odor. Fermentation was affected by several factors such as pH, temperature, nature and composition of medium, agitation rate, sugar concentration, dissolved oxygen and CO2. During fermentation the yeast S. cerevisiae contained the enzyme invertase which was responsible for hydrolysis of sucrose to glucose and fructose. Enzyme zymase further converted glucose to alcohol and carbon dioxide19,20. The evaluation parameters of AJ, A1 and Aa formulation of wine is shown below in Table 2. Wine acids are very useful components of wine which in greater quantity determine the quality of wine. They are also responsible for sour tasting and act as preservatives21. The TA in terms of tartaric acid in AJ was 1.95% is further increased up to 2.1% and 2.88% in A1 and Aa formulations respectively (Table 2). Acetic acid is responsible for the volatile acidity found in the wine during fermentation, which further produces pungent wine. Wine should have a pH in between 2.9- 4.0, pH above 4 indicates that the wine may spoiled quickly and become chemically unstable22. The pH value of AJ was 4, after fermentation it gradually decrease to 3.4 and 3 in A1 and Aa formulations respectively. From this it can be confirmed that, the increasing TA is directly proportional to decreasing pH. The INDIAN J TRADIT KNOWLE, VOL. 15, NO 2, APRIL 2016 256 Table 1 — Effect of fermentation period and temperature on formulation of Amla wine Sl. No. Group Fermentation conditions Formulation Label Fermentation period(months) Fermentation temperature (0C) 1. A Fermenter change 100ml AJ +10gm F 100ml AJ +10ml Y A1 Aa 3 3 30-35 2. B Temperature change C Duration B1 Ba C1 C2 C3 Ca Cb Cc D1 D2 D3 Da Db Dc E1 E2 Ea Eb 3 3 1 2 3 1 2 3 3 3 3 3 3 3 3 4 3 4 3-4 3. 100ml AJ +10gm F 100ml AJ +10ml Y 100ml AJ +10gm F 100ml AJ +10ml Y 4. D Juice concentration Variation 50ml AJ +10gm F 75ml AJ +10gm F 100ml AJ +10gm F 50ml AJ +10ml Y 75ml AJ +10ml Y 100ml AJ +10ml Y 5. E Addition of 100ml AJ +10gm F+5gm J Jaggery (J) 100ml AJ +10gm F+15gm J 100ml AJ +10ml Y+5gm J 100ml AJ +10ml Y+15gm J AJ - Amla juices; F – Flowers; Y – Yeast; J - Jaggery Table 2—Evaluation parameters of AJ and A1, Aa formulations Components AJ A1 TSS (0Brix) 3.9 0.5 TA (%) 1.95 2.1 VA (gm/100ml) 0.03 0.057 4 3.4 pH Free SO2 (mg/kg) ND 960 Specific gravity (gm/ml) 1.10 0.95 Viscosity (cp) 0.80 0.45 TPC (gm/100ml) 1.4 1.2 TFC (gm/100ml) 0.96 0.9 Ethanol (%) ND 8.69 DPPH (µg/ml) 7.5 8.5 Reducing sugar (gm/100ml) ND 0.02 ND – Not detected; AJ – Amla Juice; A1 and Aa – formulations Aa 0.2 2.88 0.09 3 1600 0.81 0.38 0.9 0.85 9.29 8 0.025 Amla wine decreasing total soluble solids (TSS) in A1 and Aa formulation as compared with AJ is indicates the utilization of sugar by the wine yeast to produce ethanol19. Free SO2 protecting the wine phenols from oxidation, also helps to reduced the unwanted fermentation. AA of 5.6 gm/L concentration is a powerful antioxidant present in AJ whose content decreased to 3.96gm/L and 4.06gm/L in A1 and Aa formulations but still it is present in appreciable amounts. A phenolic substance provides the taste, color, odor and thickness to the wine and also acts as preservative. Flavonoid typically comprises 30-35 30-35 30-35 approximately 50% of the phenolic content7. In vitro antioxidant activity, the IC50% value for AJ, A1 and Aa formulation were found to be 7.5µg/ml, 8.5µg/ml, 8µg/ml which was required to obtain 50% of the DPPH degradation. From the above results it can be concluded that, the IC50% value of the formulation is much closed to the IC50% value of the standard AA. So the formulation may have prominent and significant antioxidant activity. The fermentation of the wine increases the antimicrobial potential along with the growth of probiotic lactic acid bacillus (LAB) and yeast23. Wine has a long association with human artistic cultural and religious activity and it is considered as nutritious, safe and healthy drink, besides an important adjunct to the diet unlike distilled liquor24. GC analysis The presence of (%) SGA as ethanol in Amla wine was determined by using GC and compare it with 99.9% absolute ethanol which is served as a standard (Fig. 1A). The percentage of ethanol in A1 and Aa formulation were found to be 1.62% and 2.40%, respectively. In various formulations it is 0.59% (Ba) and 1.61% (B1), 0.72% (C1), 1.23% (Ca), 0.30% (C2), 0.68% (Cb) and 1.42% (C3) and 2.38% (Cc), 0.53% (D1), 0.76% (Da), 0.97% (D2), 1.25% (Db). By addition of 5g of jaggery, % ethanol was found to be ARGADE & PANDE: INFLUENCE OF DIFFERENT FERMENTATION CONDITIONS ON FORMULATION OF AMLA WINE 257 Fig.2—TLC of (A)- A1 Formulation, (B)- Aa Formulation, (C)- Standard AA, (D)- HPTLC chromatogram of Aa formulation, (E)- HPTLC chromatogram of standard AA Fig.1—(A)-Gas Chromatogram of Absolute ethanol (99.99%), (B)-Gas Chromatogram of E2 formulation, (C)-Gas Chromatogram of Eb formulation 2.78% (E1) and 3.34% (Ea) which was further improved to 8.69% (E2) and 9.29% (Eb) in the respective (Figs. 1B, C) formulations. From the results of Gas chromatogram it can be concluded that the self generated ethanol is more in the yeast (S. cerevisiae) fermentation compared to the Dhataki flowers [Woodfordia friticosa (L.) Kurz]. The percent of ethanol production was increased with increasing concentration of Amla juice and the ethanol content was observed more in the jaggery formulations. HPTLC profile The presence of Ascorbic acid in formulation A1 (Fig. 2A) and AA (Fig. 2B) was confirmed by TLC against reference standard Ascorbic acid (Fig. 2C) by using Ethyl acetate: GAA: Formic acid: Water (6:1:1:2v/v/v/v) as mobile phase with the Rf value 0.45. The HPTLC technique was used to determine the Rf value of Aa & AA and the formulation was found to be 0.48 with the peak area 1836.9 (Fig. 2D) and 0.42 with the peak area 3305.6 (Fig. 2E), respectively. Fig.3—(A)-IR spectrum of A1 formulation, (B)-IR spectrum of Aa formulation Spectral analysis by IR IR spectrum of wave number (cm-1) Vs %T gives sufficient information about the structure of compounds or number of functional groups present in the sample. The O-H stretch shows broad absorption peak at 3627-3555cm-1(Fig. 3A) in A1 formulation where as peak at 3470cm-1 in Aa formulation (Fig. 3B) it indicated the presence of alcohol and phenol. Carbonyl groups of amide were detected at 1651cm-1 INDIAN J TRADIT KNOWLE, VOL. 15, NO 2, APRIL 2016 258 and 1634cm-1 in A1 and Aa formulation respectively. The absorption of various types of bond such as O-H, N-H, C=O, =C-H, C-H might be the characteristics of presence of Alcohol, Phenol, Amide, Acetic acid, Aldehyde in the above formulations. The basic outcome of this research project is formulation and development of Amla wine. Herbal wine is described as best Yogvahi in Ayurvedic literature. The principle of Yogvahi is more or less similar to the concept of bio-enhancers in contemporary science. Therefore, it can be stated that wine has used as Anupaan to increase palatability as well as bioavailability of principal drug. Due to above invention ultimately it gives benefits to Indian farmers to boost the production of Amla fruits and they may get chance to earn good profit out of it. The health benefits of wine are mainly determined by its active ingredient alcohol. Wine has a long history of use as an early form of medication, being recommended variously as a safe alternative to drinking water, an antiseptic for treating wounds, a digestive aid, and as a cure for a wide range of ailments including lethargy, diarrhoea and pain from child birth risk of heart disease, stroke, diabetes mellitus, metabolic syndrome and early death.. The anticoagulant properties of wine may have the potential benefits of reducing the risk of blood clots that can lead to heart disease. The anti-bacterial nature of alcohol has long been associated with soothing stomach irritations and ailments like traveler's diarrhoea where it was a preferred treatment to the less palatable bismuth treatments. Acknowledgement Authors are grateful to the Principal and the Management of Sanjivani College of Pharmaceutical Education and Research, Kopargaon for providing us the excellent facilities to carry out the research work. 6 7 8 9 10 11 12 13 14 15 16 17 18 References 1 2 3 4 5 A Finose & K Devaki, Phytochemical and chromatographic studies in the flowers of Woodfordia fruticosa (L) Kurz, Asian J Plant Sci Res, 1(3) (2011) 81-85. Indian Herbal Pharmacopoeia, Revised new edn, (Indian drug manufacturers association, Mumbai), 2002, 214-221. Shah & Quadry’s, Pharmacognosy, 12th revised edn, (BS Shah Prakashan, Ahmedabad), 2004-2005: 303-304. Mukhergee P K, Quality control Herbal drugs, An approach to evaluation of Botanicals, 1st edn, (Business Horizons, New Delhi), 2002, 1-515. Soni S K, Bansal Namita & Soni Raman, Standardization of conditions for fermentation and maturation of wine from 19 20 21 Amla (Emblica officinalis Gaertn.), Nat Prod Rad, 8(4) (2009) 436-444. Annex J, Summary of current food standards, (2013) 1-21. Bhondave Prashant, Burase Raghunath, Takale Shrikant, Paradkar Anant & Patil Sanjay, Yeast consortium isolated from Woodfordia fruticosa flowers proved to be instrumental for traditional Ayurvedic fermentation, Int J Pharm Biomed Res 4(1) (2013) 37-45. Verma Neeraj, Amresh G, Sahu P K, Mishra Neelam & Rao Ch V, Anti-inflammatory and antinociceptive activity of hydroethanolic extracts of Woodfordia fruticosa (L.) kurz flowers, Der Pharm Sinica, 3(2) (2012) 289-294. Parthik Patel, Patel N M & Patel P M, WHO guidelines on quality control of herbal medicines, Int J Res Ayurveda Pharm, 2(4) (2011) 1148-1154. Lin Y & Tanaka S, Ethanol fermentation from biomass resources: Current state and prospects, Appl Microbiol Biotechnol, 69 (2006) 627-642. Mannul methods of analysis of foods, Fruit and vegetable products, Food safety and standards authority of India, (Ministry of health and family welfare government of India, New Delhi), 2012, 1-57. Singh, Mhaveer, Khan, Mohammad Ahmed, Khan, Masood Shan, Ansari, SH & Ahmad Sayeed, Quality assessment and evaluation of in vitro antioxidant potential of Phyllanthus emblica L, Indian J Tradit Knowle, 14(2) (2015) 265-272. John Owusu, Haile Ma, Zhenbin, Wang, Ronghai et al., The influence of ph on quality of Tomato (Lycopersicon esculentumMill) wine, Int J Adv Biotec Res, 3(3) (2012) 625-634. V K Parthiban, V Prakasam & K Prabakar, Changes in the biochemical constituents of carrot roots due to bacterial soft rot, Int J Appl Biol Pharm, 3 (2012) 231-238. Atanassova M, Georgieva S & Ivancheva K, Total phenolic and total flavonoid contents, Antioxidant capacity and biological contaminants in medicinal herbs, J Univ Chem Technol Met, 46(1) (2011) 81-88. Snehal Salunke, Vishal Pande, Prakash Kendre, Somnath Vibhute, Effect of Standardized Polyherbal Formulations on Blood Glucose, Body Weight, Food and Water Consumption of Rat, Pharm Sci, 21(2) (2015) 56-63. Fatmah Malekpoor, Abdollah Ghasemi Pirbalouti, Azam Salimi, Antimicrobial and Antioxidant activities and total phenolic content of Tanacetum polycephalum Schutz. Bip. As a folkloric herb in south western iran, Indian J Tradit Knowle, 14(3) (2015) 370-375. Saeed Naima, Khan Muhammad R & Shabbir Maria, Antioxidant activity, total phenolic and tatal flavonoid contents of whole plant extracts Torilis leptophylla L, BMC Comple Altern Med, 12(221) (2012) 1- 12. Meena A K, Singh Arjun & Rao M M, Evaluation of physicochemical and preliminary phytochemical studies on the fruit of Emblica officinalis Gaertn., Asian J Pharm Clin Res, 3(3) (2010) 242-243. Manwar Jagdish, Mahadik Kakasaheb & Paradkar Anant, Plackett-Burman Design: A statistical method for the ARGADE & PANDE: INFLUENCE OF DIFFERENT FERMENTATION CONDITIONS ON FORMULATION OF AMLA WINE optimization of flowers of Woodfordia fruticosa, Ferment Technol, 2(1) (2013) 1-6. 22 Patel N V & Telange D R, Qualitative and quantitative estimation of Gallic acid and Ascorbic acid in polyherbal tablets, Int J Pharmaceut Sci Res, 2(9) (2011) 2394-2398. 23 Rajkovic Milos B & Sredovic Ivana D, The determination of titrable acidity and total tannins in red wine, J Agric Sci 54 (2009) 223-246. 259 24 Sujogya Kumar Panda & Gunanidhi Sahoo, Process characteristics and nutritional evaluation of handia – A cereal based ethnic fermented food fro odisha, Indian J Tradit knowle, 13(1) (2014) 149-156. 25 Joshi V K & Chauhan Arjun, Preparation of plum wine with reduced alcohol content: Effect of must treatment and blending with sand pear juice on physicochemical and sensory quality, Indian J Nat Prod Resour, 5(1) (2014) 67-74.
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