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.