Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
Cholai, a Distilled Beverage of West Bengal: ProductCharacterization
M.Sc. Dissertation Submitted for Master Degree in Microbiology of
Sikkim University
Rituparna Sengupta, M.Sc. 4th Semester
(Roll No: 10UMSM17)
2012
Department of Microbiology
Sikkim University, Tadong 737 102, Sikkim, India
CHAPTER PAGE NUMBER
i
INTRODUCTION 1
REVIEW OF LITERATURE 2
MATERIALS AND METHODSCulture Media Used 8Reagents 8Traditional Knowledge 12Sample Collection 12Microbiological Analysis 12Characterization of Bacterial IsolatesCell Morphology 13Endospore Staining 13Biochemical AnalysispH 13Titratable Acidity 14Estimation of Reducing Sugar 14Estimation of Total Sugar 15Estimation of Alcohol 16 Statistics 18
RESULTS:Traditional Method of Preparation 19Flow Sheet of Preparation 20Mode of Consumption 20Similar Products 20Ethnic Importance 21Microbiological Analysis 21Biochemical Analysis 21Proximate Composition 22
DISCUSSION 23
CONCLUSION 25
BIBLIOGRAPHY 27Tables 31Figures 40Illustrations 49
Acknowledgement
ii
I would like to express my deepest gratitude to my academic and research advisor Professor (Dr.) Jyoti Prakash Tamang for his constant guidance and support during the completion of my work. Because of his constant help and invaluable suggestion I am able to complete my work in the stipulated time. I would also like to thank Dr. Sudarsan Tamang for his constant help and encouragement during the course of my work. In addition I would like to thank Dr. Buddhiman Tamang for his suggestions and help. I would like to thank Mr. Gagan Chettri for helping me to carry my dissertation work smoothly. I would also like to thank Mr. Shankar Prasad Sha for his timely help. I am indebted to the Department of Microbiology, Sikkim University for providing me with research facilities.
Moreover I would like to thank all the people I have come to know in Sikkim University, whose friendship I will always remember. Finally I extend my sincere appreciation to my beloved parents for their love, affection and encouragement in every aspect of my life and studies.
Rituparna Sengupta
Date: 27th May 2012
Place: Tadong
SUMMARY
iii
‘Cholai’ is a well known distilled alcoholic beverage of West Bengal (now Paschim
banga). It is an alcoholic clear distilled drink which is produced from sugarcane molasses and
sugar. The fermentation procedure may be either natural or felicitated by the addition of locally
prepared mixed starter cultures. The beverage is produced in simple homemade distillation unit
and most of the times secrecy is maintained to evade higher liquor taxes. Though the pure form
of ‘Cholai’ is not a health threat but the altered or adulterated ‘Cholai’ is highly toxic and upon
consumption it causes blindness and death. ‘Cholai’ is very high in alcoholic content, so dilution
with water is must before consumption. Three different samples were collected from three
different places of West Bengal and were subjected to microbiological and biochemical analysis.
Spore-forming bacilli were isolated from the samples. The alcohol content was found to be very
high whereas sugar content was found to be too low.
iv
INTRODUCTION
Tamang (2010a) defined ethnic fermented foods and alcoholic beverages as foods
produced by the ethnic people using their native knowledge from locally available raw materials
of plant or animal sources either naturally or by adding starter culture(s) containing functional
microorganisms which modify the substrates biochemically and organoleptically into edible
products that are culturally and socially acceptable to the consumers. He used the term ‘ethnic’ to
denote the community-based fermented foods and beverages prepared by different ethnic people
using their native or traditional or indigenous knowledge. Ancient people had developed the
culinary skills to make food recipes based on several biological, geographical and physical
factors as well as individual sensory likings (Tamang and Samuel, 2010a). Consumption of
alcoholic drinks in India has been mentioned in the Ramayana during 300–75 BC (Prakash1961).
Alcoholic beverages represent a vast diversity of products ranging from ethnic, fermented
beverages, alcoholic drinks, and distilled alcoholic products to wine and beer (Stewart, 1987).
The ethnic alcoholic beverages of any ethnic people of Asia and Africa have a strong ritualistic
importance (Tamang, 2010b).
Alcoholic beverages are produced mainly by the hydrolysis of a sugar source (mainly
starch) by the microbial fermentation by molds and yeasts. After that it may be distilled or not.
Sometimes the fermentation is natural and sometimes it is facilitated by adding microbial culture
from the previous batch to quicken the fermentation process.
Cholai is a distilled alcoholic beverage of West Bengal (now Paschimbanga). The present
paper deals with the traditional knowledge of processing of ‘cholai’ , its microbiological analysis
and product characterization.
1
REVIEW OF LITERATURE
Recently Tamang (2010b) has classified alcoholic beverages of the world into 10 types:
1) Non-distilled and unfiltered alcoholic food beverage produced by amylolytic starters (eg.
kodo ko jaanr and bhaati jaanr of India and Nepal, makgeolli of Korea, lao-chao of
China, tapé of Indonesia)
2) Non-distilled and filtered alcoholic beverage produced by amylolytic starters (eg. saké of
Japan, krachae or nam-khaao or sato of Thailand basi of Philippines, yakju and takju of
Korea)
3) Distilled alcoholic beverage produced by amylolytic starter (eg. raksi of India, Nepal and
Bhutan, shochu of Japan, soju of Korea)
4) Alcoholic beverage produced by human saliva (eg. chicha of Peru)
5) Alcoholic beverage produced by mono-fermentation (eg. beer)
6) Alcoholic beverage produced from honey (eg. tej of Ethiopia)
7) Alcoholic beverage produced from plants (eg. pulque of Mexico, toddy and kanji of
India)
8) Alcoholic beverage produced by malting (germination) (eg. Bantu beer of South Africa,
pito of Nigeria, tchoukoutou of Benin)
9) Alcoholic beverages prepared from fruits without distillation (eg. wine, cider)
10) Distilled alcoholic beverages prepared from fruits and cereals (eg. whisky, brandy, rum)
Some of the similar distilled alcoholic beverage that has been studied and documented are as
follows:
2
Whiskey:
Whiskey is a type of distilled alcoholic beverage. It is produced by the fermentation of
malted barley or cereal products. Specific flavors are added to the distillate to give particular
products (Bluhm 1995). Fermentation is commercially carried out by the yeast Saccharomyces
cerevisiae. The fermentation can be carried out naturally where indigenous yeast strains ferment
the sugar source and contribute to various aromas and flavors or it can be facilitated by adding
Saccharomyces cerevisiae. Whiskey is very high in alcohol content. Whiskey is aged for several
years in wooden casks. Among the wooden cask types the oak casks are used often. Due to the
aging whiskey gets its flavor. (Koda et al., 2003) The flavour is dependent on the type of wooden
cask used and the time of aging. Whiskey is mainly composed of ethanol and water. The other
constituents in whiskey comprises of less than 1 %.
But those components are responsible for the aroma and flavor of the product.
Whiskey is thought to have originated in Irish monasteries in the 12th century, although it rose to
its current prominence after being imported to Scotland in the 14th and 15th centuries (Nicol,
2003). There are mainly two different types of whiskey, malt whiskey and grain whiskey. Malt
whiskies are made in a number of countries, including Australia, Canada, the Czech Republic,
England, France, India, Japan, New Zealand, Northern Ireland (NI), Pakistan, the Republic of
Ireland (RoI), Scotland, Sweden, Turkey, Wales and the USA (Murray, 1997). The major
producers are Scotland, Ireland, India and Japan. All malt distillers use some malted barley as the
source of fermentable sugars and required enzymes. On the other hand grain whisky is produced
from unmalted cereals (Brosnan, 2003).
3
Rum:
Rum is a fairly tasteless and neutral spirit, traditionally produced in the Caribbean and
Central America countries. It is mainly derived from the fermentation and distillation of sugar
molasses, the black treacle-like substance which remains after sugar crystallisation. Once the
alcohol is obtained from the fermentation and distillation processes, it undergoes further
processing such as percolation through carbon filters, ageing in oak barrels and blendings, which
give rum its characteristic flavour (Nicol, 2003; Persad-Doodnath, 2008). In general, rums can be
divided into two classes according to both colour and flavour: white (rums are filtered to remove
any colour gained during ageing) and dark or aged rums. Although both classes are
representatives of rum flavour, undoubtedly the aged rums possess themost balanced rumflavour
(Persad- Doodnath, 2008).
A diversity of different strains of Saccharomyces cerevisiae eventually dominate the
fermentation after the initial growth of other species within the genera Pichia, Candida, and
Kluyveromyces. (Schwan et al., 2001). Rum production from molasses fermentation may
involve contributions from Schizosaccharomyces pombe as well as S. cerevisiae (Fahrasame and
Ganow-Parfeit, 1998).
Brandy:
Brandy is a distilled beverage that is distilled from the fermented fruit juice. It is a
high-alcohol spirit distillate consumed in all regions of the world. Wine from fermented grape
juice or from other fermented fruit juices such as apricots, apples, cherries, plums, mirabelle,
sloes, peaches, bilberries, raspberries, blackberries, blackcurrants, strawberries, rowan berries,
and figs are distilled, usually twice. (Hallgarten 1979). The first distillation gives a clear liquor
4
called brouillis, which is again redistilled to give a spirit with about 70% ethanol and is called
eau de vie. In some cases, such as in armagnac-type of brandy, only single distillation is used,
giving a spirit with 50%–55% ethanol. Clear brandies such as kirsch are bottled directly, while
others such as cognac and armagnac are aged for several years in wooden casks to give
characteristic flavors and brown color. Cognacs aged anywhere between 10-20 years happen to
be excellent and light for anyone to enjoy two or more portions. The intensity, balance and finish
of a good Cognac cannot be equalled by many other distillates (Berberroglu, 2002).
Saccharomyces cerevisiae is the functional yeast in the fermentation of fruits into wines.
Raksi:
Raksi is a traditional distilled beverage of Nepal that is distilled from fermented cereal
beverages (Kozaki et al., 2000). Bhaati jaanr, poko, makai ko jaanr, kodo ko jaanr, gahoon ko
jaanr, fermented masses of buckwheat, potato, canna, and cassava roots are distilled in a large
cylindrical metallic vessel measuring 40 cm× 30 cm× 25 cm. for 2–3 hour continuously over
firewood in an earthen oven.(Tamang 2010). Raksi prepared after replacing the condensing water
five times is known as panch pani raksi, which is a common alcoholic drink. Sometimes, petals
of Rhododendron spp. are mixed during distillation to give a distinct aroma to raksi. This type of
raksi is commonly prepared in the Rhododendron growing regions of the Himalayas (Tamang et
al., 1996). The alcohol content of raksi is 22%–27% (v/v) (Thapa 2001). It is consumed directly
without any dilution with water.
Rakia of Bulgaria:
5
Rakia is a distilled alcoholic beverage that contains very high amount of alcohol (60-
70%). It is produced from the distillation of newly prepared wine from different fruits. In
Macedonia rakia is mostly made of grapes, while Serbia and Montenegro are famous for their
plum rakia (Chirilovic 2002). Bulgarian and Croatian rakia might be made as well of grapes, as
of plums. In general it can be produced of any fruits that contain sugar, e.g. apples, apricots,
pears. (Chirilovic, 2002) The word ‘rakia’ comes from Turkish raki which means ‘strong vodka,
distilled from fermented fruits and spiced with anise’ (Chirilovic, 2002). Balkan rakia is not
spiced with anise, although there exist anise flavoured beverages: Macedonian mastika and
Greek ouzo. Only in Bulgaria, as noted by Ch. Vakarelski, ‘all vodkas are twice distilled and
aromatised with anise seed’ (Vakarelski, 1965). The tradition of making rakia in the Balkans
extends to XIV century and exists in almost all the countries in the region. At the beginning it
was made only of grapes, that is of wine, and then also of other fruits (Chirilovic, 2002).
Rakia is being drunk mostly in the morning, before eating. As every beverage with
high alcohol content it improves digestion. In the morning, people drink very strong rakia from
special, tiny glasses (bigger glasses are used for rakia with less alcohol content, the biggest for
wine) (Chirilovic, 2002). Rakia is also drunk before dinner, as an aperitif and in the evenings.
Akpeteshie of Ghana:
Traditional alcoholic beverages have been consumed in Ghana and other West
African communities for centuries. Locally distilled liquor, also known as Akpeteshie in
Ghana and Ogogoro in Nigeria, is a spirit drink distilled from palm wine (Odeyemi, 1980).
This drink is of historical significance in Ghana and Nigeria because, as a local gin,
colonial administrators barred it in an attempt to control the West African liquor trade in
6
the early part of the last century (Isidore, 2001). Palm wine was originally the drink of choice
in southern Ghana, replaced by rum and schnapps during the period of the slave trade
(Akyeapong, 1996). Men, thus, have come to favor distilled spirits, seen as “hot” or
“strong,” over palm wine or beer. Drinking distilled spirits was asign of prestige in pre-
colonial Ghana and as such, a behavior controlled by the elders and the politically
powerful. Women did not consume alcohol; young men drank rarely and then only as a result
of the beneficence of the rich and the powerful (Akyeapong, 1996).
There is a simple way of making distilled liquor without requiring a special
monitoring system but this gives a relatively short shelf life period as compared to the
industrially prepared liquors (Odeyemi, 1980). Akpeteshie is essentially an unflavoured alcohol
distillate. The storage materials as well as the conditions affect its alcohol strength with
time. The beverage is regarded “flat” if it loses its sharp strength. The Akpeteshie has a
relatively short shelf life period as compared to the industrially prepared liquors (Zakpaa et
al., 2010). It can however be aged in order to improve its flavor as well as aesthetic
appeal. This is done by storing in the presence of certain plant products like the roots of some
plants. This usually imparts a bitter taste to it. Akpeteshie is distilled from fermented palm wine
or sugar-cane juice and require a simple apparatus of two tins (usually four-gallon kerosene
tins) and copper tubing. The standardized alcohol strength of Akpeteshie today is between
40 and 50% by volume (Akyeapong, 1996).
7
MATERIALS AND METHODS
CULTURE MEDIA USED:
Nutrient Agar (HiMedia)
Peptone 5.0 gram
Beef extract 3.0 gram
NaCl (Sodium Chloride) 5.0 gram
Agar 15.0 gram
Distilled Water 1 liter
REAGENTS:
Malachite green stain:
Malachite green 0.5 g
Distilled water 100 ml
Safranin counter stain:
Stock solution (2.5 % w/v alcoholic solution) :
Safranin 2.5 g
95% Ethanol 100 ml
Working Solution:
10 ml of stock solution.
90 ml of distilled water.
8
Phenolphthalein indicator: (0.1 % w/v in 95% ethanol)
Sodium hydroxide solution (NaOH, 0.1 N):
Sodium hydroxide 2.0 g
Distilled water 500 ml
Oxalic acid solution (0.1 N):
Oxalic acid 0.63 g
Distilled water 100 ml
Reagent C (Arsenomolybdate reagent)
Reagent C = Solution A + Solution B
Ammonium Molybdate 25.0 g
Concentrated Sulfuric acid (H2SO4) 21.0 ml
Disodium Hydrogen Arsenate, Heptahydrate (Na2AsO4, 7H2O) 3.0 g
Distilled Water 479.0 ml
Solution A:
25 gram of Ammonium Molybdate was dissolved in 450 ml of distilled water followed
by addition of 21 ml of concentrated Sulfuric acid to it.
Solution B:
3 gram of Disodium Hydrogen Arsenate was dissolved in 25 ml of distilled water.
Reagent C:
9
Solution B was added to Solution A slowly with stirring. Then it was diluted to 500 ml by
the addition of distilled water and mixed well. Continue stirring for 24 hours at 37º C. It
was then stored in 1000 ml Amber coloured reagent bottle with stopper. (This reagent can
be stored up to 6 months at room temperature)
Reagent D (Low alkalinity copper reagent) :
Reagent D = Reagent A + Reagent B
Reagent A:
Anhydrous Sodium Carbonate 25.0 g
Sodium Potassium Tartarate 25.0 g
Sodium Hydrogen Carbonate 20.0 g
Anhydrous Sodium Sulfate 200.0 g
Distilled water 1 Liter
Anhydrous Sodium Carbonate, Sodium Potassium Tartrate, Sodium Hydrogen Carbonate
were altogether dissolved in 300 ml distilled water. Separately anhydrous sodium sulfate was
dissolved in 500 ml of boiling distilled water. Then both the solutions were mixed together and
diluted up to 1 liter. Stored in a reagent bottle with glass stopper.(Can be stored up to 1 year in
room temperature.)
Reagent B:
Copper Sulfate (CuSO4, 5H2O) 30.0 g
Concentrated Sulfuric Acid (H2SO4) 4 drops
10
Distilled Water 200 ml
Copper sulfate was dissolved in 200 ml of distilled water containing 4 drops of concentrated
Sulfuric acid.
Reagent D:
25 ml of Reagent A was mixed with 1 ml of reagent B. Reagent D was freshly prepared before
each experiment.
Starch Solution:
Soluble Starch 1.0 g
Distilled Water 100 ml
A paste of 1.0 gram of soluble starch was prepared with distilled water. This paste was
then poured into 100 ml of boiling distilled water in a beaker with constant stirring by a magnetic
stirrer. The mixture is boiled until clear solution is obtained. It was cooled and stored in a bottle
with glass stopper. Freshly prepared volume of starch solution was used for each titration as its
aqueous solution is not very stable.
Sodium Thiosulphate solution (Na2S2O3, 0.1 N) :
Anhydrous Sodium Thiosulfate (M.W. 158.11) 3.9 g
Distilled Water 250 ml
11
Potassium dichromate solution (K2Cr2o7, 0.5 N) :
Potassium dichromate 19.75 g
Distilled water 250 ml
Documentation of traditional knowledge:
The traditional knowledge regarding the preparation procedure of cholai, mode of
consumption and its socio economic importance was collected and documented from the people
of southern part of West Bengal.
Collection of samples:
Three different ‘cholai’ samples were collected from Jagatdal, Kankinara and Titagarh
which are located in the district of North twenty four Parganas in West Bengal. The samples
were collected in air tight falcon tubes and kept in autoclaved plastic bags and sealed tightly.
Microbiological analysis:
For microbial analysis only presence of heat resistant endospore forming Bacilli was
checked as the product is distilled. Spore-forming bacilli were isolated on nutrient agar
(HiMedia), after inactivation of vegetative cells by heating at 100 C for 2 min (Tamang and
Nikkuni, 1996) and then incubated at 37 C for 24 h. Purity of the isolates is checked by
streaking again on fresh agar plates of the isolation media and sub-culturing on corresponding
broths/agar, followed by microscopic examinations. Microbiological data obtained are
transformed into logarithms of the numbers of colony forming unit (cfu) per g of sample.
12
Identified strains of microorganisms are preserved in respective media using 15 % (v/v) glycerol
at (20 C).
Characterization of bacterial isolates:
Cell morphology:
Cream coloured small colonies were isolated on nutrient agar plates after incubation period.
The colonies were round and slimy in nature.
Endo-spore staining:
For staining of endospores Schaeffer-Fulton method of endospore staining was followed.
A glass slide was taken and cleaned. A loop full of the microorganism from nutrient agar plate
was taken and a smear was made on the glass slide by adding a drop of water at the centre of the
slide. It was then air dried and heat fixed. Malachite green stain was added over the slide. Then
the slide was steamed over a boiling water bath for 15 min. The slide was washed under clear tap
water. Counterstaining was done with safranin for 30 sec. The slide was again washed with tap
water and blot dried. Finally the slide was examined under the oil immersion lens (100X ) for the
presence of endospores
Biochemical Analysis:
pH:
The pH of the sample was determined directly (AOAC, 1990) using a digital pH meter
(Thermoscientific Orion Star) after calibration with standard buffer solutions. (pH 4, 7 and 10)
(Tamang and Thapa, 2006).
13
Titratable acidity:
Titratable acidity of sample is calculated by titrating the 1 ml of liquid sample in 9 ml of
distilled water with 0.1 N sodium hydroxide (NaOH) to end point of phenolphthalein (0.1 % w/v
in 95 % ethanol) (AOAC, 1990). The NaOH solution was first standardized against oxalic acid
and its normality after standardization was found to be 0.091 (N). The titratable acidity was
expressed in terms of lactic acid (AOAC, 1990); (Tamang and Thapa, 2006)
Titratable acidity was calculated by using the following formula:
TA= (N×V1× eq. weight of lactic acid ×100× dilution factor)/(V2×1000)
N= 0.091
Equivalent weight of lactic acid= 90
V2= 10
V1= Final reading on burette
Estimation of Reducing Sugar:
For the estimation of reducing sugar was carried out as described by Thapa and Tamang
(2006) based on traditional colorimetric method of Somogyi (1945) using glucose as standard
solution. (Tamang and Nikkuni, 1996). 10 ml of capped test tubes were washed with distilled
water. Blank was prepared with 1 ml of distilled water, adding Somogyi copper reagent and
arsenomolybdate reagent according to the protocol described in the table below. Sugar standard
solution was prepared at a concentration of 1 mg/ml glucose in 100 ml of distilled water.
Aliquots of 5 to 100 µl were transferred with an accurate micropipette to different 10 ml test
tubes. 1 ml of the sample to be analyzed (S) was transferred to a 10 ml test tube. There were
14
three different samples. For each sample to be analyzed triplicate set of analysis was carried out.
To all the test tubes 1 ml of Somogyi copper solution (Reagent D) was added. All the test tubes
were capped tightly. Then the tubes were heated vigorously in a boiling water bath for 20
minutes. Then the tubes were allowed to cool for 5 minutes in running tap water. 1 ml of
arsenomolybdate reagent (Reagent C) was added to each of the tubes. The test tubes were shaked
until no bubbles were evolved.
The colour appeared after addition of 1 ml of arsenomolybdate reagent. All volumes were
diluted to 5 ml with distilled water and the test tubes were allowed to stand for 20 minutes at
room temperature. The solution from the test tubes were transferred to cuvettes and the
absorbance of the sugar standards and the samples were measured at 520 nm in a UV/Vis
spectrophotometer (Perkin Elmer Lambda 25). Before that blank solution’s absorbance was set
to zero. To calculate the concentration of sugar present in the sample, a graph of A520 vs
concentration of sugar was plotted. The sugar concentration of the samples to be analysed were
calculated from the intercept. Finally the reducing sugar content was calculated in percentage
For Sample (S) analysis: Three different unknown samples were to be analysed. They were
assigned as S1, S2 and S3. For each type of sample triplicate set of experiment was carried out. The
sets were assigned as A, B and C.
Estimation of total sugar:
Total sugar was determined by determining reducing sugar in hydrolysed sample with
HCl (AOAC, 1990). In a 300 ml conical flask fitted with condenser, 2 g of sample was blended
in 20 ml of distilled water to which 160 ml of distilled water and 20 ml of HCl (25 %) were
15
added. It was heated in vigorously boiling water bath for 3 h, cooled in a running tap water,
neutralized with 10 % NaOH using pH meter (Thermo scientific, Orion Star)) and diluted to 500
ml with distilled water. It was filtered and the filtrate was taken for determining reducing sugar
as described above (Somogyi, 1945). Total sugar was calculated in percentage.
For Sample (S) analysis: Three different unknown samples were to be analysed. They were
assigned as S1, S2 and S3. For each type of sample triplicate set of experiment was carried out. The
sets were assigned as A, B and C.
Estimation of alcohol:
For sample titration:
Alcohol content of sample was determined by dichromate oxidation method (AOAC,
1990). The 10 ml of extract was diluted to 100 ml with distilled water. From that dilution 10 ml
of extract was taken in a conical flask with stopper to which 100 ml of distilled water was added.
To that extract 10 ml of N/5 K2Cr2O7 and 10 ml of concentrated H2SO4 were added and allowed
to stand for 1 h. After this, stopper was removed and 100 ml of distilled water was added,
followed by addition of 8 % KI and immediately titrated with N/10 Na2S2O3 using freshly
prepared 1 % starch (HiMedia ) solution as the indicator as described in the table below. Alcohol
content was calculated in percentage.
For Blank Titration:
10 ml of standard Potassium dichromate (K2Cr2O7) solution (0.5 N) was pipette out into a
500 ml conical flask. 1 gram of Potassium iodide (KI) dissolved in 10 ml of water is added
16
followed by addition of 2 ml of concentrated Sulfuric acid (H2SO4). The mixture is shaken and
then allowed to stand in dark for 5 minutes covering the mouth of the flask with a watch glass to
ensure complete liberation of iodine. The watch glass and the sides of the flask were washed
down with distilled water and diluted to 80 ml. The solution was then immediately titrated
rapidly with sodium thiosulfate (Na2S2O3) solution running from a burette until the brown colour
fades to straw yellow. Then 1 ml of freshly prepared starch solution was added, shaken to obtain
a deep blue colour. The titration was continued with continuous shaking until the blue colour just
disappears leaving a bright green solution. The titration was done in triplicate to get accurate
result. To avoid aerial oxidation the titration was carried out in CO2 atmosphere by adding 2.0
gram of pure Sodium bicarbonate (NaHCO3) before addition of acid.
Standard Titration:
To find out the accuracy of the alcohol estimation of the sample a standard with known
alcoholic concentration was also taken. For this a standard Rum sample (42.8% v/v) was taken
and titrated against standard 0.1 (N)sodium thiosulfate solution and the alcohol percentage was
calculated. The calculated alcohol percentage was nearly same. (42%)
Result Calculations:
The blank titration tells that how much acid dichromate was present at the start. As no
alcohol was added the full amount of the dichromate is still present. The blank titrations were
carried out so the result can be compared with those of the sample titrations.
1) The average volume of sodium thiosulfate used for sample titration from theconcordant
sample results was determined
17
2) The average volume of sodium thiosulfate used for the blank titration from theconcordant
blank results was also determined.
3) The volume of the sodium thiosulfate solution used for the sample titration was
substracted from the volume used for the blank titration. This volume of the sodium
thiosulfate solution was used to determine the alcohol concentration.
4) The number of moles of sodium thiosulfate in this volume was calculated.
5) Using the following equations, the relationship between the moles of sodium thiosulfate
and the moles of ethanol was determined
a. as 6 mol of S2O32- is equivalent to 1 mol of Cr2O7
2-
b. and 2 mol of Cr2O72- is equivalent to 3 mol of C2H5OH
c. then 1 mol of S2O32- is equivalent to 0.25 mol of C2H5OH
6) The moles of alcohol in the sample solution was calculated using this ratio
7) As the dilution was 1:100 the result was multiplied by 100.
8) The answer is converted into percentage (g/100 ml).
By following the above procedure the percentage of alcohol for the three different samples
were calculated.
Statistics:
Standard deviation (S.D.) value of the three samples were taken to calculate the range of
reducing sugar, total sugar and alcoholic content in the samples. The formula used is -
S.D. = √{∑ ( xi−x ) 2/(n−1)};
x= mean of the three samples,
xi = SA, SB and SC of each of the three samples.
18
RESULTS
Traditional method of preparation:
MATERIALS NEEDED: Earthen pot, Sugarcane molasses (Medium quality), Sugar, Sal
ammoniac (NH4Cl), Yeast Cake ( If rapid fermentation needed).
At first the molasses along with the sugar and yeast cake is kept in a earthen pot. It is kept
without movement for 5-6 days for the fermentation to be completed. Sal ammoniac which is
called ‘Neshadol’ in local language is also added as yeast nutritive. After completion of
fermentation the fermented mash is collected and half filled in simple homemade distillation
unit. Like other alcoholic fermentation the end product contains ethanol and slight traces of
methanol. As methanol is poisonous it must be separated. To do that brewers have developed
own distillation unit with simple and common objects. The distillation unit is mainly made up of
stainless steel utensil which may have inner copper lining. As copper is known to remove the
sulfur by product produced during fermentation as sulfide.
The fermented mash is half filled in the stainless steel utensil and placed on fire. The
utensil is covered with a plate. Over that another utensil filled up with cold water is placed. The
utensil containing fermented mash contains a pipe, whose end portion is inserted inside a funnel
or bottle. The pipe is mainly bamboo made. Now as the mixture is heated steam will be
generated. This steam will pass through the pipe and condensed and collected inside the bottle
drop wise. This is called ‘chullu’ or ‘cholai ’ To maintain the temperature the water is changed
after certain interval from the topmost utensil. After 500 ml of ‘cholai’ has been collected, it is
mixed with equal amount of water to make it consumable as the alcoholic concentration is very
high. The alcohol is not aged so the colour is crystal clear.
19
For preparation of 20 liter ‘cholai’
Molasses (8 kg) + Sugar (3 kg) mixed, kept in an earthen pot Added traditionally prepared mixed starter
Fermented for 5-6 days
Fermented mash taken out and half filled in homemade distil unit
Distillation
Distilled liquor collected
Mixed with equal amount of water and bottled
Cholai
Fig 1: Flow sheet of traditional method of preparation of cholai
Mode of Consumption:
Cholai is drunk directly with or without addition of water. It is popular among the low-
income group of people who drinks after the hard work in the evening along with snacks. Cholai
is also known as poor man’s whiskey locally.
Similar products are Raksi of India and Nepal (Tamang et al. 1996), Yu of Manipur
(India) (Tamang) distilled from Atingba, Saké of Japan (Inoue et al. 1992), Soju of Korea (Lee
and Kim 1993), and Krachae or Nam-khaao or Sato of Thailand (Vachanavinich et al. 1994).
Kentucky moonshine of Kentucky (Maurer 2003), Raki of Albania (Lachenmeir et al. 2011),
Rakia of Bulgaria (Petrov, 1985), Zivania of Cyprus (Rebecca et al. 2005), Samogon of Russia
(McKee, 1999), Georgia Moon Corn Whiskey of United States (Owens et al, 2009), Akpeteshie
of Ghana (Zakpaa et al., 2010)
Ethnic Importance:
20
‘Cholai’ is also known as ‘poor people’s whiskey.’ Study says that the main consumers
are socially backward people in terms of financial status. Those people are unable to gain access
to other high priced alcoholic beverage. But they can easily access ‘cholai’ as its price is
relatively low. After all day of hard work they consume ‘cholai’ with snacks. It has became a
kind of tradition among these people.
Experimental:
Microbiological analysis:
From the three different sample of ‘cholai’ heat resistant endospore forming Bacilli was
isolated on nutrient agar plate. Upon staining endospores were seen as bright green and
vegetative cells were brownish red to pink under oil immersion lens (100 X) of light microscope.
Biochemical analysis:
Proximate Composition:
Proximate composition of ‘cholai’ is presented in the following table. The pH, acidity
expressed as lactic acid and alcohol content of the product ranged from 4.98- 6.08 %, 0.23-
0.35% and 40.27- 45.49% respectively. The range of reducing sugar content and total sugar
content was found to be 0.00104- 0.00136% and 0.00138- 0.00162% respectively which was too
low as the product is distilled.
Proximate Compositions of ‘cholai’ are listed in the table below:
21
Parameters* Sample (‘cholai’)
pH* 5.53 ± 0.55
Titratable acidity (as percent of
lactic acid)*
0.29 ± 0.057
Alcohol %* 42.93 ± 2.66
Reducing sugar %* 0.0012 ± 0.00016
Total sugar %* 0.15 ± 0.00012
*Data represent the means (± SD) of 3 samples.
22
DISCUSSION
Moonshine is an illegally produced distilled alcoholic beverage. It is distilled in nature
and also known as white lightening, hooch, mountain dew across the different part of the world.
In West Bengal it is locally known as ‘Chullu’ or ‘Cholai.’
The name moonshine depicts its secretive nature. It is brewed, distilled and sold in secret,
so the high liquor taxes can be evaded. In fact the art of illegal brewing is quite old as the
government is regulating this whole procedure from quite a long time. Any alcoholic beverage
that is produced in large scale with an intention for selling without having a government licence
is termed as illegal. In earlier times the smuggled alcohol was moved during nighttime to avoid
detection so the term ‘moonshine’ was used to indicate this smuggled alcohol and eventually it
came to be used in reference to alcohol which was produced illegally.
‘Cholai’ is produced by fermentation of molasses along with sugar and then distilling the
alcoholic mash. Yeast cakes are used in the fermentation procedure sometimes. As the distillers
have to buy the yeast cakes from local market and it is kind of costly to them they prefer natural
fermentation of the molasses along with sugar. For this they add Sal ammoniac (Natural form of
Ammonium Chloride) to the mixture to be fermented. Sal ammoniac is known as yeast nutritive
for ages. It helps in the growth of yeast like Sacharomyces cerevisiae by providing them with
nitrogen required for growth. The distillers aim in doing so is to allow the growth of indigenous
yeast that will help in the fermentation procedure. But one disadvantage of natural fermentation
is that it takes longer period. It takes about 15-20 days to ferment an amount of sugar-molasses
mixture that will be sufficient to make 20 litre of distilled alcoholic beverage. On the other hand
if yeast cakes that are bought from the market are used then the fermentation period for the
23
production of same amount of distilled spirit is reduced dramatically. It takes only 5-6 days for
complete fermentation.
The distillers in this region practice both natural and induced fermentation according
to the demand in the market. If demand in the market is higher only then they use yeast cakes in
fermentation to produce the distilled spirit quickly and to supply it quickly. But during slow
running season they do not use yeast cakes and prefer the natural fermentation though it takes
longer period. In this way they cut in the cost of production to gain a higher profit from sale.
Most of these type of liquors are made by illegal breweries. (Locally called bhatti).
The owners of the breweries sometimes do not think about quality. The only thing they think
about is profit. Moreover due to the absence of strict law and regulations the product that reaches
the consumers are sometimes compromised and adulterated. As a result several people die after
consuming these modified product due to poisoning. Most of the times the producers mix
methanol and insecticide with ‘cholai’ to gain easy profit. During mixing if the concentration of
methanol or insecticide crosses the threshold level of human body then it costs several human
lives. The recent Bengal Moonshine tragedy that happened in 14th December, 2011 is an
unfortunate example where more than 200 casualties were reported. Earlier also such incidents
happened in West Bengal and also in other parts of the country. Tamilnadu, Madya pradesh,
Gujrat are few examples. This type of incidents are very much unfortunate and unacceptable. So,
there must be strict law and regulations to control such kind of shameful incidents in the future.
Not only our government but also local residents should take active part to stop this kind of
incidents in future.
The problem is only banning of the product is not a very good solution. Because one can
not deny the fact that the poor and uneducated people will not have the luxury to buy branded
24
liquors. So, inspite of government ban they will still go for it unaware of the fact of the danger
that lies within. And most importantly the product itself is not harmful, but some people are
making it harmful. So, instead of trying to kill the tradition of producing these age old country
liquors, strict rules and regulations should be implied so that the quality is not compromised. One
important thing is to reduce the high taxes upon country liquors like ‘cholai’ so there will be no
need to compromise the quality to gain profit. Instead of stating this age old tradition as illegal
and turning a blind eye in this matter, it can be legalized and the whole procedure can be brought
under a constant vigilance to stop any immoral activity. One recent example is that of a distillery
to make South Carolina’s first legal moonshine. This will help to keep the quality intact,
moreover the hundreds of year age old tradition will be saved.
The pH, acidity expressed as lactic acid and alcohol content of the product
ranged from 4.98- 6.08 %, 0.23-0.35% and 40.27- 45.49% respectively. The range of reducing
sugar content and total sugar content was found to be 0.00104-0.00136% and 0.00138-
0.00162% respectively which was too low as the product is distilled (Pryde et al., 2011). The
very high alcoholic concentration is due to the fact that the product is distilled (Pryde et al.,
2011)
As the product is distilled, only heat resistant endospores were able to survive. So only
spore forming Bacilli was isolated from the sample.
Conclusion:
‘Cholai’ has become a very important drink among a large population in West Bengal.
Till now no work either microbiological or biochemical has been done on this topic. However
25
this is one important aspect to study because it is directly related to welfare of society. Due to
limited time period only little has been done. Future prospects of this study can be widening the
sample area and also the other aspects like effect upon human health. Moreover sociological
aspects are also needed to be studied broadly regarding laws, rules and regulations so that
Science can directly come in contact with the society for the welfare of our community people.
26
BIBLIOGRAPHY
Akyeapong, E. (1996). What’s in a drink? Class struggle, popular culture and politics of
Akpeteshie (local Gin) in Ghana, 1930-1967. Journal of African History 37: 215-236.
AOAC (1990). Official Methods of Analysis, 15th edition. Association of Official Analytical
Chemists, Virginia.
Berberroglu, H. (2002). American distiller: a source of information on the distilling process
(vol:4),The American distillation institute.
Bluhm, L. (1995). Distilled beverages In Biotechnology. Food and Feed Production with
Microorganisms, vol. 5, ed. G. Reed, pp. 447–476. Weinheim, Germany: Verlag
Chemie.
Brosnan, J. (2003). Whisky :Technology, Production and Marketing. (Eds: Russell. I., Stewart.
G., Bamforth. C. and Russell. I). Elsevier.
Chirilovic, M. (2002). From Providentia to Patoka: Rakia History. In Politika, 27 IX.
Fahrasame, L. and Ganow-Parfeit, A. (1998). Microbial flora of rum fermentation media.
Journal of Applied Microbiology 84: 921–928.
Hallgarten, P.A. (1979). Spirits and Liqueurs. Faber and Faber, London,U.K.
Isidore, S.O. (2001). Drug and Alcohol Consumption by out of school Nigerian
Adolescents, African Journal of Drug Alcohol Studies. 1(2): 99.
Koda, H., Hossain, S.J., Kiso, Y. and Aoshima, H. (2003). Aging of Whiskey Increases the
Potentiation of GABAA Receptor Response, Journal of Agriculture and Food Chemistry
51(18): 5238–5244.
27
Kozaki, M., Tamang, J.P., Kataoka, J., Yamanaka, S. and Yoshida, S. (2000). Cereal wine
(jaanr) and distilled wine (raksi) in Sikkim. Journal of Brewing Society of Japan 95(2):
115–122.
Murray, J. (1997). Jim Murray’s Complete Book of Whisky. Carlton Press.
Nicol, D.A.(2003). Batch distillation, In Whisky: Technology, Production, and Marketing,vol.
1, ed. Academic Press, pp. 155‐177. San Diego, CA: Rusell.
Nicol, D. A. (2003). Rum, In Fermented beverage production, 2nd ed, Kluwer Academic , pp.
263–287. New York.
Odeyemi, F. (1980). The quality of the Nigerian native alcoholic beverage (Ogogoro). Kemia
Kemi 7: 134–135.
Persad-Doodnath, V. (2008). From sugar to rum – the technology of rum making. In Distilled
Spirits Production, Technology and Innovation (Eds: Bryce, J.H. and Stewart, G.G).
Nottingham University Press, Nottingham, UK, pp. 159–167.
Prakash,O. (1961). Food and Drinks in Ancient India. Delhi, India: Munshi Ram Monoharlal
Publishers.
Pryde1, J., Conner, J., Jack, F., Lancaster, M., Meek, L., Owen, C., Paterson, R., Steele, G.,
Strang, F. and Wood, J. (2011). Sensory and Chemical Analysis of ‘Shackleton’s
Mackinlay Scotch Whisky, J. Inst. Brew 117(2): 156–165.
Schwan, R.F., Mendonca, A.T., da. Silva, J.J., Rodrigues, V. and Wheals, A. (2001).
Microbiology and physiology of Cachacha (aquardiente) fermentation. Antonie van
Leeuwenhoek 79: 89–96.
Somogyi, M. (1945) A new reagent for the determination of sugars. Journal of Biological
Chemistry 160: 61-62.
28
Tamang, J.P. (2010a). Himalayan Fermented Foods: Microbiology, Nutrition, and Ethnic
Values. New York: CRC Press/Taylor & Francis Group.
Tamang, J.P. (2010b). Diversity of fermented beverages. In: Fermented Foods and Beverages of
the World. (Eds: Tamang, J.P. and Kailasapathy, K). CRC Press, Taylor & Francis
Group, New York, pp. 85-125.
Tamang, J.P. and Nikkuni, S. (1996). Selection of starter culture for production of kinema,
fermented soybean food of the Himalaya. World Journal of Microbiology and
Biotechnology 12 (6): 629-635.
Tamang, J.P. and Samuel, D. (2010a). Dietary culture and antiquity of fermented foods and
beverages. In: Fermented Foods and Beverages of the World. (Eds: Tamang, J.P. and
Kailasapathy, K). CRC Press, Taylor & Francis Group, New York, pp. 1-40.
Tamang, J.P. and Thapa, S. (2006). Fermentation dynamics during production of bhaati jaanr, a
traditional fermented rice beverage of the Eastern Himalayas. Food Biotechnology 20(3):
251–261.
Thapa, S. and Tamang, J.P. (2006a). Microbiological and physico-chemical changes during
fermentation of kodo ko jaanr, a traditional alcoholic beverage of the Darjeeling hills and
Sikkim. Indian Journal of Microbiology 46 (4): 333-341.
Tamang, J.P., Thapa, S., Tamang, N. and Rai, B. (1996). Indigenous fermented food beverages
of the Darjeeling hills and Sikkim: Process and product characterisation. Journal of Hill
Research 9(2): 401–411.
Thapa, S. (2001). Microbiological and biochemical studies of indigenous fermented cereal-
based beverages of the Sikkim.himalayas. PhD thesis, Food Microbiology Laboratory,
Sikkim Government College (under North Bengal University) Gangtok, Sikkim.
29
Vakarelski, Ch. (1965). Ethnography of Bulgaria. Wroclaw, Bulgaria.
Zakpaa, H. D., Mak-Mensah, E. E. and Avio, O. A. (2010). Effect of storage conditions on the
shelf life of locally distilled liquor (Akpeteshie), African Journal of Biotechnology
9(10): pp. 1499-1509.
30
Tables:
Estimation of pH
Sample type pH Average pH
S1A 5.19
5.19 S1B 5.23
S1C 5.15
S2A 6.22
6.17 S2B 6.10
S2C 6.19
S3A 5.20
5.23 S3B 5.24
S3C 5.25
Table 1
Estimation of Titrable acidity:
Sample Type
Average volume of NaOH
solution (A+B+C)/3
S1 0.30
S2 0.37
S3 0.40
Table 2.1 Titration
Sample Type Titrable acidity
31
S1 0.025
S2 0.030
S3 0.033
Table 2.2 Titrable acidity result
Estimation of reducing sugar:
T.T.
Standard
solution
(µl)
Somogyi
Copper Reagent
(ml)
Arsenomolybdat
e reagent (ml)
Distilled
water
added (µl)
Final
volume
(ml)
T1. 5 1H
eate
d in
boi
ling
wat
er b
ath
for
20 m
inut
es. A
llow
ed to
cool
for
5 m
inut
es in
run
ning
tap
wat
er.
1 2995 5
T2. 10 1 1 2990 5
T3. 25 1 1 2975 5
T4. 50 1 1 2950 5
T5. 60 1 1 2940 5
T6. 70 1 1 2930 5
T7. 80 1 1 2920 5
T8. 90 1 1 2910 5
T9. 100 1 1 2900 5
Blan
k
__ 1 1 3000 5
Table 3.1 For glucose standard and blank: (glucose standard stock concentration 1µg/ml)
32
T.T. Sample
solution
(ml)
Somogyi
Copper
Reagent (ml)
Arsenomolybdate
Reagent (ml)
Distilled
water
added
(ml)
Final
volume
(ml)
S1A 1 1
Hea
ted
in b
oilin
g w
ater
bat
h fo
r 20
min
utes
and
allo
wed
to c
ool f
or 5
min
utes
in r
unni
ng
tap
wat
er.
1 2 5
S1B 1 1 1 2 5
S1C 1 1 1 2 5
S2A 1 1 1 2 5
S2B 1 1 1 2 5
S2C 1 1 1 2 5
S3A 1 1 1 2 5
S3B 1 1 1 2 5
S3C 1 1 1 2 5
Table 3.2 For Sample (S) analysis
Measurement of Optical Density (O.D.) at 520 nm:
For Standard sugar solutions:
T.T.
Concentration of standard
sugar solution (µg/ml)
Absorbance at 520 nm (A520)
T1. 1 0.05
T2. 2 0.11
T3. 5 0.31
T4. 10 0.57
T5. 12 0.65
33
T6. 14 0.85
T7. 16 0.91
T8. 18 1.17
T9. 20 1.22
Table 3.3
For unknown samples:
T.T. Concentration (µg/ml) Mean Absorbance at 520 nm
(A520)
S1. 10 (from graph) 0.57
S2. 13 (from graph) 0.75
S3. 12 (from graph) 0.65
Table 3.4
Results:
Sample type Concentration (µg/ml) Amount in 100 ml (mg) Percentage % (g/100ml)
S1 10 1 0.001
S2 13 1.3 0.0013
S3 12 1.2 0.0012
Table 3.5
Estimation of Total sugar:
For standard glucose solutions and blank: (glucose standard stock concentration 1µg/ml)
T.T.
Standard
solution
Somogyi
Copper Reagent
Arsenomolybdat
e reagent (ml)
Distilled
water
Final
volume
34
(µl) (ml) added (µl) (ml)
T1. 5 1
Hea
ted
in b
oilin
g w
ater
bat
h fo
r 20
min
utes
. Allo
wed
to
cool
for
5 m
inut
es in
run
ning
tap
wat
er.
1 2995 5
T2. 10 1 1 2990 5
T3. 25 1 1 2975 5
T4. 50 1 1 2950 5
T5. 60 1 1 2940 5
T6. 70 1 1 2930 5
T7. 80 1 1 2920 5
T8. 90 1 1 2910 5
T9. 100 1 1 2900 5
Blan
k
__ 1 1 3000 5
Table 4.1
For sample analysis:
35
T.T. Sample
solution
(ml)
Somogyi
Copper
Reagent (ml)
Arsenomolybdate
Reagent (ml)
Distilled
water
added
(ml)
Final
volume
(ml)
S1A 1 1
Hea
ted
in b
oilin
g w
ater
bat
h fo
r 20
min
utes
and
allo
wed
to c
ool f
or 5
min
utes
in r
unni
ng
tap
wat
er.
1 2 5
S1B 1 1 1 2 5
S1C 1 1 1 2 5
S2A 1 1 1 2 5
S2B 1 1 1 2 5
S2C 1 1 1 2 5
S3A 1 1 1 2 5
S3B 1 1 1 2 5
S3C 1 1 1 2 5
Table 4.2
Measurement of Optical Density (O.D.) at 520 nm: (For Standard sugar solutions)
T.T.
Concentration of standard
sugar solution (µg/ml)
Absorbance at 520 nm (A520)
T1. 1 0.05
T2. 2 0.10
T3. 5 0.29
T4. 10 0.50
36
T5. 12 0.58
T6. 14 0.70
T7. 16 0.78
T8. 18 0.87
T9. 20 0.97
Table 4.3
For unknown samples:
T.T. Concentration (µg/ml) Mean Absorbance at 520 nm
(A520) [ (A+B+C)/3]
S1. 14 (from graph) 0.70
S2. 16 (from graph) 0.78
S3. 16 (from graph) 0.78
Table 4.4
Result:
Sample type Concentration (µg/ml) Amount in 100 ml (mg) Percentage % (g/100ml)
S1 14 1.4 0.0014
S2 16 1.6 0.0016
S3 16 1.6 0.0016
Table 4.5
Alcohol estimation:
Titration of blank i.e. 10 ml of 0.5 (N) Potassium dichromate solution by 0.1 (N) Sodium
thiosulfate solution:
37
Number
of
titration
Volume of Potassium
dichromate solution (ml)
Volume of Sodium
thiosulfate solution (ml)
Average volume of
Sodium thiosulfate
solution (ml)
1. 10.0 51.2
51.2 2. 10.0 51.3
3. 10.0 51.1
Table 5.1
Sample Titration: (by 0.1 N Na2S2O3 solution):
Number of
titration
Sample
No.
Volume of 0.1 (N) Na2S2O3
solution (ml)
Average volume of Na2S2O3
solution (ml)
1 S1A 12.3
12.4 2 S1B 12.5
3 S1C 12.4
1 S2A 10.1
10.2 2 S2B 10.2
3 S2C 10.3
1 S3A 12.5
12.4 2 S3B 12.4
3 S3C 12.4
Table 5.2
38
Standard Titration: (Rum sample)
Number
of
titration
Volume of standard
Rum sample (ml)
Volume of sodium
thiosulfate solution (ml)
Average volume of
Sodium thiosulfate
solution
1. 10.0 20.5
20.4 2. 10.0 20.3
3. 10.0 20.4
Table 5.3
Results:
Sample Type Alcohol percentage % (g/100
ml)
S1 41.4
S2 46.0
S3 41.4
Table 5.4
39
Digital Figures:
Figure 1: (From left to right) Reagent C, Reagent A, Reagent B and Reagent D
Figure 2 :Isolated Bacilli colonies on Nutrient Agar plate after re-streaking
40
Figure 3: Green endospores of Bacilli are seen under light microscope (100X)
Figure 4: Bacilli endospores seen under phase contrast microscope (40 X)
41
Figure 5: Standard, Blank and Sample solutions + Low alkalinity copper reagent (Reagent D)
during reducing sugar estimation
Figure 6: Same solutions after addition of Arsenomolybdate reagent (Reagent C) during reducing
sugar estimation. Colour development can be seen clearly.
42
Figure 7: ‘Cholai’ solution after standing for 1 hour before titration during the estimation of
alcohol content
Figure 8: ‘Cholai’ solution with starch indicator during alcohol estimation.
43
Figure 9: ‘Cholai’ solution after completion of titration by sodium thiosulfate solution during
alcohol estimation
Figure 10: Dark brown colour formation during blank titration due to the liberation of Iodine
during the estimation of alcohol
44
Figure 11: Dark brown colour fades to straw yellow during titration with sodium thiosulfate
solution during alcohol estimation
Figure 12: Dark blue colour formation after addition of starch indicator during blank titration
during alcohol estimation.
45
Figure 13 : Bright green solution after the completion of blank titration during alcohol estimation
Figure 14: Sugarcane Molasses
46
Figure 15: Earthen Pot
Figure 16: Fireplace for distillation unit to run
47
Figure 17: Simple Homemade Distillation Unit
Figure 19: Cholai
48
Illustrations:
0 5 10 15 20 250
0.2
0.4
0.6
0.8
1
1.2
1.4
f(x) = 0.0612908496732027 x − 0.0166503267973845R² = 0.991255883776067
(20,1.22)(18,1.17)
(16,0.91)(14,0.85)
(12,0.65)
(10,0.57)
(5,0.31)
(2,0.11)
(1,0.05)
Concentration of standard sugar solution (µg/ml)
Image: Graphical representation of standard sugar concentration vs absorbance at 520 nm
for estimation of reducing sugar content
49
A 520
0 5 10 15 20 250
0.2
0.4
0.6
0.8
1
1.2
f(x) = 0.0481372549019609 x + 0.0122549019607838R² = 0.998116222840488
Series2Linear (Series2)
Concentration of sugar standard (µg/ml)
Abso
rban
ce a
t 520
nm
Graphical representation of standard sugar concentration v/s absorbance at 520nm (A520)
for estimation of total sugar content.
50