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

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‘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

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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