COMPARITIVE STUDY OF THE

FERMENTATION OF VARIOUS FOOD

MATERIALS
INTRODUCTION:
Fermentation is typically the conversion of carbohydrates to alcohols and carbon
dioxide or organic acids using yeasts, bacteria, or a combination thereof, under
anaerobic conditions (absence of oxygen) by the action of enzymes. Enzymes are
complex organic compounds, generally proteins. They are highly specific with
regard to their substrates. Fermentation in simple terms is the chemical
conversion of sugars into ethanol. Ethanol fermentation, also referred to as
alcoholic fermentation is the biological process in which sugars such as glucose,
fructose, and sucrose are converted into cellular energy and thereby produce
ethanol and carbon dioxide as metabolic waste products. All ethanol contained in
alcoholic beverages is produced by means of fermentation induced by yeast.
Wine is produced by fermentation of the natural sugars present in grapes and
other kinds of fruit. Ethanol fermentation occurs in the production of alcoholic
beverages and ethanol fuel, and in the leavening of bread dough. Fermentation is
used in preservation techniques and in production of foods such as yogurt,
cottage cheese (paneer), dhokla, idli, chocolates, cheese etc. ‘Fermentation’ has
been derived from the Latin word ferver, which means ‘to boil’, as during
fermentation, there is a lot of frothing in the liquid due to evolution of carbon
dioxide. This gives it the appearance as if it is boiling!

Yeasts are unicellular eukaryotic microorganisms classified in the kingdom Fungi,

Yeast size can vary greatly depending on the species, typically measuring 3-4 µm
in diameter, although some yeasts can reach over 40 µm. Most yeasts reproduce
asexually by mitosis, and many do so by an asymmetric division process called
budding. Yeasts do not form a single taxonomic or phylogenetic grouping. The
term yeast is often taken as a synonym for Saccharomyces cerevisiae.

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Natural fermentation precedes human history. The earliest evidence of
winemaking dates from eight thousand years ago, in Georgia, in the Caucasus
area. Seven-thousand-year- old jars containing the remains of wine have been
excavated in the Zagros Mountains in Iran. There is strong evidence that people
were fermenting beverages in Babylon circa 3000 BC, ancient Egypt circa 3150 BC,
pre-Hispanic Mexico circa 2000 BC, and Sudan circa 1500 BC. Ancient fermented
food processes were developed long before man had any knowledge of the
existence of the microorganisms involved.

When studying the fermentation of sugar to alcohol by yeast, Louis Pasteur

concluded that the fermentation was catalyzed by a vital force, called “ferments”,
within the yeast cells. The “ferments” were thought to function only within the
yeast cells. The “ferments” were thought to function only within living organisms.
Nevertheless, it was known that yeast extracts (Yeast extract is the name given to
processed yeast products made by extracting the cell contents (removing the cell
walls)) can ferment sugar even in the absence of living yeast cells. While studying
this process in 1897, Eduard Buchner found that sugar was fermented even when
there were no living yeast cells in the mixture; by a yeast secretion that he termed
zymase, i.e., fermenting activity of yeast is due to active catalyst of biochemical
origin. In 1907 he received the Nobel Prize in Chemistry for his research and
discovery of “cell-free fermentation.”

Main uses of fermentation

The primary benefit of fermentation is the conversion of sugars and other
carbohydrates, e.g., converting juice into wine, grains into beer, carbohydrates
into carbon dioxide to leaven bread, and sugars invegetables into preservative
organic acids.

Food fermentation has been said to serve five main purposes:

 Enrichment of the diet through development of a diversity of flavors,

aromas, and textures in food substrates.

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  Preservation of substantial amounts of foods through lactic acid,
alcohol, acetic acid, and alkaline fermentations
 Biological enrichment of food substrates with protein, essential
amino acids, essential fatty acids, and vitamins
 Elimination of antinutrients
 A decrease in cooking time and fuel requirement

OBJECTIVE
In this project, time taken for fermentation of various fruit / vegetable juices had
to be compared. Fermentation is one of the oldest methods of processing food
into a form that is suitable for preservation.

In fermentation technology, we stress in understanding the various process in

fermentor and how various intrinsic factors influence the fermentation process.
Fermentation technology being an industrial microbiology subject are geared in
producing maximum amount of high economical fermentation products. The
objective of this project is to compare the rates of fermentation of different fruit
and vegetable juices. The information gained from this experiment may be used
by wineries to determine which fruit juice ferments best. But it is difficult to
understand and control the fermentation process as it involves various
components such as effect of substrates, products inhibition, conditions and
complex microbial interactions. Fermentation is affected by several factors
including the temperature, salt concentration, pH, oxygen availability and nutrient
availability. The rate of fermentation can be controlled by manipulating any of
these factors.
Temperature
Different yeasts tolerate different temperatures. For Saccharomyces cerevisiae, it
is around 35-400C. A variation of just a few degrees from this temperature alters
the activity of the microbes and affects the quality of the final product.

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Nutrients i.e. Sugar content
All bacteria require a source of nutrients for metabolism. The fermenters require
carbohydrates, in this case sugars glucose and fructose. The energy requirements
of microbes are very high. Limiting the amount of substrate available can reduce
the rate of fermentation.

Effect of oxygen
If oxygen is present, some species of yeast will oxidize pyruvate completely to
carbon dioxide and water. Thus, these species of yeast will produce ethanol only
in an anaerobic environment. However, many yeasts such as the baker’s yeast

Saccharomyces cerevisiae, or fission yeast Schizosaccharomyces pombe, prefer

fermentation to respiration. These yeasts will produce ethanol even under
aerobic conditions.

Hence the rate of fermentation varies.

The fermentation process is not only complex but always in a state of flux.
Process, we are therefore in a situation to always be adaptive and reactive to
these changes so that throughout the fermentation process we are always
sustaining the conditions in a narrow window of optimal fermentation conditions.

In order to help us do this we need to know fermentation kinetics. When we talk

about fermentation kinetics we are talking about fermentation models. Kinetics
and modellings are very useful to us as tools to make fermentation predictions
and enhancing our experimental designs to be more focused to the specific
problems such as the rate limiting steps or product inhibition.

The study of fermentation kinetics helps us by providing clear quantitative data

for us to understand the process and improve the process accordingly. Peering
into observation ports might be good advertising gimmick for fermentation
technology but do not really help much in understanding the process or even to
control and predict the fermentation outcome. Subjective observations will rarely
help in producing optimum fermentation process and thus affect profitability
studies and making decisions.

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Its numbers that count!

Thus the importance of the study of fermentation kinetics or models.

The first step in the study of fermentation kinetics is to understand the various
processes involved in the whole process. Such questions such as inputs and
outputs, the metabolic pathways involved and type of products or side products
formed. The various individual reactions involved and what factors control the
metabolite levels. Then only after all the relevant data are obtained do we start
formulating the models.

THEORY
Fermentation is the slow decomposition of complex organic compounds into
simpler compounds by the action of enzymes. Enzymes are biological molecules
that catalyze (i.e, increase the rates of) chemical reactions. Fruit and vegetable
juices contain sugar such as sucrose, glucose and fructose. The chemical
equations below summarize the fermentation of sucrose, whose chemical
formula is
C12 H22 O11. One mole of sucrose is converted into four moles of ethanol and four
moles of carbon dioxide:

C12H22O11 + H2O + Invertase  2 C6H12O6

Glucose + Fructose

C6H12O6 + Zymase  2 C2H5OH + 2CO2

Glucose + Fructose

Sucrose is hence first converted to glucose and fructose with the enzyme
invertase, while enzyme zymase converts glucose and fructose to ethyl alcohol.

Invertase
Invertase (systematic name: beta-fructofuranosidase) is an enzyme that catalyzes
the hydrolysis (breakdown) of sucrose. Related to invertases are sucrases.
Invertases and sucrases hydrolyze sucrose to give the same mixture of glucose

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and fructose. Invertases cleave the O-C (fructose) bond, whereas sucrases cleave
the O-C (glucose) bond.

For industrial use, invertase is usually derived from yeast. It is also synthesized by
bees, who use it to make honey from nectar. Optimum temperature at which the
rate of reaction is at its greatest is 600 C and an optimum pH of 4.5.

Invertase
C12H22O11 + H2O C6H12O6 + C6H12O6
Sucrose Glucose Fructose

Zymase
Zymase is an enzyme complex (“mixture”) which catalyzes the fermentation of
sugar into ethanol and carbon dioxide. They occur naturally in yeasts. Zymase
activity varies among yeast strains.

Zymase
C6H12O6 + C6H12O6 2C2H5OH + 2CO2
Glucose Fructose Ethanol

Chemical test: Fehling’s solution

To test for the presence reducing sugars to the juice, a small amount of Fehling’s
solution is added and boiled in a water bath. During a water bath, the solution
progresses in the colors of blue (with no glucose present), green, yellow, orange,
red, and then brick red or brown (with high glucose present). A colour change
would signify and the presence of glucose.

Sucrose (table sugar) contains two sugars (fructose and glucose) joined by their
glycosidic bond in such a way as to prevent the glucose isomerizing to aldehyde,
or the fructose to alpha-hydroxy-ketone form. Sucrose is thus a non-reducing
sugar which does not react with Fehling’s solution.(Sucrose indirectly produces a
positive result with Benedict’s reagent if heated with dilute hydrochloric acid
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prior to the test, although after this treatment it is no longer sucrose.) The
products of sucrose decomposition are glucose and fructose, both of which can
be detected by Fehling’s as described above.

By comparing the time required for completion of fermentation of equal amounts

of different substances containing starch the rates of fermentation can be
compared.

Addition of yeast
In wine making, yeast is normally already present on grape skins. Fermentation
can be done with this endogenous “wild yeast,” but this procedure gives
unpredictable results, which depend upon the exact types of yeast species
present. For this reason, a pure yeast culture is usually added, this yeast quickly
dominates the fermentation. Baker’s yeast is the common name for the strains of
yeast commonly used as a leavening agent in baking bread and bakery products,
where it converts the fermentable sugars present in the dough into carbon
dioxide and ethanol. Baker’s yeast is of the species Saccharomyces cerevisiae,
which is the same species commonly used in alcoholic fermentation, and so is also
called brewer’s yeast.

Pasteur’s salt
Pasteur’s salt solution is prepared by dissolving ammonium tartarate, 10.0 g;
potassium phosphate, 2.0 g; calcium phosphate, 0.2 g; and magnesium sulphate,
0.2 g dissolved in 860 ml of water.

The Pasteur’s salts in solution act as a buffer to any acids the yeast may create.
Since yeast only converts sugar (most likely sucrose or glucose) to ethanol under
anaerobic conditions, and it is unreasonable to assume that there will be no
oxygen present in the laboratory, some acetic acid is created as a result. The
Pasteur salts act as buffers to the acidity so that the proteins in the yeast do not
become denatured.

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 PROCEDURE

1. 5.0 ml of apple juice was taken in a clean 250 ml conical flask and
diluted with 50 ml of distilled water
2. 2.0 gram of Baker’s yeast and 5.0 ml of solution of Pasteur’s salts
were added to the above conical flask.
3. The contents of the flask were shaken well and the temperature of the
reaction mixture was maintained between 35-400C.
4. After 10 minutes 5 drops of the reaction mixture were taken from the
flask and added to a test tube containing 2 ml of Fehling reagent. The
test tube was placed in a boiling water bath for about 2 minutes. The
colour of the solution or precipitate was then noted.
5. Step 4 was repeated after every 10 minutes until the reaction mixture
stopped giving any red colour or precipitate.
6. This time taken, i.e. time taken for the completion of fermentation was
noted.
7. All the above steps were repeated by taking 5 ml each of grape juice,
black grape juice, sweet lime juice, orange juice and carrot juice.

Precautions:
 All apparatus should be clean and washed properly.
 The flask should not be rinsed with any of the solution.

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 OBSERVATION

Volume of fruit juice taken = 5.0 ml

Volume of distilled water added = 50.0 ml
Weight of baker’s yeast added = 2.0 g
Volume of solution of Pasteur’s salts = 5.0 ml

Time Colour of reaction mixture on reaction with Fehling solution

( in
minutes ) Apple Juice Sweet lime Carrot Orange Tomato Juice
Juice Juice Juice
10 Red Red Red Red Red
20 Red Red Red Red Brownish
Red
30 Red Red No Change Red Brown
40 Red Red No Change Brown Dark Brown
50 Brownish Greenish No Change No Change No Change
Red Brown
60 Brown No Change No Change No Change No Change
70 No Change No Change No Change No Change No Change

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Graph:

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RESULT

The time taken for fermentation of carrot juice was well before the rest of the
juices, it’s recorded time being 30 minutes. This means that carrot juice has the
highest sucrose content from the various samples taken. After 50 minutes orange
and tomato juices gave positive test for fermentation with Fehling’s solution. For
sweet lime juice time taken for fermentation was 60 minutes and for apple juice it
was 70 minutes.

REFERENCE:

1.Wikipedia - The free encyclopedia - (http://en.wikipedia.org)

2.Comprehensive Practical Chemistry

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