SHREE SWAMINARAYAN

PUBLIC SCHOOL
GANDHINAGAR

CHEMISTRY INVESTIGATORY PROJECT

On

To compare rate of fermentation of

following fruit juices:-
1.Apple juice,
2.Orange juice,
3.Carrot juice
2021-22 (CLASS-XII)

SUBMITTED BY: HONEY MODI

BOARD’S ROLL NO: ___________________

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 CERTIFICATE

This is to certify that Honey Modi of class XII science has

prepared the investigatory project of chemistry entitle To
compare rate of fermentation of following fruit
juices:-1.Apple juice, 2.Orange juice, 3.Carrot juice during
academic year 2021-2022. She has prepared the project under
my guidance.

__________________ _____________________ __________________

Ext. Examiner PGT Chemistry Principal’s Sign

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 ACKNOWLEDGEMENT
I Honey Modi would like to express my sincere gratitude to
my chemistry teacher Mrs. Poonam Matai for her vital
support, guidance and encouragement without which this
project would not have come forth from my side. Special
thanks goes to my teacher who helped me a lot in completing
the project by giving interesting ideas, thoughts and made this
project accurate. I wish to thank my parents for their
undivided support and interest who inspired and encouraged
me to go on our way, without which I would be unable to
complete my project.

And at last but by no mean the least I would like to thank the
almighty God who made all the things possible.

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 Table of contents

1.Introduction —---------------------------------- 6
2.Objective —------------------------------------- 8
3.Scope and limitations —----------------------- 11
4.Principal/theory—------------------------------ 13
5.Experiment—----------------------------------- 16
6.Aim—-------------------------------------------- 16
7.Requirement—---------------------------------- 16
8.Procedure—-------------------------------------- 17
9.Observation—----------------------------------- 18
10.Result—---------------------------------------- 19
11.Bibliography—-------------------------------- 19

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 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.

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

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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 in
vegetables 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.
→ 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 vitamin
→ Elimination of antinutrients
→ A decrease in cooking time and fuel requirement

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 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.

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

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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.
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

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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.

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 SCOPE AND LIMITATIONS

SCOPE

The scope of this project is as wide as the scope of process of

fermentation. This project aspires to explore one of the innumerable
applications of the biochemical concept of breakage of highly ordered
large molecules into smaller ones by the action of microorganisms or
enzymes. Some of the applications include:

THE PRODUCTION OF ALCOHOL

Beers, wines and spirits are all produced by fermenting various
carbohydrates. Yeasts do this naturally to sugars; a property that has been
utilized by humans for thousands of years. Ethanol is also produced
industrially on a large scale for use as a biofuel. This has traditionally
involved a two step fermentation procedure using aerated tanks containing
the yeast Saccharomyces cerevisciae and substrate carbohydrates.

THE PRODUCTION OF CITRIC ACID

Citric acid is a useful product in both the food and pharmaceutical
industries; it is used in food as a preservative and to produce an acidic,
sour taste in soft drinks and other beverages. In the pharmaceutical
industry it can be used as buffering agent and to clean equipment. Citric
acid is formed by the fermentation of a molasses substrate by the fungus
Aspergillus Niger. The biochemical pathway involved includes the
production of pyruvate in glycolysis, followed by its conversion to citric
acid via the condensation of acetyl co-enzyme A and oxaloaecetate.

ACETIC ACID PRODUCTION

In the presence of the Acetobacter bacterium and oxygen, fermented
carbohydrates, ciders or wines can be converted to vinegar (acetic acid).
The result is usually is usually a 5 % solution of acetic acid. Acetic acid is
used in diluted form in the food industry as a condiment and pickling

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agent. It is also employed in industry as a solvent and an important reagent
in many organic synthesis reactions.

A VERSATILE REACTION
Fermentation certainly produces a diverse range of chemicals and is
obviously a key reaction in many industries. The one thing all these
processes have in common is an initial culture containing carbohydrates
and a particular species of microorganism.

LIMITATIONS
One of the limitations of fermentation as a process is its requirement for
multiple reagents. Secondly, in many cases the time taken is quite long and
this creates a need for catalyst. Without catalysts, the reaction is extremely
slow. The limitation of our project is the slight error in the result and the
project is limited to the fermentation of the juices with Baker’s yeast and
not under normal conditions i.e. without adding Baker’s yeast.

Owing to the different criterion on which the rate of fermentation depends,

if the experiment is not carried out in the optimal temperature range, the
rates will turn out to be different than the actual rates of the juices that
have been taken.

It is not possible to get the exact theoretically estimated value due to

impurities in the reagents as well as the compounds.

Another point to be noted is that the rates calculated from this experiment
is just one case and this can’t actually access the rate of fermentation of the
fruit. An average needs to be taken to access its actual value.

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 PRINCIPLE/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 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

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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 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

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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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 Experiment
Aim:
To compare the rates of fermentation of some fruit/vegetable juices and
determine the substance which has the highest rate of fermentation
amongst the various samples taken.

Requirement:-
a. Chemical Requirement
- Pasteur’s salts
-Yeast
-Fehling’s reagent

b. Apparatus Requirement
-Conical flasks
-Test tubes Beaker
- Bunsen burner, tripod stand and watch glass

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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 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

Colour of Reaction mixture on reaction with fehling solution.

Time(in Apple juice Orange juice Carrot juice

minutes)
10 Red Red Red
20 Red Red Red
30 Red Red No Change
40 Red Brown No Change
50 Brownish Red No Change No Change
60 Brown No Change No Change
70 No change No Change No Change

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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 juices gave positive test for fermentation with Fehling’s
solution. For apple juice it was 70 minutes.

BIBLIOGRAPHY
Wikipedia - The free encyclopedia - (http://en.wikipedia.org)
Comprehensive Practical Chemistry

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