CHEMISTRY

PROJECT
2023-24
Topic:
“TO COMPARE THE RATES OF
FERMENTATION OF THE FOLLOWING
FRUIT OR VEGETABLE JUICES”

Session
2024-25

Guided By: SUBMITTED BY:

Mr. Satyaveer Aditya Kumar
Singh Sisodiya
 CERTIFICATE

This is to certify that Aditya kumar

Singh student of Class 12th has
successfully completed the investigation
Project on the Topic " TO COMPARE THE
RATES OF FERMENTATION OF THE
FOLLOWING FRUIT OR VEGETABLE
JUICES " in fulfilment of curriculum of
CENTRAL BOARD OF SECONDARY
EDUCATION leading to the award of
annual examination of the year 2024-25.

Mr. Satyaveer Singh

(Internal Examiner)
 ACKNOWLEDGEMENT

It gives me great pleasure to express my

gratitude towards our chemistry teacher Mr.
Satyaveer Singh sir for her guidance, support
and encouragement throughout the duration
of the project on the topic " to compare the
rates of fermentation of the following
fruit or vegetable juices"

Then I would like to thank my parents and

friends who have helped me with their
valuable suggestions and guidance has been
helpful in various phases of the completion of
the project. Without their motivation and help,
the successful completion of this project
would not have been possible. I hope you will
like this project.

Thanking you!
Aditya kumar Singh
 INDEX

1. INTRODUCTION
2. OBJECTIVE
3. SCOPE AND LIMITATION
4. PRINCIPLE/THEORY
5. EXPERIMENT
6. AIM
7. REQUIREMENT
8. PROCEDURE
9. OBSERVATION
10. RESULT
11. BIBLIOGRAPHY
 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 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 catalysed 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 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.

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 modelling 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 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.
 SCOPE AND LIMITATION
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
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 need to be taken
to access its actual value.
 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:
C 12 H 22 O11 + Invertase → 2C 6 H 12 O6

Glucose + Fructose
 C 6 H 12 O6 + Zymase → 2 C2 H 5 OH + 2 C O2
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 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.
C 12 H 22 O11 + H2 O Invertase C H O + C H O
6 12 6 6 12 6

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.
C 6 H 12 O6 + C 6 H 12 O6 Zymase 2 C2 H 5 OH + 2 C O2
→

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 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.
 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:
1. Chemical Requirement
a. Pasteur’s salts
b. Yeast
c. Fehling’s reagent
2. Apparatus Requirement
a. Conical flasks
b. Test tubes
c. Beaker
d. Bunsen burner, tripod stand and watch glass
PROCEDURE
1. 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.
3. 2.0 gram of Baker’s yeast and 5.0 ml of solution of Pasteur’s salts
were added to the above conical flask.
4. The contents of the flask were shaken well and the temperature of
the reaction mixture was maintained between 35-400C.
5. 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.
6. Step 4 was repeated after every 10 minutes until the reaction
mixture stopped giving any red colour or precipitate.
7. This time taken, i.e. time taken for the completion of fermentation
was noted.
8. 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:
1. All apparatus should be clean and washed properly.
2. The flask should not be rinsed with any of the solution.
 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 Colour of reaction mixture on reaction with
Fehling solution
(In Apple OrangeJuic CarrotJuice
minutes) Juice e
10 Re Re Re
d d d
20 Re Re Re
d d d
30 Re Re No
40 d d Change
No
Re Brow
d n Change
50 Brownish No No
Red Change Change

Carrot juice

Orange juice

Apple juice

0 10 20 30 40 50 60 70 80