COMPARATIVE STUDY OF RATE OF FERMENTATION

OF FRUIT / VEGETABLE JUICES

CHEMISTRY INVESTIGATORY PROJECT REPORT

SUBMITTED BY

ANKIT SHARMA
IN PARTIAL FULFILMENT OF THE

CBSE GRADE XII

IN

CHEMISTRY
AT

, MG RLY COLONY
KENDRIYA VIDYALAYA
BANGALORE- 560023
2020-2021
 CERTIFICATE

This is to certify that ANKIT SHARMA of Grade XII, KV MG RLY

COLONY, BANGALORE with register number
____________________ has satisfactorily completed the project in Chemistry on
COMPARATIVE STUDY OF RATE OF FERMENTATION OF
FRUIT/VEGETABLE JUICES in partial fulfilment of the requirements of
All India Secondary School Certificate Examination (AISSCE) as prescribed by
CBSE in the year 2020-2021.

Signature of the Signature of the

Candidate Teacher In-Charge

Signature of the Signature of the

Principal External Examiner
 ACKNOWLEDGEMENT

I would like to thank my teacher, Mr. R DHARMARAJ and Lab

Assistant Mr. NAGARAJ for guiding me through this project and for
their valuable inputs which provided me with a constant nudge for
improvement.

It is imperative to thank our Principal, Mr. D.B PATHAK for

providing me the opportunity to work on this project.

This project and reading-up on the same has provided me with an in depth
understanding of the topic.It has nurtured my scientific temperament and curiosity.

Table of Contents
 INTRODUCTION.................................................................................................................. 1
 OBJECTIVE......................................................................................................................... 3
 SCOPE AND LIMITATION................................................................................................... 5
 PRINCIPLE/THEORY .......................................................................................................... 7
 EXPERIMENT .................................................................................................................... 10
 Aim: ................................................................................................................................... 10
 Requirement: .................................................................................................................... 10
 PROCEDURE .................................................................................................................... 12
 OBSERVATION ................................................................................................................. 15
 RESULT............................................................................................................................. 16
 BIBLIOGRAPHY................................................................................................................ 17
 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

1
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.

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

2
 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.

3
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.

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.

4
 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.

5
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.

6
 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

7
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 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.

8
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.

9
 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

10
• Test tubes

• Beaker

• Bunsen burner, tripod stand and watch glass

11
 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.

12
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.

13
 • PRECAUTIONS
All apparatus should be clean
and washed properly.
• The flask should not be rinsed
with any solution.
• The type of hazards that might
occur can be classified into the
following areas:
• Microbiological hazards
• Chemical hazards
• Physical hazards

14
OBSERVATION

15
 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.

16
 BIBLIOGRAPHY

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

•Practical Chemistry Comprehensive - XII

• Class XII Chemistry Text Book

•Internet

o www.google.com
o http://fermentationtechnology.blogspot.com

17