COMPARATIVE STUDY OF RATE OF FERMENTATION

OF FRUIT/VEGETABLE JUICES

A CHEMISTRY PROJECT REPORT

SUBMITTED BY SUBMITTED TO
NIYAMATULLA ANSARI SANIYA FIRDOUS
IN PARTIAL FULFILMENT OF THE
CBSE GRADE XII
IN
CHEMISTRY
AT

AZAMGARH PUBLIC SCHOOL

KOTILA,CHECKPOST,AZAMGARH

UP,INDIA

2018-2019
 Preface
As a part of the course curriculum and to deepen and widen practical
knowledge in the concept of "FERMENTATION OF
FRUIT/VEGETABLE JUICES" students are required to make project
report on FERMENTATION OF FRUIT/VEGETABLE JUICES.

Completing the project helped us know more about the concept

FERMENTATION OF FRUIT/VEGETABLE JUICES. Working with
other team members taught us the importance of cooperation, coordination,
and synergy. I hope you will find our project report interesting. All
constructive criticism and feedback is cordially invited.
 ACKNOWLEDGEMENT

I would like to thank my teachers, Ms. SANIYA Ma'am 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, Mrs.Huma Waseem for providing

me the opportunity to work on this project.

It goes without saying that my classmates, Belal, Ahmad, Divyansh for their
help in due course of this project. My
parents have also played a part in helping me in this project. My thanks goes
out to them also.

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.

Signature of the

Candidate
 CERTIFICATE

This is to certify that NIYAMATULLA ANSARI of Grade XII, AZAMGARH

PUBLIC SCHOOL, UP with register number
____________________ has satisfactorily completed the project in Chemistry on

"COMPARATIVE STUDY OF RATE OF FERMENTATION OF

FRUIT/VEGETABLE JUICES" in partial fulfillment of the requirements of
All India Secondary School Certificate Examination (AISSCE) as prescribed by
CBSE
in the year 2018-2019.

Signature of the Signature of the

Candidate Teacher In-Charge

Signature of the Signature of the

Principal External Examiner
 CERTIFICATE

This is to certify that NIYAMATULLA ANSARI of Grade XII, AZAMGARH

PUBLIC SCHOOL, UP with register number
____________________ has satisfactorily completed the project in Chemistry on

"COMPARATIVE STUDY OF RATE OF FERMENTATION OF

FRUIT/VEGETABLE JUICES" in partial fulfillment of the requirements of
All India Secondary School Certificate Examination (AISSCE) as prescribed by
CBSE
in the year 2018-2019.

Signature of the Signature of the

Candidate Teacher In-Charge

Signature of the Signature of the

Principal External Examiner
 ABBREVIATION

Sl.No Abbreviation Expansion

1 C Centigrade
2 g gram
3 ml milliliter
4 O Oxygen
5 C Carbon
 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
 ABBREVIATION

Sl.No Abbreviation Expansion

1 C Centigrade
2 g gram
3 ml milliliter
4 O Oxygen
5 C Carbon
 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.
 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 needs 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:

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 60 0 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.

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:

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
 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.
 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
Apple Sweet lime Carrot Orange Tomato
minutes )
Juice 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
Graph
 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.
 BIBLIOGRAPHY

Wikipedia - The free encyclopedia

Comprehensive Practical Chemistry

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