COMPARATIVE STUDY OF RATE OF FERMENTATION

Ayush S Agarwal STD.XII SEC.-B

Roll.No.–10
2020-21
 ACKNOWLEDGEMENT

I would like to express my gratitude to my chemistry

teacher, Mr. Vinesh Madhavan and our principal Mr. P
Vamshi Krishna for furnishing me with this golden
opportunity to work on the project pertaining to the
Determination of ions in toothpaste.

Secondly, I would like to thank my parents for providing

me with adequate resources to work on this project.

Working on this project has been a very insightful journey

and gave me an opportunity to understand the chemistry
behind day to day substances like vegetable juices.
Table of Contents

S.No. CONTENTS
1. Introduction

2. objective

3. Scope and limitation

4. Principle/theory

5. Experiment

6. aim

7. requirement

8. Procedure

9. observation

10. result

11. biblography
 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
1
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

2
• 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

3
 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.

4
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.

Its numbers that count!

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

5
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.

6
 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

7
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.

8
 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.

9
 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.

10
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.

11
 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

12
b. Apparatus Requirement

• Conical flasks

• Test tubes

• Beaker

13
• Bunsen burner, tripod stand and watch glass

14
 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.

15
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.

16
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.

17
Precautions:

• All apparatus should be clean and washed properly.

• The flask should not be rinsed with any of the solution.

18
 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 Sweet lime Carrot Orange Tomato
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

19
Graph

20
 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.

21
 BIBLIOGRAPHY

1. www.intechopen.com
2. www.hindawi.com
3. www.seminarsonly.com
4. www.onlinelibrary.wiley.com
5. www.sciencedirect.com
6. www.journals.plos.org
7. Wikipedia - (www.wikipedia.org)

22
23