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 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 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 fermenter 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 35-40C. 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/ 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 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
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 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 cerevisiae
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 oxaloacetate.

ACETIC ACID PRODUCTIONS:

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:

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 600oC and an optimum pH of 4.5.
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.

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.

Apparatus and Chemicals Required:

▪ Pasteur’s salts
▪ Yeast
▪ Fehling’s Reagent
▪ Conical Flask
▪ Test tubes
▪ Beakers
▪ Bunsen Burner
▪ Tripod stand
▪ 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,
sweet lime juice, orange juice, tomato juice and carrot juice.
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
Graph
Result:

The time taken for fermentation of carrot juice was well before the rest of the
juices, its 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.
 BIBILIOGRAPHY

● Wikipedia
● Chemistry Class 12 NCERT
Contents:
1. Introduction
1.1. Main uses of fermentation
2. Objective
2.1. Temperature
2.2. Nutrients/Sugar content
2.3. Effects of Oxygen
3. Scopes and Limitation
3.1. Scope
3.2. The Production of alcohol
3.3. The production of Citric acid
3.4. Acetic acid productions
3.5. A Versatile reaction
3.6. Limitation
4. Principle/Theory
4.1. Invertase
4.2. Zymase
4.3. Chemical test: Fehling’s solution
4.4. Addition of yeast
4.5. Pasteur’s salt
5. Experiment
5.1. Aim
5.2. Apparatus and Chemicals required
5.3. Procedure
5.4. Observation
5.5. Graph
5.6. Result
6. Bibliography