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 wine making 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 and grain flour 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 35-40*C. 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 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 theprocess 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.
 AIM AND LIMITATION

AIM
The aim 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 particular species of micro-oganisms.
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.
 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
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-fructo furanosidase) is an enzyme
that catalyzes the hydrolysis (breakdown) of sucrose. Related to
invertases are surcrases. 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*C 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 color 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 and
grain flours 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

• Busen burner, tripod stand and watch glass

 PROCEDURE

a) Take 5 gms of wheat flour in 100 ml conical flask and add 30

ml of distilled water.
b) Boil the contents of the flask for about 5 minutes
c) Filter the above contents after cooling, the filtrate obtained is
wheat flour extract.
d) To the wheat flour extract. taken in a conical flask. Add 5 ml
of 1% aq. NaCl solution.
e) Keep this flask in a water bath maintained at a temperature of
50-60 degree celsius. Add 2 ml of malt extract.
f) After 2 minutes take 2 drops of the reaction mixture and add
to diluted iodine solution.
g) Repeat step 6 after every 2 minutes. When no bluish color is
produced the fermentation is complete.
h) Record the total time taken for completion of fermentation.
i) Repeat the experiment with gram flour extract, rice flour
extract, potato extract (juice) and record the observations.
With Fehling Solution :

a) 5.0 ml of apple juice was taken in a clean 250 ml conical flask

and diluted with 50 ml of distilled water.
b) 2.0 gram of Baker’s yeast and 5.0 ml of solution of Pasteur’s
salts were added to the above conical flask.
c) The contents of the flask were shaken well and the
temperature of the reaction mixture was maintained
between 35-40*C.

d) 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.
e) The test tube was placed in a boiling water bath for about 2
minutes
f) The color of the solution or precipitate was then noted.
g) Step 4 was repeated after every 10 minutes until the
reaction mixture stopped giving any red color or precipitate.
h) This time taken, i.e. time taken for the completion of
fermentation was noted.
i) 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
Time required for the fermentation- (grain flour)
Grain flour Time of fermentation taken
(in hours)
Wheat flour 10
Gram flour 12.5
Rice flour 15
Potato extract 13

Time required for the fermentation- (with Fehling solution)

Time Color of reaction mixture on reaction with Fehling solution
(in
mins) Apple Sweet Carrot Orange Tomato
Juice lime Juice Juice Juice
Juice
10 Red Red Red Red Red

20 Red Red Red Red Brownish

Red
30 Red Red No Red Brown
Change
40 Red Red No Brown Dark
Change Brown
50 Brownish Greenish No No No
Red Brown Change Change Change
60 Brown No No No No
Change Change Change Change
70 No No No No No
Change Change Change Change Change