Beruflich Dokumente
Kultur Dokumente
CHANDRAPUR
2018-2019
Investigatory project on Comparative Study Of
Rate of Fermentation of Fruit Juices
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.
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.
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.
FUTURE SCOPE
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:
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.
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.
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
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.
4. Step 4 was repeated after every 10 minutes until the reaction mixture
stopped giving any red colour or precipitate.
5. This time taken, i.e. time taken for the completion of fermentation was
noted.
6. 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:
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