Experiment No.

6
Alcohol Fermentation
BioChem Lab, BSN1A Date Submitted: November 14, 2019
Group No. 3 Date Performed: November 7, 2019

7:00 AM – 1:00 PM
De Leon, Ana Aurea A.

I. Objectives
1. To examine the fermentation property of the different sugars.
2. To examine the substrate specificity of fermentation.
II. Introduction

All living organism obtain the energy necessary to sustain life, from the oxidation of
organic substances by molecular oxygen, in the process of respiration. Under anaerobic
conditions, many organisms, including yeast, obtain the energy from the process of fermentation.
In alcoholic fermentation, characteristic of many yeast species, the fermentation process starts
with one molecule of the six carbon sugar-glucose, and terminates with two molecules of the two
carbon alcohol-ethanol, and two molecules of CO₂.
C6 H12 O6 --------- → 2 CH3 CH2 OH + 2 CO2
The CO₂ released in the process, dissolves in water and forms a carbonic acid. This acid
dissociates to form hydrogen carbonate and hydronium ions:
CO2 + 2 H2O --------- → H2 CO3 --------- → H3O⁺ + HCO3⁻
In acidic solutions, the dissolution of CO₂ in water decreases and it is released to the air
as CO₂ gas.

III. Materials

A. Equipment
Thermometer 250-ml Beaker
Universal Clamp (4) Large Test tubes
Corks (4) 25-mL Erlenmeyer flask
Wire gauze Iron stand
Mineral Oil (4) 25 cm Glass Tubing
Funnel Clay
Tripod Hot Plate
Beam balance Stirring rod
 Reagents
Glucose Yeast solution
Sucrose Ba (OH)₂
Glycerol Fructose
Starch

IV. Procedure

1. Weigh 4 g of sugar, 2 g of dry active yeast and 0.25 g of disodium hydrogen

phosphate.

2. Monitor your distilled water to reach 40° c using the thermometer

3. Add 2 g of dry active yeast and a stir bar to your buffer solution.

4. In a test tube add 3% barium hydroxide solution with 1mL mineral oil.

5. A precipitate of Barium carbonate will form as CO₂ is produced during fermentation

stir the mixture for 2 hrs.

CO₂
CO₂

Sugar Solution and yeast

 6. Dismantle the set-up. Remove the mineral oil using medicine dropper

7. Filter the precipitate that is present in the test tube using the fluted filter paper. Dry
for 1 hr at 105°c and weigh.

V. Results

Alcohol Fermentation

Carbohydrate Formation of Amount of Rate of

gas precipitate (g) fermentation
Glycerol NAD + H₂ 0g 0g/hr
Glucose 2CO₂ 0.60g 0.30g/hr
Sucrose CH3CHO + CO2 1.50g 0.75g/hr
Fructose ADP + H+ 1.20g 0.60g/hr
Starch CH3CHO + CO2 0.90g 0.45g/hr
VI. Discussion

Alcoholic fermentation is a complex biochemical process during which yeasts convert

sugars to ethanol, carbon dioxide, and other metabolic byproducts that contribute to
the chemical composition and sensorial properties of the fermented foodstuffs.
Alcoholic fermentation is the basis for the manufacturing of alcoholic beverages such
as wine and beer.
Alcoholic fermentation is carried out by yeasts and some other fungi and bacteria.
The first step of the alcoholic fermentation pathway involves pyruvate, which is
formed by yeast via the EMP pathway, while it is obtained through the ED pathway
in the case of Zymomonas (bacteria). In the following step, the pyruvate is
decarboxylated to acetaldehyde in a reaction that is catalyzed by the enzyme pyruvate
decarboxylase.
 VII.Conclusion
Alcoholic fermentation, is a biological process which converts sugars such
as glucose, fructose, and sucrose into cellular energy, producing ethanol and carbon
dioxide as by-products. Because yeasts perform this conversion in the absence
of oxygen, alcoholic fermentation is considered an anaerobic process. Alcoholic
fermentation converts one mole of glucose into two moles of ethanol and two moles
of carbon dioxide, producing two moles of ATP in the process.

VII. Assessment

Question:

1. Explain why glycerol is not fermented under the given set of conditions.

Yeast cannot metabolize glycerol through the anaerobic fermentation. Conversion of glycerol to
ethanol will produce 1 molecule more NADH, which cannot be re-oxidized into NAD under
anaerobic condition. This coenzyme redox imbalance is the cause why glycerol is not a
fermentable carbon source.

2. How does yeast get fructose into glycolysis?

Adenine second phosphorylation reaction follows the isomerization step. Fructose 6-phosphate is
phosphorylated by ATP to fructose 1,6-bisphosphate (F-1,6-BP). The prefix bis- in bisphosphate
means that two separate monophosphate groups are present, whereas the prefix di- in
diphosphate (as in adenosine diphosphate) means that two phosphate groups are present and are
connected by an anhydride bond.

This reaction is catalyzed by phosphofructokinase (PFK), an allosteric enzyme that sets the pace
of glycolysis (Section 16.2.1). As we will learn, this enzyme plays a central role in the
integration of much of metabolism.

3. What class of glycoside enzymes must yeast possess to effect fermentation of

carbohydrates?

Many glycoside hydrolases, and specifically amylases and cellulases, have not only a catalytic
domain by which they are identified, but also other domains, some of which are second catalytic
domains. However, it is more likely that these additional domains bind solid polysaccharides.
CAZy has classified these carbohydrate-binding domains (CBMs) in much the same fashion as
the glycoside hydrolase classification (Table 2). At present, almost forty CBM families
differentiated by primary structure exist. Of these, four CBM families bind starch and a further
eleven bind cellulose. Cellulose-binding CBMs often can bind other β-linked nonglucan
polysaccharides such as xylan and chitin. Furthermore, a substantial number of CBMs do not
bind glucans at all.

4. What monosaccharides would be produced by hydrolyzing lactose, maltose, sucrose,

and starch and which of these reactions yeast seem able to carry out? Explain.

Maltose

It is formed most often by the partial hydrolysis of starch and glycogen. In the manufacture of
beer, maltose is liberated by the action of malt (germinating barley) on starch; for this reason, it
is often referred to as malt sugar. Maltose is about 30% as sweet as sucrose. Hydrolysis
reactions are catalyzed by enzymes such as maltase. The same reactions can be carried out in the
laboratory with dilute acid as a catalyst, although in that case the rate is much slower, and high
temperatures are required. The hydrolysis of maltose produces two molecules of D-glucose.

H+ or maltase

maltose−−−−−−−−→ 2D-glucose

Maltose is a reducing sugar. Thus, its two glucose molecules must be linked in such a way as to
leave one anomeric carbon that can open to form an aldehyde group. The glucose units in
maltose are joined in a head-to-tail fashion through an α-linkage from the first carbon atom of
one glucose molecule to the fourth carbon atom of the second glucose molecule.

Lactose

Lactose is a reducing sugar composed of one molecule of D-galactose and one molecule of D-
glucose joined by a β-1,4-glycosidic bond (the bond from the anomeric carbon of the first
monosaccharide unit being directed upward). The two monosaccharides are obtained from
lactose by acid hydrolysis or the catalytic action of the enzyme lactase:

Sucrose

Sucrose, probably the largest-selling pure organic compound in the world, is known as beet
sugar, cane sugar, table sugar, or simply sugar. Most of the sucrose sold commercially is
obtained from sugar cane and sugar beets (whose juices are 14%–20% sucrose) by evaporation
of the water and recrystallization. The dark brown liquid that remains after the recrystallization
of sugar is sold as molasses. The sucrose molecule is unique among the common disaccharides in
having an α-1,β-2-glycosidic (head-to-head) linkage. Because this glycosidic linkage is formed
by the OH group on the anomeric carbon of α-D-glucose and the OH group on the anomeric
carbon of β-D-fructose, it ties up the anomeric carbons of both glucose and fructose.

This linkage gives sucrose certain properties that are quite different from those of maltose and
lactose. As long as the sucrose molecule remains intact, neither monosaccharide “uncyclizes” to
form an open-chain structure. Thus, sucrose is incapable of mutarotation and exists in only one
form both in the solid state and in solution. In addition, sucrose does not undergo reactions that
are typical of aldehydes and ketones. Therefore, sucrose is a nonreducing sugar.

The hydrolysis of sucrose in dilute acid or through the action of the enzyme sucrase (also known
as invertase) gives an equimolar mixture of glucose and fructose. This 1:1 mixture is referred to
as invert sugar because it rotates plane-polarized light in the opposite direction than sucrose. The
hydrolysis reaction has several practical applications. Sucrose readily recrystallizes from a
solution, but invert sugar has a much greater tendency to remain in solution. In the manufacture
of jelly and candy and in the canning of fruit, the recrystallization of sugar is undesirable.
Therefore, conditions leading to the hydrolysis of sucrose are employed in these processes.
Starch (polysaccharides) molecules undergo hydrolysis, it forms either monosaccharides,
disaccharides or trisaccharides.

The end products depends on the strength of enzymes used and the common enzymes are,

 α-Amylase, which produces the disaccharide maltose and the trisaccharide

maltotriose
 β-Amylase, which produces the disaccharide maltose
 γ-Amylase, which produces glucose
α-Amylase is an enzyme present in human saliva and β-Amylase in potatoes and other starchy
vegetables. Starch hydrolysis is, in fact, happening in human body every time we consume
carbohydrates.

Therefore yeast could carry out Maltose, starch, lactose and other monosaccharide can react to
yeast, because yeast is able to take in solutes across their cell membrane by diffusion, facilitated
diffusion, and active transport. Simple sugars, such as glucose and fructose, are taken up by
facilitated diffusion. (Unknown, 1981) Yeast may also metabolize their sugar in the presence of
oxygen, but are unable to produce ethyl alcohol. Instead, the sugars are broken down into carbon
dioxide and water. Only under anaerobic conditions, such as alcoholic fermentation, is yeast able
to produce ethanol and carbon dioxide.

VIII. References

N.A (n.d.). Retrieved from https://www.assignmentexpert.com/homework-

answers/chemistry/inorganic-chemistry/question-28649.

N.A (n.d.) Structural Biochemistry/Proteins/Purification/Isoelectric Focusing.

Retrieved from
https://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Purification/Is
oelectric_Focusing.

Biology For Major (n.d.). Retrieved from https://courses.lumenlearning.com/suny-

wmopen-biology1/chapter/proteins/

Chatterjea (1 January 2004). Textbook of Biochemistry for Dental/Nursing/Pharmacy

Students. Retrieved from https://en.wikipedia.org/wiki/Xanthoproteic_reaction

1. Jean Sloat Morton, Ph. D., Glycolysis and Alcoholic Fermentation,

www.icr.org/pubs/imp/imp-090.htm, 1980
2. Jackson: Cornell university, Alcoholic Fermentation,
www.nysaes.cornell.edu/fst/faculty/acree/fs430/lectures/thk29ferment.html, 1981

3. World Book Inc., World Book Encyclopedia, Second Edition, 1994

4. Unknown author, Cornell University, Stuck Fermentations,

www.nysaes.cornell.edu/fst/faculty/acree/fs430/lectures/thk35yeastnutrients.html,
1981

5. T. A. Davidson, Fermentation,
www.members.tripod.com/hey_ty/booze/ferment.html, 1999

6. David Gauthier, DIY CO2- yeast, www.actwin.com/fish/aquatic-

plants/month.9706/msg00187.html, 1996

7. A. Bennett, General chemistry 309, Organic and Biochemistry: Carbohydrates and

Disaccharides
https://chem.libretexts.org/Courses/Sacramento_City_College/SCC%3A_Chem_309_-
_General%2C_Organic_and_Biochemistry_(Bennett)/Text/14%3A_Carbohydrates/14.6%3A_Di
saccharides, html 2019

IX. Conforme

Prepared by:

Ana Aurea De Leon