CELL BIOLOGY LABORATORY REPORTSubject : Cell Biology LaboratoryLecturer : Dr.rer.nat. Maruli PandjaitanInstructor : Ms. Nani Pasaribu, M. Si; Ms. Sylvia Yusri, S. SiFaculty/Class : Life Science/LS 1 ADate of Experiment : 25 September 2013Date of Lab. Report : 9 October 2013Semester : 1Time of Experiment : 14-00 – 16.00 pm

Experiment: Fermentation

NAME:

Felicia Melissa, Kristania Hadhiwaluyo, Chita Sakina Putri, Jonathan Winarno,Nabila Putri

Group : 4

S W I S S G E R M A N U N I V E R S I T Y

Campus BSD CityBumi Serpong DamaiTangerang 15321 – Indonesia

Tel.+62 21 537 6221Fax. +62 21 537 6201

sgu.info@sgu.ac.id

www.sgu.ac.id

I. Objectives To determine which fermentation of sugar, alcoholic fermentation using lactose or

starch, that produces more products in the form of carbon dioxide gas, CO2(g).

II. Theoretical Background Carbohydrates

First and foremost, carbohydrates are large biological molecules or macromolecules that

plays a significant role in living things. They play an important role as an energy source

through the process of oxidation, energy storage, precursor to other important biological

molecules, and dietary fiber in the form of cellulose. Carbohydrates also form part of the

structures of some cells and tissues. In terms of its structure, this macromolecules is also

known as “saccharides (saccharo , Greek for “sugar”) that contain large quantities of

hydroxyl groups along with aldehydes or ketones functional group. Theoretically speaking,

it can be divided into varieties of categories such as monosaccharide (as its monomer),

disaccharides, oligosaccharides, and polysaccharides.

Monosaccharide

Primarily monosaccharide is the monomer of this macromolecules, it plays an important role

as chemical energy storehouse & durable building material for biological construction, it

exists with the general formula (CH2O)n . In terms of its structure, it is composed of carbon

atom connected with single bonds as its backbone, one carbonyl group (C=O), and at least

two hydroxyl (-OH) groups. It is colorless, crystalline, and water soluble due to the presence

of polar hydroxyl group that is able to form hydrogen bond with adjacent water molecules.

Additionally due to the presence of a chiral carbon atom (4 different groups surrounding the

central carbon atom), carbohydrates exist with a stereoisomer properties which enable them

to form 2 identical molecules of carbohydrates with different arrangement due to the

difference in their positioning in space, even though they are made of the same atoms,

connected in the same sequence or in other words enantiomers. On the other hand, the cyclic

form of carbohydrates also can exist as isomer with difference in their structural

arrangement of atoms, raising many different types of monosaccharaides shown below.

If hydroxyl group of carbon 1 is below plane of ring à α-pyranose or α-glucose

If -OH above plane of ring à β−pyranose or β−glucose

Four isomers are cyclic, two of them with a pyranose (six-membered) ring, two with a

furanose (five-membered) ring. Galactofuranose occurs in bacteria, fungi and protozoa.

The monosaccharide itself

is actually divided into 2

big groups depending on

the type of functional

group that it contains and

the number of carbon

atoms that are present in

its backbone. In terms of

number of carbon atoms in

its backbone,

monosaccharide can be defined as triose (3 carbons), tetrose (4 carbons), pentose (5

carbons), hexose (6 carbons), or heptose (7 carbons) sugar. However in terms of functional

group, usually they are classified into either aldose or ketose sugars. Ketose sugars is

primarily a sugar containing a ketone functional group. It is also known as fruit sugar or

fructose. Pure, dry fructose is a very sweet, white, odorless, crystalline solid and is the most

water-soluble of all the sugars, it is found in honey, tree and vine fruits, flowers, berries, and

most root vegetables. Whereas aldose sugars is primarily a type of sugar containing

aldehyde functional group. Two main examples of aldose sugars are glucose, an important

carbohydrate in animal (including human) physiology, diet and nutrition, and galactose,

which present in mammal’s milk and exist as epimer of glucose.

Disaccharide

The second category of carbohydrates is called disaccharides. It is formed when two

monosaccharides join together to make larger molecuels through condensation reaction

shown in the picture below to form glycosidic linkage or bond between carbon-oxygen-and-

carbon between the two monosaccharides residues. They serve primarily as readily available

energy stores and different monosaccharides join together to form different disaccharides,

for example maltose, lactose and sucrose, shown in the table below.

MALTOSE

It is the least common disaccharide in na-

ture. Through condensation reaction, it is

connected by an α-1-4 glycosdic linkage.

LACTOSE

Is a disaccharide sugar derived from galac-

tose and glucose. It is known as 4-O- β -D-

galactosylpyranosyl-a-D-glucopyranoside.

Lactose is only found in milk. It is digested

by the enzyme lactase. Pure lactose is found

in whey, the watery

byproduct of cheese production. And

similar to maltose it is also connected by a

β1-4 glycosidic linkage.

SUCROSE or table sugar

Also known as table sugar. Both cyclic

carbons of glucose and fructose are tied

together in α-1-2 glycosdic linkage.

Oligosaccharide

This third category of carbohydrates has a similar structural features as polysaccharide, it is

made up monomers of monosaccharide that joined together to form a polymer however with

smaller units (3 to 10 monosaccharide units) compared to polysaccharide. This type of

carbohydrates may be found in foods such as in beans and peas. It is usually undigested until

it reaches the large intestines triggering the release of hydrogen, carbon dioxide, and

methane gas. However oligosaccharide also plays an important role as it usually attached to

lipids and proteins converting them into glycolipids and glycoprotein that is particularly important on plasma membrane from which they project.

PolysaccharideLastly the polysaccharides, this class of carbohydrates is made up of many, many sugars

(monossacharides as its monomers) hooked together to form a very large molecule.

Primarily there are three main types of polysachharides, they are glycogen, starch, and

cellulose. Glycogen is a polymer made up of α-glucose which are joined by α(1—>4) & α(1

—>6) glycosidic linkage bonds, the presence of an α(1—>6) glycosidic linkage bonds

resulted as a branched part of the molecule. Glycogen plays an important role as a chemical

energy storehouse in most animals.

Starch, similar to glycogen is a polymer made up of α-glucose, it exists as an energy

storage house in plants, and stored as densely packed granules (starch grain) found in

membrane-bound plastids within plant cells. Like lactose, an enzyme called amylase is

required to break down starch. In terms of its structure, it is composed of two different

polymers of amylose and amylopectin. Amylose is an unbranched, helical molecule of

sugars joined by α(1—>4) glycosidic linkage whereas amylopectin is a branched polymer of

α-glucose with similar structure as glycogen but with an irregular branching pattern.

Whereas cellulose, different from the starch and glycogen which is made up of polymer of

α-glucose, this carbohydrates is made up of polymer of β-glucose, which are joined by β (1

—>4) glycosidic linkage bonds. It is a major component of the plant’s cell wall and it is

exist as a long, unbranched polymer arranged in such a way to form molecular cables that

resist pulling (tensile) forces. Cellulose also plays an important role as a dietary fiber, which

is necessary to be consume by human as it aids digestion by absorbing water and pushing

food along the digestive tract, making bowel movements easier. However it is proven that

even though cellulose present as the most abundant organic material on Earth, most

multicellular organisms lack enzyme to degrade it, however this enzyme which is called as

cellulase is found at unicellular organisms such as fungi, bacteria, and protists.

Fermentation

Fermentation is primarily a process in which microorganisms such as yeast, in the case of

this investigation, breaks down organic substance like sugar into simpler substances. It

occurs in an anaerobic condition (oxygen is not present) and triggers the breakdown of sugar

molecule into ethanol and CO2. In the real life, fermentation has been playing an important

implication particularly in the food industry where fermentation using the action of

microorganisms is considered desirable and necessary to produce alcoholic beverages such

as wine, beer, and cider. The uses of this process is also important in the pastry where yeast

is used in the process of leavening of the bread, “and for preservation techniques to create

lactic acid in sour foods such as sauerkraut, dry sausages, kimchi and yogurt, or vinegar

(acetic acid) for use in pickling foods” (Wilder, B n.d.).

In the context of cellular respiration

The actual process where fermentation happen is actually found in the process of cellular

respiration. The first step in the cellular respiration is called glycolysis, where glucose is

broken down into pyruvate through series of steps and with the help of various kinds of

enzyme without the presence of oxygen. Once glycolysis has finished thus producing

pyruvate, then with the presence of the oxygen pyruvate can undergoes an aerobic

respiration through the TCA or Kerb’s cycle producing a bigger amount of energy in the

form of ATP. However, if oxygen is still not present during the process, pyruvate can

undergoes an anaerobic respiration through fermentation pathway either forming lactate or

ethanol as its product.

Anaerobic respiration is an important process of respiration despite the fact that it only

produces lesser amount of ATP than aerobic respiration, because it is the sole source of ATP

for many anaerobic bacteria, and used by many eukaryotic cells when their oxygen supply is

low. Additionally the product of anaerobic respiration which is the coenzyme NAD+ from

NADH is considered crucial as it helps to replenish the limited supply of NAD+ during

glycolysis to proceed with its net yield of 2 ATP molecules per glucose and would cease

when its supply was exhausted, resulting in cell death. The anaerobic respiration itself is

divided into two kinds of fermentation pathway, they are lactic acid fermentation in

contracting muscle and alcoholic fermentation which is found in plants or microorganism,

for example fermentation that will be conducted by the help of yeast in this investigation.

Lactic Acid Fermentation

Lactic acid fermentation occurs by converting pyruvate into lactate using the help of

an enzyme called Lactate dehydrogenase. In this process the pyruvate accept

electrons and hydrogen from NADH thus producing NAD+ which is used to replenish

the supply NAD+ in the glycolysis process. It is responsible of the sour taste in some

food such as sauerkraut and yogurt and as a temporary energy source for human

muscle cells, however this process also triggers the buildup of lactate in the muscle,

which can damage the muscle tissue.

Alcoholic fermentation

Different from lactic acid fermentation, alcoholic fermentation is a two-step process.

Firstly, pyruvate is converted into acetaldehyde with the help of the enzyme called

Pyruvate decarboxylase and water releasing CO2. Then, after acetaldehyde has

formed, this compound accept electrons and hydrogen from the coenzyme NADH

which regenerates NAD+ which is used to replenish the supply NAD+ in the

glycolysis process, forming ethanol with the help of the enzyme Alcohol

dehydrogenase.

Yeast

Yeast (Saccharomyces cerevisiae) is a microscopic, unicellular organism that belongs to the

group of organisms called fungi. There are various kinds of yeast and some of them has

been playing an important role to humans. Some of the known uses of yeast are in the

production of bread, beer, cheese, wine, and whiskey, production of diet supplement as they

are rich in B vitamins. They are also used in “genetic engineering to produce large quantities

of certain hormones and enzymes, which are used for such medical purposes as healing

wounds and reducing inflammation. Some types of yeast, however, cause disease”

(HowStuffWorks 2013) such as candidiasis.

In terms of its structure, yeast cell is relatively bigger in size compared to the cell of a

bacterium however smaller than a plant cell. It is oval or round in shape and has a thin

membrane. Their cell walls consist of “an outer layer of mannoprotein which are associated

with glucanes, an inner layer of glucannes associated with chitin, and a cytoplasmic

membrane with a high protein complex content”. Yeast are eukaryotic organisms, therefore

its cell contains similar organelles with those eukaryotic cells in general. It contains nucleus

with 16 linear chromosomes, mitochondria as its powerhouse to generate ATP, ribosomes

(with the same size as plant and animal cells), however without the presence of chloroplasts.

Yeasts are known for its ability to undergoes fermentation process (an anaerobic respiration)

in which they secrete enzymes that break down food in the form of organic matter,

particularly sugars, into nutrients they can absorb, producing carbon dioxide and ethanol.

Those organic matters that can be fermented are fructose, glucose, and other

monosaccharide (simple sugars), which are found in most fruits. The enzymes found in the

yeast chemically break down these simple sugars into products that the cell can use,

however there are other species of yeast found in nature that can also chemically break

down disaccharides (double sugars) into its monossacharide units. The application of this

process is commonly used to turn “fruit juices into wine and helps turn wort (diluted grain

mash) into beer or whiskey. The carbon dioxide produced by fermentation makes the

bubbles in beer and some kinds of wine, and causes bread to rise. As bread bakes, the

alcohol produced by fermentation evaporates” (HowStuffWorks 2013).

Primarily in the process of fermentation, yeast uses various kinds of enzymes to help them

in breaking down the sugars. Biologically speaking, enzymes are substances that are used to

increase the rate of reaction without itself undergoing a chemical change by lowering the

overall activation energy of the reaction. Theenzymes that may be found in the yeast are

sucrase (invertase), zymase, maltase (glucase), lactase, hexosephosphatase, reductase,

carboxylase, melibiase, and endo-tryptase, as well as proteolytic enzymes. However it is

proven that not all kinds of yeast containing the same enzymes, instead each type of yeast

behave differently towards the various kinds of sugar. For example, “most yeasts can invert,

and then ferment, cane-sugar, because the enzyme sucrase (invertase) is of common

occurrence in the yeasts. On the other hand, the enzyme lactase is absent from the majority

of yeasts, and hence these are incapable of fermenting milk-sugar.”

Primarily in this investigation there are two different kinds of sugars that is used in the

fermentation process, firstly is starch (a polysaccharide sugar) and lactose (a disaccharide).

In this investigation, these two different sugars are expected to produce different amount of

product in the form of CO2 (carbon dioxide gas) during the process.

YEAST AND STARCH

Theoretically, yeast cannot effectively break down the starch even though it is composed of

units of sugar molecules which is simply polymer of a of α-glucose. Therefore in the pro-

duction of fermented beverages like beer, the starch that is found in the raw material must be

broken down into its monosaccharide or disaccharides sugar units through series of steps by

the help of enzymes as yeast is unable to digest it unless it is broken down into its glucose

units. In terms of its structure, the starch itself exist as a mixture of two different polymer,

an unbranched chain form called amylose and a branched form called amylopectin, and for

yeast to ferment a starch, these two polymers must be broken down by a specific enzyme

called amylase or alpha and beta-amylase to be specific.

Amylase enzyme must be present in order to break down starch into its monomer. Alpha-

amylase catalyze the breakdown of amylopectin in starch into smaller chain of various

lengths, the smaller chain main contain a monosaccharide unit (glucose), a disaccharide

(maltose), or a larger molecules which contain many glucose units. Whereas beta-amylase

help catalyze the breakdown of amylose in starch into separate molecules of maltose, a dis-

accharide sugar. Finally once the starch has been decomposed containing only monosaccha-

ride and disaccharide, the yeast produce the enzyme maltase to break the maltose, formed by

the breakdown formed with the help of beta-amylase, into simple sugars (glucose) mole-

cules that it can ferment producing alcohol and carbon dioxide.

YEAST AND LACTOSEPrimarily, lactose is unable to be fermented by yeast in fermentation process. It is due to the

fact that the enzyme used in the breakdown of lactose into its component, galactose and

glucose, is not common in yeast and absent in major species of yeast. Thus are only able to

breakdown lactose into its component when they are genetically engineered to produce

lactase. Primarily, lactase is an enzyme that has an ability to split lactose into its sugar

monomers. It is found in the intestine of young mammals, in plants, fungi, yeasts and bacteria.

Although this enzyme is commonly absent from the yeast, through the advance of genetic-

related technologies, a species of yeast has been genetically modified and biotechnology

created in such a way that they will have the ability to break down lactose which is called

Kluyveromyces lactis.

Kluyveromyces lactis or diary yeast is a species of a scientifically and biotechnologically non-

Saccharomyces important yeast that may be found in milk-derived products.

Since the 1950s, K. lactis has been used as a source of lactase (beta-galactosidase), an

enzyme that degrades milk suga(lactose) and is necessary for production of lactose-free

dairy products. And these days, it is commonly used for genetic studies and in the food

industry due to is ability to break down lactose into its constituent sugar components.

Therefore yeast is unable to decompose lactose into simple sugar as the enzyme is

commonly absent in major species of yeast found commercially, whereas yeast still will be

able to break down starch into simple sugars even though it is not effective as breaking

monosaccharide (simple sugars) or disaccharide sugars (except lactose) it is due to the fact

that the polymer of starch which are amylose and amylopectin must be broken down into

shorter chain by various types of enzymes before it can undergoes fermentation by yeast.

III. Apparatus and Materials Apparatus

Materials

IV. Procedure

V. Data

VI. Discussion

VII. Conclusion

VIII. References http://www.bio.brandeis.edu/classes/bio18/glycogen.gif

http://biochemable.wordpress.com/tag/amylose/

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http://themedicalbiochemistrypage.org/carbohydrates.php

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http://classes.ansci.illinois.edu/ansc438/milkcompsynth/milksynth_lactosesynth.html

http://www.rsc.org/Education/Teachers/Resources/cfb/carbohydrates.htm

http://www.elmhurst.edu/~chm/vchembook/546maltose.html

http://www.elmhurst.edu/~chm/vchembook/546lactose.html

http://www.elmhurst.edu/~chm/vchembook/546sucrose.html

http://www.elmhurst.edu/~chm/vchembook/547glycogen.html

http://www.elmhurst.edu/~chm/vchembook/547starch.html

http://www.elmhurst.edu/~chm/vchembook/547cellulose.html

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http://hyperphysics.phy-astr.gsu.edu/hbase/biology/celres.html#c3

http://hyperphysics.phy-astr.gsu.edu/hbase/biology/ferment.html

http://www.healingnaturallybybee.com/articles/ferment1.php

http://www.exploreyeast.com/article/what-yeast

http://science.howstuffworks.com/life/fungi/yeast-info.htm

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http://www.ask.com/question/yeast-ferment-lactose

http://answers.yahoo.com/question/index?qid=20100315124900AA74F8E

http://genolevures.org/klla.html

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1364.2006.00049.x.pdf?

v=1&t=hnjtf67w&s=ba1dc60b8c217596cd0fc950b79f601decd03b58

Cell Biology Laboratory Module