LABORATORY MANUAL

SBK3033 FOOD SCIENCE

DR. WONG CHEE FAH

DEPARTMENT OF BIOLOGY
FACULTY OF SCIENCE AND MATHEMATICS

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PREFACE

This course will offer you opportunities to improve your applied science skills in your
future undertakings. The goal of this class is for students to make scientific measurements
of some of the important chemical reactions occurring in foods.
Students successfully completing this class will:
1. Recognize the important reactions in food chemistry and their consequences.
2. Be familiar with methods to measure these reactions.
3. Be capable of reporting their results in an appropriate format.
4. Be capable of designing and conducting an experiment to understand a simple food
science problem.

More importantly, food science experiments very often do not work out as we planned. In
chemistry labs the chemicals are pure, the conditions are controlled and you can be
expected to get a “right” answer. In food science we often have poorly defined starting
materials and many reactions occurring in parallel under non-ideal conditions.
Unsurprisingly the data we get is often noisy and hard to interpret. Wherever you go in life
you will be trying to make difficult decisions on poor data. The machine is broken – you
don’t have a good schematic or time to pull it apart but its making a squeaking noise and
drawing too much power. Can you infer the cause and propose a solution without a more
thorough diagnosis? Developing this type of skill should be a benefit of this class.
Come to the lab prepared – know what you will be asked to do and have an idea of the
results you expect. Be observant – what do your samples look like? What do they smell
like? Do you think your measurement technique is capable of giving meaningful results for
your sample? Be thorough – record your observations. Make sure you get the data you
were asked for but note your other observations. Be imaginative – once you have your
observations can you construct a scientific explanation including not only what you
expected (and why) but also what happened (and why). Have fun – remember there isn’t a
final!

Academic Integrity: The measurements you make as a working scientist will be used by
yourself and others to make decisions that will affect the profitability of companies and the
lives of individuals. It is therefore essential that you truthfully and accurately report both
what you did and what you saw and measured. You are encouraged to work together
within your research group to conduct laboratory exercises and discuss the interpretation
of results but the actual presentation of the report is your individual responsibility and you
are not permitted to copy each others’ work.

Safety Considerations: You will be working with materials and techniques that could be
harmful to you and others in the lab. Instructors will attempt to inform you of unusual

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risks but it is your responsibility to follow typical good laboratory safety practices (the
Penn State manual is available online http://www.ehs.psu.edu/safety/ Safety_Manual.doc).
If you are unsure about the safety of a procedure or your capacity to perform it safely, ask
an instructor before proceeding.

• You must wear a lab coat, safety glasses, and close toed shoes at all times
• Dispose of waste carefully. Many reagents can be washed down the sink with plenty
of water but organic solvents and some other materials must be put in designated
storage bins for safe disposal. If in doubt, ask your instructor.
• Other classes use the lab. At the end of each exercise you must leave all your
glassware clean and put away and the bench surfaces cleaned down.
• No mouth pipetting
• Label all reagents (name/date/contents)
• No eating or drinking allowed in the laboratory

Pre-Labs. To get the most of the lab you must come prepared. This will include carefully
reading the lab manual and the relevant sections of food science textbook. You should
arrive at each lab knowing what you plan to do, why, and what results you can expect.

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CONTENT

Page

Lab 1. Starch analysis ...................................................................................................................................... 5

Lab 2. Protein analysis ..................................................................................................................................... 7

Lab 3. Lipid analysis .......................................................................................................................................... 13

Lab 4. Vitamin C analysis ................................................................................................................................. 20

Lab 5. Fermentation process .......................................................................................................................... 22

Lab 6. Microbial contamination ..................................................................................................................... 27

References .............................................................................................................................................................. 32

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LAB 1. STARCH ANALYSIS
Starch is largely a mixture of amylose and amylopectin molecules. Both of these are glucose
polymers with α1-4 linkages but amylopectin also has α1-6 branch points and is a much
larger molecule. Plants use starch as a long-term energy store by locking the amylose and
amylopectin into semicrystalline granules. A cross section of a starch granule and the
arrangement of amylopectin within it are shown in the Figure. The location of the amylose
is unknown but presumed to be in the amorphous region.
A starch granule will gelatinize if heated in the presence of water. The heat and water
increases the molecular mobility of the amorphous regions (i.e., cause a glass to rubbery
transition) and the added freedom allows the crystalline region to melt. The granule looses
crystal structure, gains water and expands to many times its original size. The swollen
granules can often overlap one another leading to a great increase in solution viscosity.
Excess heating or shear can rupture the weak swollen granules leading to a decrease in
paste viscosity.
On cooling there is a progressive realignment and recrystallization first of amylose and
then the amylopectin molecules as double helices. Recrystallization acts as linking points in
the polymer matrix and can lead to the formation of a solid gel.

Experiment 1.

(a) Label four test-tubes 1-4.

10% glucose solution into tube 1

1% starch solution into tube 2

(b) Put about 20 mm (depth) of
1% albumen solution into tube 3

water into tube 4

(c) To each tube, using a dropping pipette, add three drops of iodine solution. Shake the
tube (sideways, not up and down) to mix the contents. Look for any colour changes apart
from the yellow colour of iodine itself. Copy the table below and record the results in your
notebook.

Substance Colour change after adding iodine

10% glucose solution

1% starch solution

1% albumen solution

water

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Discussion

1. The substances selected for testing are examples of three of the principle chemical
substances in cells, sugar (glucose), starch, protein (albumen). With which of these
substances did iodine react to give a colour change?

2. Does your result indicate that there is, for example, no sugar and no protein that
will give a colour change with iodine?

3. What experiments would you have to carry out in order to give a confident answer
to question 2?

4. Does the result indicate that starch will always react with iodine solution to give a
colour change?
5. What was the point of the water in tube 4?

Experiment 2. Testing food for the presence of starch

PRECAUTION. Experiment 1 showed that the starch test is sensitive, so if you use a test-
tube for more than one experiment, make sure it is thoroughly cleaned out each time or
else traces of starch from one food sample may remain on the sides and give a positive
result for a sample which, in fact, contains no starch. For the same reason, the mortar and
pestle must be washed between each test.

(a) Label six test-tubes 1~6. Copy the table given below into your notebook.

(b) Crush a 1 cm cube, or equivalent quantity, of the first food sample in a mortar with
about 10 cm3 water (50mm in a test-tube).

(c) Pour the mixture into a clean test-tube and, using a test-tube holder, heat the mixture
in a small flame of a Bunsen burner till it boils for a few seconds, shaking the tube gently all
the time.

(d) Cool the tube under a running tap.

(e) Add five drops of iodine solution.

(f) Record your results in the table in your notebook and repeat the experiment with the
next food sample.

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 Food sample Colour change with iodine Interpretation of result
1 Potato
2 Onion
3 Bread
4 Banana
5 Apple
6 Dried milk

crush with pour into boil cool under add iodine

water test-tube tap

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LAB 2. PROTEIN ANALYSIS
The goal of this experiment is to demonstrate some of the functional roles of proteins in
foods and how these can be modified through ingredient interactions.
Proteins are important functional ingredients in foods. We depend on them to form gels
and to stabilize emulsions, foams and films. In the present work we will investigate some of
the structures that can be made from whey protein isolate (WPI). Whey protein is a
mixture of globular proteins found in milk. They are a by-product of cheese manufacture
and widely sold and used as a food ingredient.
Whey protein is a mixture of globular proteins each with established primary, secondary,
tertiary and quaternary structures maintained by non-covalent interactions. We are
concerned with the inter-protein interactions that will hold the molecule in a given
conformation and intra-protein interactions that may lead to aggregation. The most
important of these here are:
• Hydrophobic interactions. Hydrophobic amino acids will try to avoid water by
either coiling into the core of the polymer or adsorbing into a non-polar solvent.
• Electrostatic interactions. Proteins have ionizable amino acids that may carry a
positive or negative charge depending on the pH. Like charges repel.

We can adjust the magnitude of the electrostatic interactions by titrating the protein
through its isoelectric point and the hydrophobic interactions by denaturing the protein.
Globular proteins can be thermally denatured by heating above a characteristic
temperature. The globule unfolds and exposed some of the hydrophobic core amino acids
to the aqueous solvent. If the structure does not regenerate, there will be a pressure to
aggregate to reduce the hydrophobic interaction. Based on these simple rules of protein
behavior we can try to understand the functional properties of whey proteins in film.
Firstly to form a solid gel, a protein must aggregate and form a continuous (i.e.,
percolating) structure throughout the container. This can be seen as a partial precipitation
of the protein because there are strong protein-protein interactions yet not so strong that
they exclude interaction with solvent.
Experiment 1. The test for protein

1. Label four test-tubes 1-4.

1% starch solution into tube 1

10% glucose solution into tube 2

2. Put about 20 mm (depth) of
1% albumen solution into tube 3

water into tube 4

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 3. Pour into each tube, about 5 mm dilute sodium hydroxide. (CARE *)

4. Add to this about 5 mm dilute copper sulphate solution. Shake the tube sideways to
mix the contents.

5. Return the tubes to the rack, leave for a few seconds and record the resulting
colours in your notebook.

Table 1.

Substance Reaction with copper sulphate and sodium hydroxide

1
2
3

* CARE. Sodium hydroxide is caustic and dissolves clothing, skin and bench tops. It is
destructive rather than dangerous so if any is spilt on the bench, neutralize it at once with
an equal volume of dilute hydrochloric acid and wipe dry. If spilt on clothing do the same
but follow with a wash in as much water as possible. If spilt on the skin do not add acid but
wash under the tap until the 'soapy' feeling is removed.

Discussion

1. The substances selected for testing are examples of three of the principal types of
chemical substances in cells; starch, sugar (glucose), protein (albumen).
2. With which of these samples did the reaction give a purple colour ?

3. The substance which gave a purple colour was a single example of its class of
substances. Would you expect all other samples of this class to give the same reaction?

4. What was the point of using test-tube 4 with the water?

NOTE This test is called the ‘biuret’ test. ‘Biuret’ is the name of the compound which gives
the purple colour.

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Experiment 2. Protein characteristics

In Part 1, you will precipitate casein from milk using an acid. This method is used to make
cottage cheese. In Part 2, you will coagulate casein from milk using the enzyme rennin. This
method is used for manufacturing cheese. In Part 3, you will coagulate soy protein from
soymilk, using magnesium sulfate. This method is used to make tofu.

Materials Required
Distilled white vinegar (acetic acid), 5% acidity
Hot plate/Bunsen burner
Pasteurized whole milk
Beakers
Soymilk
Graduated cylinder
Rennet tablets (Junket)
Balance
Epsom salt (magnesium sulfate)
Thermometer
Cheesecloth
Foil
Rubber bands
Hammer
Stirring rod/wood Popsicle sticks
Eyedroppers
Heatproof gloves
Heatproof pad
Weigh boats

Experimental Procedure

Part 1. Precipitation of casein from milk with an acid (vinegar)

1. Weigh the empty beaker and record the weight. Weigh and record the weight of 120
milliliters (1/2 cup) of milk in the beaker. Record the weight of the milk in the data
table (weight of beaker with milk – weight of beaker = weight of milk).

2. Place the beaker with the milk on a hot plate. Heat the milk to 21°C (70°F). Turn off
the hot plate and remove the beaker.

3. Add 11 milliliters (2 teaspoons) of vinegar to the warm milk and stir for 2 minutes,
then allow the milk to sit for 5 minutes. The casein will precipitate into heavy white
curds.

4. Cut out a piece (2-3 layers) of cheesecloth large enough to cover the top and 2
inches down the sides of a beaker. Using the rubber band, fasten the cheesecloth
over the top of the beaker. Pour the curdled milk into the beaker, collecting the
curds (casein) in the cheesecloth and allowing the vinegar and whey to drain off
into the bottom of the beaker.

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 5. Gather up the cheesecloth with the casein and rinse in cool water by dipping into
another beaker containing water.

6. Squeeze the casein until almost dry, then spread out the cheesecloth to let the
casein dry for 5 minutes.

7. Weigh the precipitate. (Do not weigh the cheesecloth with the precipitate). Record
your results.

Part 2. Enzymatic coagulation of the casein from milk with rennet

1. Place 1/2 of a crushed rennet tablet into a beaker. To crush the tablet, place it
between two pieces of foil and hit the tablet with a hammer.

2. Weigh the empty beaker and record the weight. Weigh and record the weight of 120
milliliters (1/2 cup) of milk in the beaker. Record the weight of the milk in the data
table (weight of beaker with milk – weight of beaker = weight of milk).

3. Place the beaker with the milk on a hot plate. Heat the milk to 43°C (110°F). Pour
the hot milk over the rennet tablet, stir for 2 minutes, and allow the milk to sit on
the lab bench for 5 minutes.

4. Collect the curds by pouring the curds and liquid into a beaker covered with
cheesecloth (2-3 layers) (see step 4 in Part 1). Gather up the cheesecloth and
squeeze out the liquid whey from the curds. Spread out the cheesecloth to allow the
curds to dry for 5 minutes.

5. Weigh the curds. (Do not weigh the cheesecloth with the curd.) Record your results.

Variations:

Test the effect of low temperature on the activity of rennet. Repeat the experiment with
cold milk at 4°C (40°F). Record your results.

Test the effect of high temperatures on the activity of rennet. Repeat the experiment with
hot milk heated to 70°C (160°F). Record your results.

Part 3. Coagulation of protein from soymilk using a salt (magnesium sulfate)

1. Weigh the empty beaker and record the weight. Weigh and record the weight of 120
milliliters (1/2 cup) of soymilk in the beaker. Record the weight of the soymilk in
the data table (weight of beaker with soymilk – weight of beaker = weight of
soymilk).

2. Place the beaker with the soymilk on a hot plate.

3. Bring the soymilk to a boil and turn off the heat. Monitor this step closely; do not
allow the soymilk to boil over the top of the beaker.

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 4. Add 1.6 grams (1/4 teaspoon) of Epsom salt (magnesium sulfate, a mineral salt) to
the hot soymilk and stir.

5. Wait until the curds are floating in an almost clear liquid.

6. Fasten a piece (2-3 layers) of cheesecloth over the top of a beaker with a rubber
band. Pour the soy curds and liquid into the beaker, collecting the curds in the
cheesecloth and allowing the liquid to drain into the bottom of the beaker.

7. Gather up the cheesecloth and squeeze out as much water as possible. Spread out
the cheesecloth to allow the curds to dry for 5 minutes.

8. Weigh the curds. (Do not weigh the cheesecloth with the curd.) Record your results.

Variations:

Test to see if rennet can coagulate soy protein. Add 1/2 rennet tablet to 120 milliliters (1/2
cup) of warm soymilk (43°C). Record your results.

Test to see if Epsom salt can coagulate casein. Add 1.6 grams (1/4 teaspoon) of Epsom salt
to 120 milliliters (1/2 cup) of boiled milk. Record your results.

Table 1– milk and soymilk curds

Weight of Weight of Describe the curd (color,
milk/soymilk curd texture)
Milk + acid
Milk + rennet
Soymilk + Epsom
salt
weight of beaker with milk – weight of beaker = weight of milk

Questions

1. Compare the weights of the curds from the milk (acid and rennet) with that from
the soymilk.

2. Why did the casein that was coagulated with the rennet weigh more than the casein
that was precipitated with the acid?

3. Compare the amount of acid casein precipitated from the whole milk with the
amount of soy protein coagulated from the soymilk. How do your results compare
with the Nutrition Facts label for each product?

4. How did the biuret test indicate the presence of proteins?

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 LAB 3. LIPID ANALYSIS
Lipids include fats, oils, waxes, cholesterol, other sterols, and most steroids. In the body, fat
serves as a source of energy, a thermal insulator and cushion around organs, and an
important cellular component. The fat-soluble vitamins are A, D, E, and K. You are probably
most familiar with the nutritional aspects of dietary fats and oils. Since fats have 2.25 times
the energy content of carbohydrates and proteins, most people try to limit their intake of
dietary fat to avoid becoming overweight. The food industry has a big market for low-fat
and non-fat foods. Just take a look around your local grocery stores!
Lipids are classified as organic compounds that are soluble (dissolvable) in organic
solvents, but only sparingly soluble in water. Lipids are biologically important for making
barriers (membranes of animal cells), which control the flow of water and other materials
into a cell.
Fats and oils make up 95% of food lipids and phospholipids, and sterols make up the other
5%. Traditionally, fats were considered to be solid at room temperature, and oils were
considered to be liquid. However, this designation is often used to distinguish between fats
and oils from animals and plants, respectively. Animal fats are found in meats (beef,
chicken, lamb, pork, and veal), milk products, eggs, and seafood (fish oil). Plant (vegetable)
oils come from nuts (peanuts), olives, and seeds (soybean, canola, safflower, and corn). We
use lipids for flavor (butter and olive oil), to cook foods (oils and shortening), to increase
the palatability of foods by improving the texture or “mouthfeel” (cakes, creamy ice cream),
and in food processing (emulsifiers).
Fatty acids are generally long, straight chains of carbon atoms with hydrogen atoms
attached (hydrocarbons) with a carboxylic acid group (COOH) at one end and a methyl
group (CH3) at the other end. These long, straight chains combine with the glycerol
molecule (see Figure 1A) to form lipids (glycerol lipids).
Figure 1
A. Glycerol molecule is the backbone of a
glycerol lipid. The triacylglycerol
contains three fatty acids attached at the
oxygen atoms of glycerol.
B. Configuration of a cis double bond.
C. Configuration of a trans double bond.
D. Linoleic acid is an essential fatty acid
containing two double bonds. It is
needed for growth and health.
E. Stearic acid is a saturated fatty acid
found in foods from animal and plant
sources.
F. Milk fat triacylglycerol molecule
illustrating the ester bonds between
fatty acids and glycerol.

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Most naturally occurring fatty acids contain an even number of carbon atoms. The 18-
carbon fatty acids are the most abundant in our food supply; examples are linoleic acid (an
omega-6 fatty acid) found in corn oil and linolenic acid (an omega-3 fatty acid) found in
flaxseed oil. Linoleic and linolenic acids are considered essential fatty acids because they
are needed for normal physiological functions and our body cannot make them. We need to
get these fatty acids from food sources. These fatty acids are found in the vegetable oils
used in several different food products.
Structure of Lipids
Most of the carbon-carbon bonds in fats are single bonds, which allow the carbons to freely
rotate, making the attached groups chemically identical. However, the number of
unsaturated bonds (double bonds) may vary from one to many in the hydrocarbon part of
the fatty acid. Since double bonds do not allow free rotation between the attached carbons,
any attached chemical groups are fixed in their respective positions.
There are two possible orientations for groups attached to the carbons in a double bond. If
they are on the same side of the double bond (close together), they are in the cis
conformation. The opposite of the cis conformation is the trans conformation, where the
residues at ends of the double bond are farther apart. Double bonds in natural vegetable
oils and in animal fats are mostly in the cis conformation (see Figures 1B and 1C);
however, a few exceptions are known where the trans conformation is present.
The presence of the double bonds is responsible for the liquid properties of native
vegetable oil. Because the cis double bonds are “kinked”, they disrupt the physical
interactions between fatty acid molecules, preventing them from packing together tightly
to form crystals (see Figure 1D, structure of linoleic acid). This disruption keeps the fatty
acid molecules from associating with each other, resulting in a liquid structure. If the
double bonds are removed by adding hydrogen (hydrogenation), the kinks are removed,
allowing the fatty acid molecules to more easily associate with each other (see Figure 1E,
structure of stearic acid). The result is crystallization (solid fat) at room temperature.
Depending on how the various fatty acid chains associate, the crystalline structure of the
solid fat can have different appearances, such as a smooth, shiny solid or a rough, puffy
solid. These crystalline forms also have different light-reflection characteristics and
physical hardness. This difference in physical properties is used when making shortening,
which is crystallized into a very white, soft crystalline form at the factory. However,
upon melting and re-solidification, it becomes more translucent and grayer, due to the
formation of a different crystal structure.
Nomenclature for Fats
If all the bonds are single, the fatty acid molecule is saturated, because the maximum
number of hydrogen atoms is associated with the carbon atoms. Some examples are tallow
(beef fat), lard (pork fat), and butter (milk fat). If there is a double bond among the carbon
atoms, the fatty acid molecule is unsaturated. Examples of unsaturated fats are canola oil,
corn oil, cottonseed oil, and soybean oil. If there are multiple double bonds (two or more),
it is called polyunsaturated. You may recall seeing the saturated, unsaturated, and
polyunsaturated terms with respect to nutritional aspects of oils. Corn and soybean oils are
some of the most important food sources of polyunsaturated fatty acids in our food supply.
Shown below are the shorthand notation used to describe some important food sources of
18-carbon (C18) fatty acids.

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  C18:0 is a fully saturated 18-carbon fatty acid called stearic acid.
 C18:1 has one double bond, between carbons 9-10, (18:1n9) counting from the
 COOH end, and is called oleic acid.
 C18:2 has two double bonds, between carbons 9-10 and 12-13, counting from the
 COOH end, and is called linoleic acid (9,12-octadecadienoic acid or 18:2n6).
 C18:3 has three double bonds at carbons 9-10, 12-13, and 15-16, counting from the
 COOH end, and is called linolenic acid (9,12,15-octadecatrienoic acid or 18:3n3).
The number of fatty acids joined to the glycerol molecule also plays a part in how the
molecule is named. If only one fatty acid is connected, the general name for the molecule is
a monoacylglycerol. If two are joined, the molecule is called a diacylglycerol, and if three
are joined, a triacylglycerol. The bond between the fatty acid and the glycerol also has a
special name. It is called an ester bond (see Figure 1F). The carboxyl end (COOH) of the
fatty acid molecule attaches to one of the -OH groups of the glycerol molecule. Because of
this combination, an -OH group and -H are left, which combine to form a water molecule.
Since triacylglycerols have three fatty acids, you can get mixed-fatty-acid triacylglycerols,
in which there are different fatty acids on each of the glycerol bonds. Naturally occurring
soybean oil is a mixed triacylglycerol, containing saturated, monounsaturated, and
polyunsaturated fatty acids. Soybean oil contains more monounsaturated and
polyunsaturated fatty acids than saturated fatty acids.
Surfactant is a short term for surface-active agent. Polar lipids, like lecithin in soybean oil,
serve as specialized surfactants known as emulsifiers. By interacting with water on one
end of the molecule and repelling water on the other end, emulsifiers keep fat globules
dispersed in water or water droplets dispersed in fat. Lipid surfactants are important to
our own cellular functions, as well as useful in stabilizing specific food products. Lecithin
is a phospholipid, which functions as a surfactant. Lecithin and other phospholipid
emulsifiers are found in food from animal and plant sources. The food sources of lecithin
are eggs, milk, cheese, and soybean oil. These chemical properties of lecithin are used in
the food industry to prevent fats from separating out of chocolate, mayonnaise, peanut
butter, and salad dressings.
The fats that you see in raw beef, chicken, and pork are known as visible fats. These fats are
in plain view and are solid at room temperature. Vegetable oils are also visible fats. The
fats that are in snack foods, cookies, desserts, and candy are known as invisible fats.
Although you cannot see them, they can add extra calories to your diet.

Experiment 1. Test for lipid

NOTE: All apparatus must be dry. All flames must be extinguished.

1. Label four test-tubes 1-4.

2. Into tubes 1 and 2 pour about 20 mm (depth) alcohol (propan-2-ol).
3. To tube 1 add one drop of vegetable oil, and shake the tube sideways until the oil
dissolves in the alcohol.

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 4. In tubes 3 and 4 pour about 20 mm water.
5. Pour the contents of tube 1 into tube 3 and the contents of tube 2 into tube 4.
6. Record your results as below:

Result when added to water

1 Oil dissolved in water 3

2 Alcohol alone 4

CLEANING THE TUBES. Keep the oily tubes separate from the others and clean them with
hot water and liquid detergent.

Discussion

1. What was the only difference between the contents of tubes 1 and 2 ?

2. What was the visible difference between tubes 3 and 4 after adding the contents of
tubes1 and 2 to the water in them?

3. How would you attempt to explain the appearance of the liquid in tube 3?

4. What difficulties can you foresee in using this test with samples which contain both
lipids and water?

5. Water and alcohol mix in all proportions. What precautions must be taken in
selecting the alcohol for use in this experiment and why is this precaution
necessary?

6. How would you design an experiment to find out the sensitivity of this test for fats?

Experiment 2: Characteristics of lipid in food – visual observation

In this experiment, we will be extracting and examining the fat in chocolate, potato chips,
and sunflower seeds. In chocolate, sugar and cocoa are dispersed in a crystallized fat
matrix. To keep the fat from separating out of the chocolate, an emulsifier called lecithin is
used. The fat in the potato chip is mostly on the surface of the chip from the frying process.
The fat in the sunflower seed is in the seed itself. The cooking oils that we use come
primarily from nuts and seeds. Examples of these fat sources are corn, soybean, and peanut
oils.

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Materials Required
Chocolate chips (semi-sweet)
Balance or scale
Sunflower seeds
Microwave
Potato chips
Paper towels
Acetone
Foil
100-mm Petri dishes
Hammer
100-and 600-milliliter beakers
Safety goggles
Graduated cylinder
Latex or rubber gloves

Experimental Procedure

A. Visual evidence of invisible fats from foods

Part 1. Chocolate Chips

1. Measure out 2 grams of chocolate chips and place on a paper towel.
2. Microwave for 40 seconds on high.
3. Fold the paper towel over the chocolate chips and gently press the chocolate chips
flat with your fingers.
4. Allow it to sit for 5 minutes. Open up the paper towel. Record your results.

Part 2. Potato Chips

1. Measure out 2 grams of potato chips and place on a paper towel.
2. Microwave for 25 seconds on high.
3. Fold the paper towel over the potato chips and crush the chips with a hammer.
4. Allow it to sit for 5 minutes. Open up the paper towel. Record your results.

Part 3. Sunflower Seeds

1. Measure out 2 grams of sunflower seeds and place on a paper towel.
2. Microwave for 25 seconds on high.
3. Fold the paper towel over the sunflower seeds and crush the seeds with a hammer.

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 4. Allow it to sit for 5 minutes. Open up the paper towel. Record your results.

Table 1 – visual observations of fat

Food Describe what you see on the paper towel

Chocolate chips

Potato chips

Sunflower seeds

B. Quantitative measurement of invisible fats from foods

Part 1. Extraction of Fat from Chocolate Chips

1. Weigh out 5 grams (9 chips) of chocolate chips. Crush the chocolate between two
sheets of foil with a hammer.
2. Label the beakers that you are using to put the food in, one each for chocolate chips,
potato chips, and sunflower seeds. Record the weights of the labeled beakers.
3. Using the beaker that is labeled for chocolate chips and place the crushed chocolate
chips in the beaker. Record the weight with the crushed chocolate chips.
4. Add 10 milliliters of acetone to the crushed chocolate chips in the beaker.
5. Swirl for 1 minute in a hood, or stir with a glass rod (in a well ventilated area).
6. Carefully decant the acetone into the Petri dish, making sure the chocolate remains
in the beaker.
7. Add 10 milliliters of acetone to the chocolate and repeat steps 5 and 6.
8. Allow the acetone in the Petri dish to dry overnight in a hood (or a well ventilated
area) to visualize the lipid that was extracted.
9. Allow the beaker with the chocolate to dry overnight. Weigh the beaker with the
chocolate.

Part 2. Extraction of Fat from Potato Chips

1. Weigh out 5 grams of potato chips. Break into dime-size pieces with your fingers.
2. Repeat steps 2-9 in Part 1.

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Part 3. Extraction of Fat from Sunflower Seeds
1. Weigh out 5 grams of sunflower seeds. Crush the seeds between two pieces of foil
with a hammer.
2. Repeat steps 2-9 in Part 1.

Table 2 – extraction of lipids

Weight Weight of Weight of Weight

Weight of % Lipid
Food of beaker with beaker with lost from
raw food extraction
beaker raw food dried food food

Chocolate
chips

Potato
chips

Sunflower
seeds
(weight of beaker with raw food) – (weight of beaker) = weight of raw food
(weight of beaker with raw food) – (weight of beaker with dried food) = weight lost from food
(weight lost from food / weight of raw food) x 100 = % lipid extracted

Table 3– description of fats

Food Color Texture Odor Viscosity

Chocolate chips

Potato chips

Sunflower seeds

Questions
1. How can you tell that the dark wet spot on the paper towel is fat and not water?

2. Rank from most to least the percentage of lipid extracted from all three foods. Look
at the Nutrition Facts label on the packages of all three foods and rank them. Did
your ranking agree with the ranking of the product labels?
3. Determine which lipids contained saturated and unsaturated fatty acids in this
experiment, based on your descriptions of the fats in the Petri dishes.

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LAB 4. VITAMIN C ANALYSIS
Vitamin C is ascorbic acid: one of its chemical properties is that it is a 'reducing agent', ie.
removes oxygen from or adds hydrogen to other chemicals. DCPIP is dichlorophenol-
indophenol, a blue dye which is decolourized by ascorbic acid on account of its reducing
properties.

Experiment 1. Determination of vitamin C content in fruit juices

1. Copy the table below into your notebook.
2. Label six test-tubes 1-6.
3. Use a 2 cm3 syringe to put 1 cm3 DCPIP solution into each tube.
4. Use a fresh syringe to draw up 2 cm3 of 0.1 % ascorbic acid solution (ie. pure
vitamin C solution).
5. Draw the solution into the pipette until the bottom of the plunger is against the 2.0
cm3 mark on the barrel of the syringe.
6. Squeeze the plunger of the syringe carefully so that you add one drop at a time of
ascorbic acid solution to the DCPIP in tube 1.
7. Shake the tube and go on adding the ascorbic acid from the syringe until the blue
dye just changes to a colourless liquid *. (If you empty the syringe before the DCPIP
goes colourless, refill it and add 2 cm3 to your final volume.)
8. Note the position of the plunger in the syringe and subtract the reading from 2.0 (or
simply count the divisions from the top mark on the barrel to the bottom of the
plunger). This will tell you the volume of ascorbic acid solution you have added (Fig.
2). Write this volume into the appropriate space in the table (not forgetting to add 2
cm3 for each time you refilled the syringe.)
9. Wash the syringe and repeat the experiment with one of the fruit juices, using
thenDCPIP in tube 2.
10. Repeat the experiment with an acidified solution or sodium sulphite, which is not a
fruit juice or a vitamin, but is a reducing agent.

*Note. The acid in the fruit juice may turn the DCPIP from blue to red but it is the point at
which the dye becomes colourless which counts. Coloured fruit juices, however, will not
give a colourless solution. The end point is when the blue (or red) colour disappears and
the original colour returns.

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Table 1
Volume needed
Tube Liquid to decolourize
DCPIP
1 0.1% ascorbic acid
2 Fresh lemon juice
3 Fresh orange juice
4 Canned or bottled orange juice
5 Fresh grapefruit juice
6 Fresh grape juice
7 Orange squash
Orange juice that has stood in an open beaker for
8
………days

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LAB 5. FERMENTATION PROCESS
Yeasts are unicellular fungi that are versatile laboratory microorganisms. They grow
rapidly and have simple nutritional requirements. When yeast degrade nutrients in the
absence of oxygen they use the process of glycolysis to produce energy in the form of ATP.
For millennia, humans have usedthe alcoholic fermentation capability of yeast of the genus
Saccharomyces to produce breads, crackersand a variety of fermented beverages including
beer and wine. The general equation for thefermentation reaction is:
Substrate + Glycolytic Enzymes → Ethyl Alcohol (C2H5OH) + CO2 + ATP
Notice the substrates and the products in the reaction. What chemicals do you think would
be appropriate substrates for yeast to degrade? The glycolytic enzymes are the enzymes
of glycolysis which function under anaerobic conditions.
Potential substrates for this work include sucrose, a disaccharide composed of glucose
(C6H12O6) and fructose, an isomer of glucose. Sodium chloride (NaCl) is table salt and
sodium lauryl sulfate (C12H25O4SNa) is an ionic detergent. Detergents are molecules with
both positively and negatively charged extremities. Starch or amylose is a polymer of
glucose produced by green plants. Sodium saccharin (C7H4NNaO3S.2H2O) is a sugar
substitute that is many times sweeter than sucrose.
Ethyl alcohol (C2H5OH) is a by-product of the fermentation of sugar by yeast. Now look at
the products and try to separate them into useful substances and waste products. What
difference might these make in the rate of fermentation? You need to know more about the
chemicals you test to understand their impact on the fermentative activities of the cell.
Carbon dioxide gas accumulates as a waste product of fermentation in yeast and cellular
respiration in many kinds of cells, including yours. Fermentation releases two molecules of
the gas from the anaerobic (not requiring molecular oxygen) degradation of a substrate,
usually glucose, as well as two molecules of ethanol plus usable energy for cell function.
Cellular respiration, an aerobic (requiring molecular oxygen) process, liberates six
molecules of carbon dioxide as well as several water molecules and energy. More energy is
released by cellular respiration than by fermentation because glucose is completely
oxidized in the process. Thus, carbon dioxide is a waste product of the energy-releasing
mechanisms of the cell. Logically, then, carbon dioxide is an indicator of the rate of
substrate degradation in an organism. More carbon dioxide will be released from an
organism as the rate of fermentation or cellular respiration increases.
What factors in the yeastís environment might change the rate of fermentation or
respiration, and thus, the rate of carbon dioxide release? To answer this question, you need
to know that both respiration and fermentation are controlled by enzymes within the cells.
As a waste product, carbon dioxide can affect activities of the organism. Increased carbon
dioxide levels stimulate more rapid breathing rates in humans which clears carbon dioxide
from the system. For an aviator without an oxygen supply, the partial pressure of carbon
dioxide (PCO2) decreases from 40 mm Hg to 24 mm Hg in the alveoli from 0 to 40,000feet
above sea level. During that same change in altitude the partial pressure of oxygen (PO2)
decreases dramatically from 104 mm Hg to 12 mm Hg. An unacclimated individual will
breathe more rapidly attempting to take in more oxygen beginning at about 8,000 feetand

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will reach a maximum breathing rate between 16,000 and 23,000 feet. The individualmay
also show other symptoms of hypoxia including mental slowness, sleepiness, nausea,
giddiness or headache. On the other hand, an aviator breathing pure oxygen can ascend to
47,000 feet before showing the effects of hypoxia.

Experiment 1. Fermentation of glucose using yeast

Beer and wine are produced by fermenting glucose with yeast. Yeast contains enzymes
that catalyse the breakdown of glucose to ethanol and carbon dioxide. In this
experiment, a glucose solution is left to ferment. Students then test for fermentation
products. This experiment takes time. The solution needs to ferment between lessons,
especially if you are distilling the final solution to produce ethanol.
Materials

Eye protection
Conical flask (100 cm3)
Boiling tube
Measuring cylinder (50 cm3)
Access to a balance (1 dp)
Cotton wool
Sticky labels
Warm water 30–40 °C
Glucose (Low Hazard), 5 g
Yeast (as fast acting as possible), 1 g
Limewater

Procedure

1. A source of warm water is required. Larger conical flasks can be used, but this
dilutes the carbon dioxide concentration, and makes testing for carbon dioxide with
limewater more difficult.

2. Put 5 g of glucose in the conical flask and add 50 cm3 of warm water. Swirl the flask
to dissolve the glucose. (Fig.1)

3. Add 1 g of yeast to the solution and loosely plug the top of the flask with cotton
wool.

4. Wait while fermentation takes place.

5. Remove the cotton wool and pour the invisible gas into the boiling tube containing
limewater. Take care not to pour in any liquid as well.

6. Gently swirl the limewater in the boiling tube and note what happens.

7. Replace the cotton wool in the top of the flask.

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 Figure 1

Experiment 2. Product of fermentation

1. Remove the cotton wool and note the smell of the solution.

2. Carefully decant or filter the solution into your distillation flask. (Significant
quantities of yeast will produce foaming and this can be carried over into the
product.)

3. Collect the fraction between 77–82 °C. (Ethanol boils at 78 °C.) This fraction should
burn easily compared with the non-flammable original solution.

4. The ethanol must be burnt or poured away immediately. It must not be kept or
used.

Figure 2

Yeast has an enzyme called zymase and this catalyses the fermentation process.

Glucose zymase → Ethanol + carbon dioxide

C6H12O6 (aq) → 2C2H5OH(aq) + 2CO2(g)

SBK3023 Food Science and Nutrition Lab Manual Page 24

Experiment 3. The effect of temperature on fermentation rate

You are provided with a boiling tube containing a suspension of yeast in a solution of
glucose. DO NOT SHAKE THE TUBE at any stage of the experiment.
1. Set up a clamp stand, clamp, tripod, gauze and water bath as shown in Fig. 3. Clamp
the boiling tube securely in the water bath.
2. Fit the manometer and syringe assembly as shown in the diagram ensuring that the
bung makes an air-tight seal in the mouth of test- tube and the tap is open (turned
upwards).

Coloured liquid

termometer

Figure 3

3. Use a dropping pipette to fill the reservoir of the manometer about three quarters
full of coloured liquid. If the liquid does not flow into the capillary, close the tap
(horizontal) and raise the syringe plunger. Air bubbles in the column can be
expelled by using the syringe to push the liquid and trapped air back to the
reservoir.
4. Turn the tap downwards, push the syringe plunger to the bottom of the barrel and
open the tap (vertically upwards). Move the marker on the capillary tube to indicate
the level of liquid.
5. Record the temperature of the water bath, note the time and close the tap
(horizontal). Leave the tap closed for one minute and keep withdrawing the syringe
plunger to bring the liquid back to the marker. After one minute bring the liquid
level exactly back to the mark, open the tap (upwards) and record in your table the
volume indicated on the syringe.

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 6. This figure represents the volume of carbon dioxide given out by the yeast in one
minute.
7. Turn the tap downwards, push the syringe plunger to the bottom and close the tap
(horizontal).
8. Take two more readings of the volume of gas produced in one minute and calculate
the average of the three.
9. Check that the tap is open (upwards) and use a small Bunsen flame to raise the
temperature of the water bath by about 5 °C. Leave the apparatus for two minutes
after heating has stopped.
10. Record the new temperature and take three more one-minute readings as before,
but this time record the temperature at the end of the three readings to obtain the
mean temperature during the experiment.
11. Repeat the operation from (g) raising the temperature about 5 °C each time until
you reach 45 or 50 °C. At temperatures between 30-40 °C the three readings should
be made in rapid succession to avoid volume changes due to cooling; at 40-50 °c you
should add 0.1 cm3 to all readings to compensate for the cooling effect.
12. Plot a graph of carbon dioxide output (vertical axis) against temperature.

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LAB 6. MICROBIAL CONTAMINATION
Microbial contamination of food has been a problem for humans throughout history.
During most of the 5 million years of our evolution hominids ate grossly contaminated
food; food covered with dirt, chewed on by rats and mice, infested with maggots and
various microbes, rotten to various degrees etc. Clearly we are not as delicate as we may
imagine ourselves. On-the-other-hand spoiled and rotten food probably did in a large
number of our ancestors and surely made a lot more of them often extremely
uncomfortable. We are only the third generation to have the benefits of safe and secure
food preservation methods.

Prior to the end of WWII (1945) frozen food was a rarity and far too expensive for the
common folk (who didn't have a freezer to put it in anyway). While canning had been
around since the early 1800's, its mechanism of preservation was not understood nor was
the significance of the canning temperature appreciated until the late 1800s. The bottom
line is that a lot of ate a lot of rather bad food; stuff we would today gag at and toss in the
nearest dumpster.

Maintaining a safe food supply is a never ceasing struggle. It was only since the 1930s
when a book by Sinclair Lewis exposed the filthy conditions in the country's slaughter
houses that the Federal Government (USA) began to regulate food safety. Even today there
are those who feel that our food supply is over regulated and that there is little reason for
concern about the safety of our food supply. What do you think? Many of you have worked
in the food service industry. Do you think that removing the safety rules and regulations
would result in a significant change in the quality of our food?

In the past few years the Northwest region has suffered more than its share of serious food
borne bacterial disease caused by a new strain of E. coli, O157:H7. This virulent pathogen
has killed many people and left a number severely injured for life. This bacterium has been
found most commonly in ground meats like hamburger. However, it has also been detected
in a variety of other foods including apple cider, fruit juices and produce. Although a lot of
research has been done to pinpoint the source of this organism, it is still not known. Beef
cattle seem to be one of the prime carriers of E. coli, O157:H7, but other animals may also
be involved. The danger is such that anyone who eats poorly cooked ground meat must
be considered at risk for contracting this disease.

Most of you have been told by your mother to store foods in the refrigerator and to wash
your hands before handling food, either for eating or in preparing them. In this exercise
you will estimate the bacterial contamination in some hamburger purchased at a local
supermarket. You will see the effect on improper storage on the numbers of bacteria in the
food and you will observe how high salt or sugar concentrations inhibit the growth of
many bacteria.

The objectives of this lesson are:

1. To learn the spread-plate count technique.

SBK3023 Food Science and Nutrition Lab Manual Page 27

 2. To learn how to estimate the bacterial contamination of hamburger and the effect of
its storage conditions upon the number of bacteria.

3. To understand the preservative nature of high osmotic pressure.

Below are some microbes associated with food; some good, some very bad characters.

Figure 2. A fungi such as you might find

on spoiled food. The HYPHAE are the
single fungi filaments. The MYCELIUM is
Figure 1. E. coli O157:H7. It doesn't look the total mass of hyphae. Note the
like a killer does it? reproductive spores on the end of these
hyphae.

Figure 4. A population of yeast cells

with daughter cells forming
Figure 3. Cells of lactobacillus. These reproductive buds. Note the bud scars
are present in the sauerkraut you've left behind after the bud leaves; the cells
made. on the far right seems to have given
"birth" to a lot of buds--maybe it's a
"grandmother" yeast!

SBK3023 Food Science and Nutrition Lab Manual Page 28

Experiment 1. Bacterial Counts in Variously Stored Meat

Materials

Samples of hamburger that have been stored properly and improperly.

Media containing a high concentration of salt or sugar.

Procedure:
1. Obtain a 1:10 dilution of the sample of the hamburger from the instructor (20 gm +
180 ml of sterile water). Record the history of the samples; a sample that has been
stored in the refrigerator since its purchase and the other sample that was left out
at room temperature overnight.

2. Obtain, per pair, 4 plates of count media, a 100 ml dilution blank, disposable plastic
pipettes, a spreading rod and a beaker of ethanol.

3. Label the plates as follows:

o Undiluted, 1 ml, 1:10

o Undiluted, 0.1 ml, 1:100

o Diluted, 1 ml, 1:1000

o Diluted, 0.1 ml, 1:10,000

4. Remove 1.0 ml from the 1:10 sample and transfer it into the dilution bottle and mix
thoroughly. This will give you a 1:1000 dilution of the original sample.

5. Place the dilutions on the respective plates. Work backwards starting with the
MOST HIGHLY diluted sample first.

o Add 0.1 ml of the sample from the dilution bottle (1:1000 dilution) to the
center of the #4.4 plate (1:10,000 dilution) and then 1.0 ml of the same
sample to the #4.3 plate (1:1,000 dilution). Using the same pipette repeat
the process with the UNDILUTED sample to the #4.2 & #4.1 plates
respectively.

6. Remove the glass spreading rod from the ethanol breakers and pass it through the
flame. DO NOT HOLD IT IN THE FLAME. The ethanol will catch fire. Allow it to
burn off AWAY FROM THE BEAKER. Hold it for about 30 seconds until it cools.

7. Successively spread the plates in EXACTLY the following order: the 1:10,000 plate
first; the 1:1,000 plate second; the 1:100 plate third and the 1:10 plate last. Do not
flame the spreading rod between plates.

SBK3023 Food Science and Nutrition Lab Manual Page 29

 o Allow the plates to sit a room temperature until the liquid soaks into the
agar, then place them in the incubator.

8. Incubate the cultures at 37oC until the next lab and then count the number of
colonies per plate. Plates with greater than 300 isolated colonies are to be labelled
TNTC (= Too Numerous To Count). Prepare a table relating the dilution to the
number of colonies and calculate the number of bacteria per gm in the original
sample. Place your result on the board with your team names under the appropriate
sample heading. Compared your results with others testing the same sample and
with the results from the other sample.

9. Write down your conclusions.

Experiment 2. Effect of High Osmotic Conditions on the Growth of Bacteria

1. Obtain 4 broth tubes per pair with the following concentrations of salt or sugar:
Salt- 0, 1%, 5%, 15% or sugar- 0, 5%, 25%, 50% (percentage by weight; i.e. for a
50% sugar solution add the dried medium (for 100 ml of medium) to 50 gm of
sucrose and add enough water to bring the solution to 100 ml final volume).

2. Label each tube.

3. Inoculate each tube with 10 drops of the original meat suspension (allow the large
chunks of meat to settle out first).

4. Mix well.

5. Incubate as described by the Instructor.

6. Estimate the amount of growth in each tube following incubation, assuming that the
growth in the control tube was 4+ and no growth is (-).

7. Record these results in the table below.

8. Gram stain a sample form a tube showing growth.

Osmotic Agent Percent Growth

0 0 4+
SUGAR 5
SUGAR 25
SUGAR 50
SALT 1
SALT 5
SALT 15

SBK3023 Food Science and Nutrition Lab Manual Page 30

Questions:

1. Explain why cuts of meat like steak are not considered as dangerous for bacterial
infection as is hamburger.
2. What are the symptoms of E. coli, O157:H7 infection?

3. What is the treatment for an E. coli, O157:H7 infection?

4. How can you tell if your hamburger is cooked properly? (Sending it to a lab for
testing is not the correct answer)

5. Using information LEARNED PREVIOUSLY in the course, determine how you might
test specifically for E. coli, O157:H7 bacteria in the meat so that you could rapidly
detect only a few 100 bacteria per gram?

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REFERENCES
http://www.ehs.psu.edu/
http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab25.html
http://www.accessexcellence.org/AE/AEPC/IFT/unit_two.php
http://quiz2.chem.arizona.edu/preproom/Demo%20Files/vapor_pressure_of_a_pure_liqui
d.htm
http://msucares.com/pubs/publications/p2469.pdf

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