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Oil and Fats Technology

BWD 30503

Laboratory Manual
Food Technology Programme
Faculty of Applied Sciences and Technology
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Laboratory Manual
BWD 30503
Oil & Fat Technology

LECTURER
Prof Madya ChM Dr. Norhayati binti Muhammad

LABORATORY DEMONSTRATOR
Madam Nur Fazira binti Abdul Rahim

LABORATORY ASSISTANT
Cik Fatin Nur Ain binti Kemat

STUDENT NAME: _____________________________

MATRIC NO.:__________________
SEM/YEAR: ____________________
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TABLE OF CONTENT

Table of Content 3
Laboratory Procedure Important Notes 4
Introduction Oils & Fats 6
Experiment 1 Physical and Chemical Characteristics of Fats and Oil 7
Experiment 2 Determination of Melting Point of Fats 14
Experiment 3 Extraction of Fats and Oil: Determination of Total Fats 16
Experiment 4 Butter Making Process: Changes in the Properties of 24
Fats and Oil
Experiment 5 Esterification of Oil and Fats 27
Experiment 6 Formulation and Stabilization of Low-Fat Spreads 35
Margarines and Mayonnaise Based on Locally
Available Fats
Experiment 7 Visit to Oil and Fats Processing Industries 39
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LABORATORY PROCEDURE

Important Notes

Attendance
You must arrive on time in the laboratory. Late arrival may result in a serious delay to your
starting practical work and may well mean that you do not have time to finish the experiment. On
arrival, you must sign the attendance sheet, and at the end of the day before you leave the
laboratory. Bags should be left in the designated area outside of the laboratory. Do not leave
valuable tems in this area.

Experiment Stations
On arrival in the laboratory, you should proceed to the experiment station where you will carry
out your practical work for that day. You should carry out the experiment in a group of four/five.
It is you and your group responsibility to ensure that the apparatus/glassware at your station is
cleaned and put away tidily at the end of the day. A member of laboratory staff can give you
advice on how to clean awkward items. Failure to leave complete and clean apparatus/glassware
in your bench will result in mark deduction. If you break any glassware you should report and get
replacement from the technical staff immediately.

Protective Clothing
The minimum protective clothing required in the laboratory consists of a pair of shoes and
fastened lab-coat worn at all times. The shoes must be able to protect your feet from chemicals
and sharp objects. Without these, entry to the teaching laboratories is forbidden. Hand glove
should be used when necessary.

Report Submission
You are required to have a laboratory notebook to be used for making rough notes for your
practical reports. Raw data obtained during the experiment must be clearly written in the
laboratory notebook. This laboratory notebook will be checked from time to time. Laboratory
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report must be prepared in group and handed in for marking no later than seven working days
after you have completed the experiment.

Preparation of Test Samples

i. Liquid oils
Use clear sediment free liquid directly after inverting container several times. If liquid
sample contains sediment, release all sediment from walls of container and distribute
uniformly throughout the oil. Stir vigorously and filter to obtain clear filtrate. For
determinations in which results might be affected by possible presence of water (e.g. iodine
value) dry sample by adding anhydrous sodium sulfate (NaSO4) in the proportion of 1 - 2 g
per 10 g sample and hold it in oven at 50°C.

ii. Solid and semi-solid fats

Soften sample if necessary by gentle heat taking care not to melt it. When soft enough mix
thoroughly for determination of moisture and volatile matter. For other determinations melt
in drying oven at a temperature at least 10°C above the melting point. If clear, proceed
directly. If turbid or contains sediment filter test sample inside oven. For determinations in
which results might be affected by possible presence of water (e.g iodine value) dry sample
by adding anhydrous Sodium Sulphate in the proportion of 1-2 g per 10 g sample and hold
(keep) it in oven at 50°C. Stir vigorously and filter to obtain clear filtrate. To retard rancidity
keep oils and fats in cool place and protect from light and air.
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INTRODUCTION

Oils & Fats

Oils and fats may also simply be called "fats" which may be of plant or animal origin. They are
almost ubiquitous in food processing - whether naturally occurring in foods or added as
ingredients for functional benefits and, despite the impression given by several sources to the
contrary; they remain an essential part of the human diet. Oil and fats in food play a key role in
determining the nutritional value, product quality and consumer appeal of the products in which
they are incorporated. Not only they are important in food nutrition, fats and oils also provide
functional roles in food, such as lubricity, structure, flavor, mouth feel and much more. They are
often delivered in the form of emulsions that make up many traditional foodstuffs which include
butter, margarine, salad dressing, mayonnaise, sour cream, cream cheese, oils, lard, and others.
There is no a single all-purpose fat or oils. Each type is manufactured for their specific
purpose, be it in cooking, baking and any other usage. Oil bearing materials used as raw materials
in fats and oils manufacturing may be of either plant (olive oil, sunflower oil, walnut oil, almond
oil and many others) or animal (duck fat, cow fat and other land animal fats; or whale fat, seal fat
and other marine animal fats) origin. The types of fats and oils used in formulations have specific
impacts on sensory, nutritional and functional aspects of finished products. They are principally
triglycerides, which is either saturated, short chained fatty acids in solid form (most animal fats)
or unsaturated, long chained fatty acids in liquid state (vegetable fats).
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EXPERIMENT 1

Physical and Chemical Characteristics of Fats and Oils

1.1 Solubility, specific gravity, and refractive index

Introduction
Fats may be characterized in a number of ways. They are soluble in organic solvents but not in
water. They have a specific gravity of less than 1.0. They may also be characterized by their
refractive indexes, which may correlate to specific structural features such as unsaturation or
chain length.

Objective
To illustrate the solubility, specific gravity and refractive index of fats

Materials
Corn oil, 250 ml
Soybean oil, 250 ml
Sunflower oil, 250 ml
Canola oil, 250 ml
Olive oil, 250 ml
Chloroform, 20 ml
Toluene, 20 ml
Alcohol, 20 ml
Sulphuric acid, 10 ml
n-hexane, acetone

Apparatus
Graduated measuring cylinders, 10 ml, 50 ml
Test tubes, 50 ml
Pycnometer
Refractometer
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Important note
When working with organic solvents, be sure to work in a fume hood and use gloves. The
procedure can be completed within 30-45 minutes.

Procedure
1. In a large test tube (50 ml), place 20 ml of water and 5 ml of vegetable oil. Shake
vigorously and observe.
2. Place in each of three large test tubes (50 ml), respectively, 20 ml of toluene, 20 ml of
alcohol, and 20 ml of chloroform. To each tube add 5 ml of vegetable oil. Shake
vigorously and observe.
3. Nearly fill the graduated measuring cylinder with vegetable oil at a temperature of 60°C.
Use pycnometer to determine the specific gravity of the oil. Refer to the “guide” section
for the use of pycnometer.
4. Determine the refractive index of each of the oils. Refer to the “equipment guide” section
for use of the refractometer.

Guide on the use of pycnometer

Pycnometer washing
1. Wash the pycnometer and its stopper with soap solution using a brush to remove dirt and
rinse with water.
2. Fill the pycnometer with 10 ml sulphuric acid and shake it and leave it for 1 hour. Note:
the solution with turns yellow as it still has dirt inside. Empty the pycnometer and wash
with tap water.
3. Finally, rinse the pycnometer with deionized water.

Pycnometer standardization
1. Take some cool distilled/deionized water and heat it for 15 minutes on a hot plate.
2. Fill the pycnometer with the boiled water and placed it in the water bath at 25°C for 30
minutes (without its stopper).
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3. Now attach the stopper tightly and dry the outer surface with tissue paper and weigh the
filled pycnometer, W3.
4. Discard the water and rinse the pycnometer with 10 ml ethanol and also 10 ml n-hexane
and acetone to remove alcohol soluble
5. Place the pycnometer in hot oven (100°C) for 15 minutes to dry it.
6. Cool the pycnometer in a desiccator for a while and then weight it, W2.

Pycnometer with samples

1. Fill the dry pycnometer with samples
2. Placed in the water bath at 25°C for 30 minutes (without its stopper).
3. Now attach the stopper tightly and dry the outer surface with tissue paper and weight the
filled pycnometer, W1.
4. Calculate its specific gravity as in the formula below:
Specific gravity = (W1-W2)/(W3-W2)

Guide on the use of refractometer

The refractometer needs to be calibrated each time before use as follows:
1. Prepare distilled or tap water.
2. Clean the prism surface
3. Place approximately 0.3 ml of water onto the prism surface.
4. Press the START key, "---" will appear, and then the measured Brix value (%) as well as
the temperature of the sample stage at the time the measurement was taken will be
displayed.
5. If the display indicates "0.0%", zero setting does not need to be performed. Wipe the water
off of the prism surface with a lint-free tissue that is ready to use.
6. If the indicated value is not "0.0", press the ZERO key while the water is covering the
prism
7. After blinking 2 times, "000" will be displayed on the LCD screen. If the display reads
8. "AAA", add more water onto the prism surface and press the ZERO key again.
9. When "000" is displayed, zero setting has been successfully completed.
10. Dry the prism surface by wiping with a tissue and ready to use.
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Measurement of sample will be taken as follows:

1. Clean the prism surface.
2. Place approximately 0.3 ml of sample onto the prism surface.
3. Press the START key, "---" will appear, and then the measured Brix value (%) as well as
the temperature of the sample stage at the time the measurement was taken will be
displayed
4. The measured value will remain displayed for approximately 2 minutes. To turn off the
display, press and hold down the START key for approximately 2 seconds.
5. Remove the sample by wiping it off with a tissue. Use water to remove any remaining
sample. Dry off any excess moisture

Guided Questions
1. In which solvents did the oil dissolve? Why?
2. What quality of oil is demonstrated by specific gravity?
3. Relate the structures of the various oils to their refractive indices.

1.2 Water-Absorbing Capacity of Fats

Introduction
In certain food systems, fats must be mixed and remain mixed. The extent to which fats can absorb
water is called the water-absorbing capacity and is important in food systems such as cakes. Fats
differ in their ability to absorb water, largely based on differences in their composition.

Objective
To demonstrate the water-absorbing capacity of commercial fats
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Materials
Rendered chicken fat, 50 g
Rendered beef fat, 50 g
Hydrogenated shortening, 50 g
Margarine, 50 g
Soft margarine, 50 g
Butter, 50 g

Apparatus
Burettes, 5 ml
Electric mixer

Important note: Add water slowly and gradually as you are beating the fats. The procedure can
be completed within 30-45 minutes.

Procedure
1. Transfer 50 g of each fat at room temperature to a small bowl of an electric mixer. While
beating at slow speed, run water into the fat from a burette at a uniform rate (about 20
ml/min) until separation occurs.
2. Record the volume of water taken up by 50 g of each fat.
3. Graph the results.

Guided Questions
1. What factors influence the emulsifying capacity of fats?
2. Why is the emulsifying capacity of a fat important?
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1.3 Plasticity of Fats

Introduction
In addition to the incorporation of water into fats (Experiment 1.2), it is occasionally important
to incorporate air into fats, as in the creaming of fat and sugar in cake mixes. This incorporation
of air is largely responsible for the leavening that these products contain. The ability to incorporate
air is related to fat crystal size. Generally, the smaller the fat crystals, the more air that can be
incorporated.

Objective
To illustrate the creaming ability of various fats

Materials
Chicken fat, 50 g
Hydrogenated shortening, 50 g
Margarine, 50 g
Soft margarine, 50 g
Butter, 50 g
Sugar (fine granulated), 25 g

Apparatus
Electric mixer

Important note
The procedure can be completed in 1 hour.
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Procedure
1. Using the same fats as in Experiment 1.2
2. Determine the relative amounts of air whipped in during the creaming operation as
follows:
i. Transfer 50 g of each fat to the small bowl of an electric mixer. Add 25 g of fine sugar
(granulated) gradually over a period of 2 min.
ii. Then continue to beat for another 3 min. Transfer the creamed mixture to a previously
weighed measuring cup and determine the weight of 1 cup of creamed fat.
iii. Take the weight of a cup measure full of water to determine the specific gravities of
the creamed fats. See Equipment Guide section for specific gravity. Graph your
results.

Guided Questions
1. What determines the creaming ability of a fat?
2. Why is the creaming ability of a fat important?

1.4 Oxidative Rancidity

Introduction
Because of their chemical structure, unsaturated fats and oils are subject to oxidative breakdown,
so called oxidative rancidity. Oxidative rancidity results from more complex lipid oxidation
processes. The processes are generally considered to occur in three phases: an initiation or
induction phase, a propagation phase, and a termination phase. In other words, this reaction is a
free radical chain reaction that involves abstraction of reactive allylic hydrogen from the fatty
acid chain followed by a series of reactions with oxygen, rearrangements, and chain cleavage to
produce odoriferous compounds. In this experiment, carotene is used as a marker of fat rancidity
reactions.

Objective
To study factors (light, temperature, antioxidants, pro-oxidants) that affect the rancidity of fats.
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Materials
Rendered beef/chicken fat, 50 g
Carotene, 10 mg
Chloroform, 1 ml
0.01% CuSO4, 25 ml
0.001% BHA or ascorbic acid, 25 ml
0.5% hemoglobin, 25 ml
Saturated salt solution, 25 ml
Turnip greens extract, 80 ml
Green onion tops extract, 80 ml
White potatoes extract, 80 ml
Apples extract, 80 ml

Apparatus
Filter papers, 7 cm diameter discs
Petri dishes with filter paper discs

Important note
If the group or groups doing this experiment also have other experiments assigned, this one should
be started first due to the length of time that must be allowed for the oxidation reaction to take
place. The procedure can be completed within 2 hours.

Procedure
1. To 50 g of rendered beef/chicken fat, add 10 mg carotene dissolved in a little chloroform.
2. Using plastic forceps, bone forceps, or forceps coated in polyethylene, dip small filter
papers (7-cm diameter is convenient) in the melted fat and allow to drain for 20 sec.
3. Transfer to Petri dishes and treat as follows:
i. Effect of temperature and light on fat oxidation
a. Cover the dish and store in the dark at room temperature.
b. Cover the dish and store in the light (direct sunlight if possible).
c. Cover the dish and store in the refrigerator.
d. Cover the dish and store in an incubator at 60°C.
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ii. Antioxidants and pro-oxidants: For these experiments, saturate small filter discs with
the test solutions; place several discs of each test solution on a filter paper saturated
with the carotene–beef fat mixture. Invert the bottom of the Petri dish containing the
papers over the Petri top containing water, using a separate Petri dish for each variation.
Store the dishes in an incubator at 40°C. Solutions to be tested include:

a. Water control
b. Dilute copper sulphate solution (0.01% CuSO4)
c. Commercial antioxidant (0.001% BHA/ascorbic acid)
d. Saturated salt solution
e. Extracts of selected vegetables (prepared by heating 20 g chopped vegetables
(turnip greens, green onion tops, white potato peel, apples) with 80 ml H2O to
the boiling point. Decant and cool before using).
(NOTE: One cannot visualize fats going rancid. Carotene, which is a highly
unsaturated hydrocarbon similar in structure to a fatty acid, turns from bright orange
to colorless as it is oxidized. Therefore, the rate of bleaching observable with the
carotene can be used as an index to the rate of oxidative rancidity of the fat).
4. Compare the treatments for their influence on oxidative rancidity as determined by the rate
of bleaching.
5. Compare the odor of fat in bleached vs. non-bleached paper. Outline the events (with
reactions) that occur as fats undergo oxidative rancidity.
6. Discuss the results of each treatment using this outline.

Guided Questions
1. What are the factors which cause fat oxidation?
2. How can the fat oxidation be minimized?
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EXPERIMENT 2

Determination of Melting Point of Fats

Introduction
The physical properties of a compound, such as melting point and boiling point can provide useful
information which can help in the identification of a sample or to establish its purity. The
temperature at which a solid melts and becomes a liquid is the melting point. Each fat has a
different melting temperature which is dictated by its chemical composition.

Objective
To determine the melting point of different fats

Materials
Chickem fat, 10 g
Shortening, 10 g
Margarine, 10 g
Soft margarine, 10 g
Butter, 10 g

Apparatus
Capillary tube with both ends open
Paper tissue
Thermometer (0-100ºC)
Elastic band
Retort stand and clamp
250 cm³ beaker
Bunsen burner
Tripod
Gauze
Small pan of water on a hotplate
Glass rod
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Eye protection

Important note
The procedure can be completed within 30-45 minutes.

Procedure
1. Stick a capillary tube along a block of fat. Wipe the outside of the tube.
2. Attach a thermometer to the capillary tube with an elastic band, so that the bulb of the
thermometer is next to the plug of fat.
3. Using a retort stand, clamp the tube and thermometer in a beaker of cold water, so that they do
not touch the sides or bottom of the beaker.
4. Heat the water gently with a Bunsen burner, stirring occasionally.
5. Observe the fat and note the temperature at which it becomes clear and begins to run out of the
tube.

Guided Questions
1. Among the fats tested, which one has the highest melting point?
2. What is the importance of knowing the melting point of fats?
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EXPERIMENT 3

Extraction of Fats and Oil: Determination of Total Fats

Introduction
Fats and oil are produced by pressing, spinning, smelting, extraction or combination of above
processes. Fats extracted from organic materials are called raw fats. Aside from fats extracted raw
fats also contain other non-fatty lipophilic substances. Extraction for this purpose is described as
the separation of substance from one phase, in which it is dissolved or suspended, to another
liquid phase. This process is possible, because substance is distributed between two phases in
certain proportions. However, extraction is only efficient when substance dissolves much better
in one of two phases, i.e. value of distribution constant significantly exceeds one.

Objective
To learn the method of extracting fats and oil and to determine the fatty acid content, iodine value
and peroxide value of the extracts.

3.1 Chicken and Beef Fat Rendering

Materials
Chicken with skin/fat
Beef with fat
Distilled water

Apparatus
Beaker
Hot plate
Knife
Thermometer
Measuring cylinder
Weighing scale
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Separating funnel

Procedure
1. Place chicken skin (250 g) in a beaker with water (1:2, w/w).
2. Boil on a hot plate for 40 min.
3. After removing skin residues, cool down the liquid to room temperature.
4. Separate fat layer using a separating funnel.
5. Calculate the percentage of total fat obtained according to the equation:
𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑐𝑐ℎ𝑖𝑖𝑖𝑖ℎ𝑒𝑒𝑒𝑒 𝑓𝑓𝑓𝑓𝑓𝑓 (𝑔𝑔)
% 𝐹𝐹𝐹𝐹𝐹𝐹 = 𝑥𝑥 100
𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑐𝑐ℎ𝑖𝑖𝑖𝑖ℎ𝑒𝑒𝑒𝑒 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 (𝑔𝑔)

6. Store at room temperature prior to be used in the next experiment (fatty acid content,
iodine value and peroxide value).
7. Repeat methods 1-6 for beef fat rendering processes.

3.2 Integrated Wet Process (IWP) Extraction of Coconut Oil

Coconut Oil can be produced by two common methods, i.e. heated (hot methods) processes non-
heated (cold methods) processes. Hot methods apply extreme pressure or high heat to extract the
oil from the nut. Cold methods obtain oil without applying heat or pressing the coconut meat
mechanically, hence the more common term "cold pressed".

3.2.1 Hot Extraction of Coconut Oil

Materials
Coconut, 1/2
Water, ~250 ml

Apparatus
Blender
Cutter
Chopping board
Analytical balance
Beaker
Strainer
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Cheesecloth or clear napkin

Measuring cylinder

Important note
The procedure can be completed within 1-2 hours.

Procedure
1. Remove the coconut meat from the shell and weigh.
2. Cut the coconut meat into small pieces.
3. Put the small pieces coconut meat into the blender and add a little water( 150-250 ml).
4. Blend the coconut meat until it turn into a soft mushy paste.
5. Squeeze the blended coconut meat to extract the coconut milk.
6. Pour “once squeeze” coconut meat into a strainer or cheese cloth and repeat process
until finish.
7. Put coconut milk into a beaker and boil on high heat to remove the water until the oil is
appears (NOTE: continous stirring the boiling coconut milk to avoid burning).
8. Strain the oil by using dry cheesecloth.
9. Weigh the oil
10. Calculate the percentage of oil.
𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 (𝑔𝑔)
% 𝑂𝑂𝑂𝑂𝑂𝑂 = 𝑥𝑥 100
𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 (𝑔𝑔)
11. Store the obtained oil at room temperature prior to be used in the next experiment (fatty
acid content, iodine value and peroxide value).

3.2.1 Cold Extraction of Coconut Oil

Materials
Coconut, ½
Water, ~250 ml

Apparatus
Blender
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Cutter
Chopping board
Analytical balance
Beaker
Strainer
Cheesecloth or clear napkin
Measuring cylinder
Mixer
Centrifuge tube

Procedure
1. Remove the coconut meat from the shell and weigh.
2. Cut the coconut meat into small pieces.
3. Put the small pieces coconut meat into the blender and add a little water (150-250 ml).
4. Blend the coconut meat until it turn into a soft mushy paste.
5. Squeeze the blended coconut meat to extract the coconut milk.
6. Refrigerate the obtained coconut milk for 24 hours.
7. Stir the coconut milk using mixer until the coconut butter is forms.
8. Separate the water from the coconut butter.
9. Soak the coconut butter in the water bath at 37oC.
10. Centrifuge the solution until the oil separate from the aqueous layer.
11. Then, filter the obtained virgin coconut oil (VCO) through cheesecloth.
12. Weigh the oil
13. Calculate the percentage of oil.
14. Store at room temperature prior to be used in the next experiment (fatty acid content,
iodine value and peroxide value).
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3.3 Fatty Acid Content

Materials

Oil/fat obtained from Exp. 3.1 and 3.2

Fatty acid methyl ester (FAME), purified standard
n-Hexane
Sodium methoxide

Apparatus

Gas Chromatography Instrument

Small vial
Micropipette
Vortex mixer

Procedure

1. Weigh approximately 50 mg of extracted oil.

2. Mix with 0.95 ml n-hexane in a vial.

3. Shake the vial to dissolve the oil.

4. Then add 0.05 ml sodium methoxide to the solution using micropipette.

5. Mix thoroughly the solution with vortex mixer for five second or until the mixture separate
into two layers (the upper layer is clear and the bottom layer is sodium glyceroxide
precipitate).

6. Extract the clear upper layer solution with micropipette/dropper and put in a clean vial.

7. Analyse the obtained solution by gas chromatography instrument (GC), equipped with
automatic on-column injector, a polar capillary column and a flame ionization detector
(FID), with Helium as the carrier gas at a flow rate of 5.4 ml/min. Identify the retention
time of fatty acid methyl esters by comparing it with those of purified standard.
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8. Measure the weight fractions of the FAMEs base on the percentage represented by the
area of corresponding peak relative to sum up the area of all peaks (AOCS Method Ce 1-
62 (1998)).

3.4 Iodine value

Materials

Oil/fat from Exp. 3.1 & 3.2

Cyclohexane
Wijs solution
Potassium iodide solution, 10%
Starch solution, 1%
Sodium thiosulphate solution. 0.1 M

Apparatus

Conical flask
Aluminium foil
Burette
Measuring cylinder
Stirrer
Weighing scale

Procedure

1. Weigh 1 g of oil accurately into 500 ml conical flask.

2. Add 3 ml cyclohexane into conical flask and swirl to ensure that it is completely dissolve.
3. Add 5 ml Wijs solution into conical flask containing test sample and swirl to mix.
4. Cover the whole conical flask by aluminium foil.
5. Store the flasks in dark at room temperature for 60 minutes.
6. Remove the flasks from dark and add 4 ml potassium iodide solution and mix.
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7. Add 30 ml of distilled water into the mixture and gradually titrate with 0.1 M sodium
thiosulphate solution with mechanical stirring.
8. Continue the titration until yellow colour had almost disappear.
9. Add 1 ml of 1% starch solution into the flask and continue titrating until blue colour disappear.
9. Record the volume of titrant used.
10. Conduct the blank sample in the same manner as test sample without oil.
11. Calculate the iodine value of each sample as follows:
12.69(B − S)N
𝐼𝐼odine value =
W

Where,
B =Volume in ml of sodium thiosulphate solution required for the blank
S= Volume in ml of sodium thiosulphate solution required for the sample
N= Concentration of the standard sodium thiosulphate solution (M)
W=Weight in g of the sample

3.5 Peroxide value

Materials
Oils from Exp. 3.1 and 3.2
Acetic acid
Chloroform
Potassium iodide
Sodium thiosulfate solution, 0.01N
Starch solution, 1%
Distilled water

Apparatus
Aluminum foil
Hot plate
Conical flask
Weighing scale
Burette
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Procedure
1. Determine peroxide value (PV) according to AOAC method 965.33.
2. Weigh 5 g of oil accurately into 250 ml conical flask.
3. Add 30 ml of 3:2 acetic acid chloroform solvent mixture in conical flask and swirl to dissolve.
4. Add 0.5 ml of saturated potassium iodide into mixture.
5. Cover the opening of conical flask by aluminum foil.
6. Keep the mixture 1 minutes in darkness with continuously shaking and add 30ml distilled
water.
7. Titrate the sample slowly with 0.01 N sodium thiosulfate solution with vigorous shaking until
yellow color is almost gone by using hot plate.
8. Add 0.5 ml 1% starch solution and continue titration, with mechanical stirring to release all
iodine from chloroform layer, until blue color disappears.
9. Record the volume of titrant used.
10. Conduct blank sample as the same manner as test sample without oil and record the volume
of titrant for blank used.
11. Calculate the peroxide value of each sample as follows:

(S − B) × N × 1000
Peroxide value =
W

Where,
Peroxide value = mEq peroxide per kg of sample
S = volume of titrant (ml) for sample
B = volume of titrant (ml) for blank
N = Normality of sodium thiosulphate solution (mEq/ ml)
1000 = conversion of units (g/ kg)
W = sample mass (g)

12. The peroxide values should less than 10 meq/kg, which is the limit allowed by the
standards of the Codex Alimentarius for edible oil and fat (Codex Alimentarius).
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Reference

AOCS Method Ce 1-62 (1998).

Codex Alimentarius (2015). Standard for edible fats and oils not covered by individual standards. CODEX
STAN 19-1981.

Lee, H.J., Lee, M.R., & Wu. H.Y. (2012). Application of poultry fat in cosmetics researches. Taiwan
Livest Res. 45:227–38.

Liang, K. L. and Fa, J. T. (2017). Influence of rendering methods on yield and quality of chicken fat
recovered from broiler skin. Asian-Australas J Anim Sci. 30(6):872-877.

Zunairah Ahmad, Rosnani Hasham, Nur Farah Aman Nor, and Sarmidi, M. R. “Physico-chemical and
antioxidant analysis of virgin coconut oil using West African tall variety.” Journal of Advanced Research
in Materials Science 13, no. 1 (2015): 1-10
 27

EXPERIMENT 4

Butter Making Process: Changes in the Properties of Fats and Oil

Introduction
Butter is a water-in-oil emulsion resulting from an inversion of the cream, an oil-in-water
emulsion; the milk proteins are the emulsifiers. Unhomogenized milk and cream contain butterfat
in microscopic globules. These globules are surrounded by membranes made of phospholipids
(fatty acid emulsifiers) and proteins, which prevent the fat in milk from pooling together into a
single mass. Butter is produced by agitating cream, which damages these membranes and allows
the milk fats to conjoin, separating from the other parts of the cream. Variations in the production
method will create butters with different consistencies, mostly due to the butterfat composition in
the finished product. Butter contains fat in three separate forms: free butterfat, butterfat crystals,
and undamaged fat globules.

Objectives
1. To learn the process involved in butter making
2. To determine the changes in the properties of fats and oil during the process
3. To determine the importance of temperature in the formation of butter

Materials
2 cups heavy cream (preferably organic and not ultra-pasteurized)
2 tablespoons plain yogurt (optional)
1/4 teaspoon of salt (optional)
Ice cubes
Sturdy sieve
Cheesecloth or clean napkin
Bowl for catching buttermilk
Hand held mixer
Plastic wrap or kitchen cloth
Spatula or wooden spoon
Clean containers for butter and buttermilk
 28

Waxed paper or parchment paper (optional)

Bowl
Measuring cups and spoons
Whisk
Clean kitchen cloth
Hot air oven
Water bath

Important note
Be sure you are use quality cream for your butter. Since it is practically the only ingredient, the
quality of cream will make a lot of difference. Do not use ultra-pasteurized cream as the high
heat used in this type of pasteurization destroys much of the cream's flavor. The procedure can be
completed in 2-3 hours.

Procedure
1. Transfer the cream in a glass bowl, cover and incubate at air-conditioned room
temperature (17-23°C) for about an hour before churning.
2. Place a sturdy sieve over a bowl and line with a few layers of cheesecloth.
3. Place the cream in the bowl and cover the top with plastic wrap or a kitchen towel to
prevent splattering. Do the same with procedure in varying temperature by placing the
bowl inside the water bath while mixing.
4. Turn on the mixer to medium-high. The cream will first whip into peaks (at around 2
minutes) and then become grainy (around 3 minutes).
5. Keep whipping until the solid mass (butter) and liquid (buttermilk) are separated.
6. The mixture will splatter heavily in the final stages of churning, so be sure the plastic wrap
is secure.
7. The process may take a little longer, up 15 minutes.
8. Pour the buttermilk through the cheesecloth and strainer, holding the butter solid back.
9. Allow the buttermilk to strain through, then plop in the butter.
10. Gather the cloth around the butter and press it hard with your fist. Do this several times to
get as much buttermilk out of the butter as possible.
 29

11. Rinse out the bowl used for buttermilk, then remove the butter from the cloth and place it
in the bowl.
12. Add 1/2 cup of ice water to the bowl, and using a spatula, press the butter into the ice
water. Note: It will quickly become cloudy with buttermilk.
13. Pour off the cloudy water, add another 1/2 cup of ice water to the bowl, and keep pressing.
Repeat until the water is clear. This may take up to 6 washings.
14. The butter will firm up towards the end, so you may find it easier to use your hands.
15. Sprinkle the salt over the butter and knead in. Again, your hands may be the best tool here.
16. Store the butter. Pack the butter into a jar with a cover, or roll it into a log using waxed
paper or parchment paper.
17. The butter is ready for tasting.
18. Keep your product into two parts (A:refrigerated; B:non-refrigerated) for two days to
determine the free fatty acid, iodine and peroxide value (Experiment 5.3, 5.4 & 5.5).

Questions
1. What properties of fats changed during the process?
2. At what temperature does the butter formed the fastest?
 30

EXPERIMENT 5

Esterification of Oil and Fats

Introduction
The process of esterification in oils and fats industries normally known as transesterification. This
process includes chemical reactions where an ester is reacted with alcohol (alcoholysis), acid
(acidolysis), or another ester (interesterification or ester exchange), to generate a new ester.
Generally, the reactions can be summarized as follows:
Alcoholysis
O O

R O C R' R" OH R" O C R' R OH

Acidolysis
O O O O

R O C R' HO C R''' R O C R''' HO C R'

Interesterification

O O O O

R O C R' R" O C R''' R O C R''' R" O C R'

5.1 Interesterification Reaction

Introduction
Interesterification involves the exchange of acyl groups between TAGs in same or different
oils/fats. In this experimen, two types of oil or fats are added in ratio of 1:1. The reaction is carried
out in the presence of lipase enzyme as biocatalyst at a temperature of 70°C in a solvent-free
system. The reaction is stopped by separating the enzyme from the reaction mixture via filtration.
The product obtained from the reaction is then dissoved in mixture of acetone:acetonitrile and
analyzed by HPLC.

Objectives
To esterified the TAGs in palm oil and sunflower oil
 31

Materials
Palm oil (PO)
Sunflower oil (SFO)
Triacylglycerol (TAG) standard (tripalmitin/triolein)
sn-1,3 specific immobilized lipase (Lipozyme 1M)

Apparatus
Beaker, 100 ml and 500 ml (for oil bath)
Burettes, 25 ml
Thermometer
Retort stand
Buchner flask
Cone flask 3 x 100 ml
Round bottom boiling flask 25 ml (or 50 ml)
Analytical balance
Sintered glass filter funnel
Pipette 25 ml
Hot plate with stirrer
Filter pump (aspirator)
Test tube

Procedure
1. Put 2.0 g mixture of PO: SFO (1:1) into a 25 ml boiling flask
2. Immerse the flask into the oil bath set at 70°C. Allow it to reach equilibrium for 5
minutes while stirring
3. After reaching the equilibrium temperature, start the reaction by adding 0.2g Lipozyme
1M. Let it stirred until end of practical session.
4. At the end of experiment, stop the reaction by separating the enzyme from the reaction
mixture through filtration by using sintered glass funnel
5. Collect the product into a 100 mL beaker
6. Cover the beaker by parafilm and place it in a safe place for HPLC analysis
 32

5.2 Triacylglycerol analysis: High Performance Liquid Chromatography (HPLC)

Introduction
Triacylglycerol (TAG) in vegetable oils can be separated according to the equivalent carbon
number (ECN) by using reversed phase high performance liquid chromatography (HPLC) system
equipped with reflective index (RI) detector. Generally, ECN is defined according to the
following equition:
ECN = CN – 2n,
where, CN = the number of carbon
n = the number of double bond
The composition of TAG is determined by comparing the standard TAG retention times. The
comparison of TAG is reported as the percentage of the peak area.

Objectives
1. To determine the TAG profile of the samples
2. To compare the TAG profiles of the sample obtained before and after interesterification

Materials
Palm oil (PO)
Sunflower oil (SFO)
Product of interesterification (Exp. 5.1)
Mixture of acetone:acetonitrile (63.5:36.5 v/v)

Apparatus
HPLC system, Shimadzu
C18 column, 150mm x 4.8 mm x 3µm
Syringe, 50 µl

Procedure
1. Weight accurately 0.50 ± 0.01g of sample.
2. Dissolve the sample in acetone-acetonitrile mixture (63.5:36.5).
3. Transfer into a 10 mL flask, make the final voleme to 10 mL.
 33

4. Make sure th e HPLC system is ready to use, with the injector is in “LOAD” mode.
5. By using a syringe, take approximately 25 µL sample, then inject into the injector.
6. Turn the spinner on syringe from “LOAD” to “INJECT” mode (This procedure will
transfer the sample from sample loop into the column)
7. Carry out the analysis until the chromatogram is complete.
8. Repeat the sample for other oils samples.

Questions
1. Do you obtain a good separation for the three oils?
2. Compare the TAG profiles obtained and discuss why they are different or similar?

5.3 Determination of Free Fatty Acid

Introduction
Fatty acids which separated from triglyceride molecules, are called ‘free fatty acids” (FFA).
formation of FFA in the oil of stored oil-seeds is a natural occurance. A small amout of FFA is
also formed during seed crushing and subsequent handling and storage of the crude oil. Amoutn
of FFA in crude oil vary with the oil species.

Objectives
To calculate the free fatty acid (FFA) content of the oils before and after interesterification.

Materials
Palm oil
Sunflower oil
Product of interesterification from Exp 5.1
0.02 N methanolic NaOH solution
Phenolpthalein indicator (1% in ethanol)
 34

Important note
Neutralized Ethanol:Diethyl ether can be obtained by transfer 50 ml ethanol:diethyl ether into a
conical flask and heat in water bath at 40°C. Add ~0.5 ml phenophthalein and neutralise it with
0.1 N NaOH until the pink colour remains unchanged.

Procedure
1. Weight approximately 50 mg sample (record the actual reading) into a 100 ml conical
flask
2. Add 25 ml of neutralized Ethanol:Diethyl ether (1:1, v/v) and shake slowly the flask to
dissove it.
3. Add 3 drops of phenophthalein indicator and titrate with 0.02 N methanolic NaOH until
the colour turn into pink.
4. Calculate the free fatty acid content as oleic acid, according to the equation:
28.2 𝑥𝑥 𝑁𝑁 𝑥𝑥 𝑉𝑉
% 𝐹𝐹𝐹𝐹𝐹𝐹 𝑎𝑎𝑎𝑎 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 =
𝑊𝑊
Where: N = normality of NaOH solution
V = NaOH volume that being used
W = sample mass in g
5. Repeat the experiment for other samples.

5.4 Iodine value

Materials

Oil/fat from Exp. 3.1 & 3.2

Cyclohexane
Acetic acid
Wijs solution
Potassium iodide
Starch solution
Sodium thiosulphate solution
 35

Apparatus

Conical flask
Aluminium foil
Burette
Measuring cylinder
Stirrer
Weighing scale

Procedure

1. Melt the fat sample by heating at maximum of 15oC which is above the melting point.
2. Filter melted fat sample through the syringe filter (0.45µm).
3. Weigh accurately 0.1-0.5g of fat sample into each two dry 500ml glass stoppered flasks.
4. Pipette 25 ml of Wij’s solution into the flasks.
5. Let flasks stand for 30 minutes in the dark with occasional shaking.
6. After incubation in dark, add 20 ml of potassium iodide solution (10% w/v) to each flask.
Shake thoroughly. Add 100ml of freshly boiled distilled water and cooled water to washing
down any free iodine on stopper.
7. Titrate the iodine in flasks with standard sodium thiosulfate, adding it gradually until the
yellow color almost disappear (solution become light yellow).
8. Then add 1-2ml of starch (1% w/v) indicator and continue the titration until the blue color
entirely disappear.
9. Record the volume of titrant used.
10. Repeat the same procedure for blank solution (omitting only the fat sample). Record the
volume of titrant used.
11. Calculate the iodine value of each sample as follows:
12.69(B − S)N
𝐼𝐼odine value =
W

Where,
B =Volume in ml of sodium thiosulphate solution required for the blank
S= Volume in ml of sodium thiosulphate solution required for the sample
 36

N= Normality of the standard sodium thiosulphate solution

W=Weight in g of the sample

5.5 Peroxide value

Materials
Oils from Exp. 3.1 and 3.2
Acetic acid
Chloroform
Potassium iodide
Sodium thiosulfate solution, 0.01N
Starch solution, 1%
Distilled water

Apparatus
Aluminium foil
Hot plate
Conical flask
Weighing scale
Buret

Procedure
1. Determine peroxide value (PV) according to AOAC method 965.33.
2. Weigh 5 g of oil accurately into 250 ml conical flask.
3. Add 30 ml of 3:2 acetic acid chloroform solvent mixture in conical flask and swirl to dissolve.
4. Add 0.5 ml of saturated potassium iodide into mixture.
5. Cover the opening of conical flask by aluminium foil.
6. Keep the mixture 1 minutes in darkness with continuously shaking and add 30ml distilled
water.
7. Titrate the sample slowly with 0.01 N sodium thiosulfate solution with vigorous shaking until
yellow color is almost gone by using hot plate.
8. Add 0.5 ml 1% starch solution and continue titration, with mechanical stirring to release all
iodine from chloroform layer, until blue color disappear.
 37

9. Record the volume of titrant used.

10. Conduct blank sample as the same manner as test sample without oil and record the volume
of titrant for blank used.
11. Calculate the peroxide value of each sample as follows:

(S − B) × N × 1000
Peroxide value =
W

Where,
Peroxide value = mEq peroxide per kg of sample
S = volume of titrant (ml) for sample
B = volume of titrant (ml) for blank
N = Normality of sodium thiosulphate solution (mEq/ ml)
1000 = conversion of units (g/ kg)
W = sample mass (g)

12. The peroxide values should less than 10 meq/kg, which is the limit allowed by the standards
of the Codex Alimentarius for edible oil and fat (Codex Alimentarius).

Question
1. What is the application of interesterification reaction in oil and fat industries?
2. What are the advantagees of using enzyme as a catalyst over chemical catalyst in fat/oil
modification reaction?
 38

EXPERIMENT 6

Formulation and Stabilization of Low Fat Spreads Margarines and Mayonnaise Based on
Locally Available Fats

6.1 Production of Margarine

Introduction
Margarine and spread preparation universally formulated to contain 52% - 80% oil and water
soluble ingredients. It is a water-in-oil emulsion, in which water droplets are kept separated by
the fat crystals. Producer categorized margarine according to demand by different consumers,
based on hardness and melting point. A hard and medium plastic characteristic is for bakery
margarine while medium plastic and soft is for table margarine. Table margarine is further
classified into the distinct categories of tub and brick margarine. Tub margarine has low solids at
low temperature, thus enabling it to be spreadable directly from the refrigerator. In addition, the
fat blends should melt completely above 37°C for good oral melt down.

Objectives
To produce margarine from palm oil

Materials
Fat soluble:
Palm oil, 79.44g
Emulsifier, 19.86 g
Lecithin, 0.4g / approximately 1 capsules
Beta-carotene, 0.003 ml
Butter flavour, 2 ml
Water soluble:
Water, 9.25g
Salt, 0.75 g
Yellow food colouring (optional)
 39

Apparatus
Mixing bowl
Hand mixer
Analytical balance
Beaker
Refrigerator
Plastic container

Important note
All solid ingredients should be dissolved before adding into the main mixture. The procedure
can be completed in approximately 1 hour.

Procedure
1. Add palm oil, lecithin, emulsifier, beta carotene and butter flavor, stir for 5 minutes.
2. Dissolve salt in water.
3. Put the water mixture into the oil mixture. Stir continuously until homogenous mixture
is form.
4. Collect the thick and homogenous mixture into small plastic container and keep in
refrigerator at 5oC-7oC for 2 days.
5. Observe the texture of your margarine.
6. Determine fatty acid content of your product (refer Exp. 3.3).

Questions
1. Propose the method to enhance the quality of your margarine?
2. Discuss the advantages of using palm oil to produce margarine compare to other oils.
 40

6.2 Production of Mayonnaise

Introduction
Mayonnaise is an oil-in-water emulsion, which is a mixture of two liquids that normally can't be
combined. Emulsifying is done by slowly adding one ingredient to another while simultaneously
mixing rapidly. This disperses and suspends tiny droplets of one liquid through another.
Emulsifiers are liaisons between the two liquids and serve to stabilize the mixture. Eggs and
gelatin are among the foods that contain emulsifiers. In mayonnaise, the emulsifier is egg yolk,
which contains lecithin, a fat emulsifier.

Objectives
To produce mayonnaise from palm oil

Materials
Egg yolk, 1
Vinegar, 30 ml
Palm oil, 300 ml
Olive oil (or other vegetable oil of your choice), 200 ml
Salt (optional)
Sugar (optional)
Apparatus
Mixing bowl
Hand mixer
Beaker
Measuring cylinder
Measuring spoons
Plastic container
 41

Important note
The emulsion will not form unless the oil is added VERY slowly. You will know you have formed
an emulsion when the mixture turns white and thick. The procedure can be completed in
approximately 1-2 hours.

Procedure
1. Add egg yolk and 15ml of vinegar to a mixing bowl.
2. Beat vigorously until slightly thick.
3. Add oil, 15ml at a time, while continuously beating the mixture, until 80ml has been
added.
4. Add 15ml of vinegar and continue to beat mixture.
5. Repeat steps 2 and 3 until all liquids have been added.
6. Keep in refrigerator 5°C-7°C for 24 hours.
7. Observe the texture of your mayonnaise.
8. Determine fatty acid content (refer Exp 3.3) of your product.

Questions
1. How to enhance the quality of your mayonnaise?
2. Explain the method to produce low fat mayonaise.
 42

EXPERIMENT 7

Visit to Oil and Fats Processing Industries

Introduction
Oils and fats processing involves a series of processes in which physical and chemical changes
are made to the raw material. For example, oils and fats are the raw materials for liquid oils,
shortenings, margarines, and other specialty or tailored products that are functional ingredients in
food products prepared by food processors in food industry.

Objectives
1. To get exposure to the extraction methods of oils and fats food products in industry.
2. To describe the steps of the production of selected oils and fats food products in industry.
3. To determine the process control involved in each unit operation.

Important note
Tasks agitation between group member and steps of the extraction, production and process control
involved of selected oils and fats food product in industry should be clearly explained in your
report.