FOOD3002 Practical: Lipids in Foods

Lipid chemistry
Lipids are a large class of compounds grouped by their solubility in organic solvents.
Chemically, we distinguish between acyl-lipids and isoprenoid lipids. The majority of acyl lipids
contain fatty acid esters. Examples of acyl lipids in foods are triglycerides in plant seeds (e.g.,
sunflower seeds) or in animal products (e.g., dairy products), as well as membrane lipids (e.g.,
phospholipids, present in all plant and animal products). β-sitosterol is an example of an
isoprenoid lipid present in plant-based foods, while cholesterol is an isoprenoid lipid present
in animal-based foods.

Lipid oxidation
A fat or oil is rancid if it has an unpleasant taste or smell as a result of decomposition. Rancidity
is caused chiefly by oxidation and hydrolysis, resulting in the breakdown of the lipids into short-
chain aldehydes and fatty acids. This phenomenon is striking in animal products, including
butter and meat, but also occurs during the storage and heating of vegetable oils. In meat,
oxidation of lipids is one of the major causes of quality and flavour deterioration and thus of
reduced shelf-life of meat products.

In both fats and oils, polyunsaturated fatty acids (PUFAs) are particularly susceptible to
oxidation, which proceeds at a rate around 60 times faster than that for monounsaturated fatty
acids. When using vegetable oils for frying, oils with low levels of PUFAs are more stable for
extended use, and the preferred choice for industrial frying.

The lipids in meat that are subject to oxidation include triacylglycerols in fatty deposits and
phospholipids in cell membranes. After animal slaughter, muscle cells can lose their anti-
oxidative capacity, allowing free radicals to attack the cellular fatty acids. The oxidation of
lipids proceeds more rapidly when the meat structure is disrupted by common processing
techniques such as grinding, chopping, freezing, and heating, which can increase exposure
of cellular components to oxygen. The main products of oxidation include aldehydes, organic
acids and ketones, depending on the type of lipids present.

Hydrolysis of lipids can be prevented in some foods by processing. For example, oats are
stabilised by steaming to extend their shelf-life; this inactivates the enzyme lipase which would
otherwise facilitate germination through hydrolysis of storage lipids.

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Please ensure you have read the Risk Assessment and Safety Data Sheets for this class,
which are available on Canvas.

Learning outcomes
At the end of this practical you should be able to describe:
• how to determine the peroxide value of lipids by titration
• the use of starch as in indicator in titrations for which iodine is generated
• the effect of heating on the production of peroxides in lipids
• how to determine the cholesterol content of a sample such as egg yolk
• the advantages in using a microplate reader for chemical analyses involving absorbance
or fluorescence
• the reason why some chemical analyses do not require production of a standard curve

Peroxide value of oils

A peroxide is a compound containing an oxygen-oxygen single bond or the peroxide anion,
O22-. Peroxides are generated according to the following chemical equation:

R-H + O2 ® ROOH

Peroxide value (POV) is used as a measure of the degree of oxidation of a fat or oil; it is
defined as the milliequivalents of peroxide per kilogram of lipid. POV is determined by titration
to measure the amount of peroxide or hydroperoxide in the sample. Potassium iodide (KI) is
added to a known amount of oil or fat, reacting with the peroxides present. The iodine liberated
is titrated with a standardized solution of sodium thiosulfate using a starch indicator. The
calculated amount of KI required to react with the peroxide present is used to determine the
POV.

Exercise 1: Determination of the peroxide value of oils

In this experiment, you will determine the POV of two cooking oil samples. The first oil sample
is untreated (fresh), whereas the second is the same type/brand of oil pre-treated with high
heat for 10 min. Oils (by definition) are liquid at room temperature. If you were performing the
same experiment on a fat (e.g., from meat or butter), you would need to first melt the sample
by gentle heating. The principle and execution of the method is otherwise the same for oils
and fats.

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 1. Pre-weigh two 250-mL Erlenmeyer flasks. Accurately weigh ~3 g untreated oil and ~2
g pre-heated oil of the same type (e.g., olive oil) separately into the two flasks,
respectively. Note these weights for your calculations. Close the flasks immediately
with a glass stopper.
2. Add 50 mL of a solvent consisting of glacial acetic acid:cyclohexane (3:2) in the fume
cupboard. Wear gloves and eye protection!
3. Add 1 mL saturated KI solution (freshly prepared) and allow to react for 60 s (time
accurately) while shaking.
4. Slowly add 100 mL deionized water with gentle shaking. Ensure the solution is well
mixed before proceeding.
5. Titrate with 0.01 M sodium thiosulfate solution (the titrant).
6. When the titrand (the sample; recall the difference between titrant and titrand)
becomes a pale straw colour (which will require ~5 ml titrant – be careful not to go to
the end-point!), add 1 mL starch solution (1% w/v) as an indicator. The solution will
turn blue. Continue the titration (with shaking) until the blue colour disappears, which
represents the endpoint.
7. Conduct a blank titration under the same conditions. Expect no more than 0.5 mL
(maybe just a few drops) of 0.01 M sodium thiosulfate to be used for the blank titration;
the volume of titrant required for the oil samples will be much higher.

To determine the POV, use the formula below:

meq (V! − V" ) ∗ c ∗ 1000 ∗ T
POV % +=
kg 𝑚
POV = peroxide value
V1 = volume of sodium thiosulfate solution used to titrate the sample
V0 = volume of sodium thiosulfate solution used to titrate the blank
c = molar concentration of the sodium thiosulfate solution
T = titre of the thiosulfate solution (i.e., the number of moles of peroxide corresponding
to one mole of sodium thiosulfate)
m = weight of the oil sample in grams

Cholesterol content of egg yolk

Egg yolk is an oil-in-water emulsion comprising ~33% lipid, ~50% water and ~16% protein.
The cholesterol content of egg yolk is probably the highest of all foods (except perhaps
mammalian brain tissue).

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The cholesterol assay is a method for quantification of free cholesterol by a colorimetric
method. The cholesterol is extracted from the egg yolk as the cholesterol ester, which is first
hydrolysed by cholesterol esterase and then oxidized by cholesterol oxidase to yield hydrogen
peroxide (H2O2) as a co-product, which reacts with a chromogenic reagent to produce a
coloured product in proportion to the amount of cholesterol.

Exercise 2: Determination of cholesterol content in egg yolk

1. Separate the white and yolk of a hen’s egg into separate beakers and weigh both the
white and the yolk. The white will no longer be required.
2. Using a glass stirring rod gently mix the yolk to disperse it.
3. Using a wide-bore Pasteur pipette, weigh approximately 10 mg (0.01 g; a very small
amount!) of the yolk into a clean, microcentrifuge tube. Record the mass to the nearest
mg (i.e., 0.001 g).
4. Add 200 μL of chloroform:isopropanol (7:11 v/v) and thoroughly vortex the resulting
suspension.
5. Centrifuge the tube for 5 min at 14,000 rpm.
6. Transfer the liquid phase – do not disrupt the pellet – to a clean microcentrifuge tube
and place in the 60°C water bath for 20 min to evaporate the chloroform.
7. Leave the tube in the fume cupboard for a further 10 min (the mixture will not go to
dryness, and you should assume the residual volume is 130 µL).
8. Mix the residual liquid containing the lipids with 200 μL cholesterol assay buffer (it does
not matter if the solution becomes cloudy) and mix by vortexing.
9. Transfer 5 μL of the cholesterol extract to a well in a microplate.
Steps 10 and 11 will be performed for you by your demonstrator.
10. Add 45 μL of the cholesterol assay buffer and 50 μL of the Reaction Mix to the same
well in the microplate and incubate the microplate at 37°C for 60 min protected from
light.
11. Measure the absorbance at 570 nm using a microplate reader.

Exercise 3: Demonstration of Soxhlet apparatus

The Soxhlet method (AOAC Method 920.39C for Cereal Fat; AOAC Method 960.39 for Meat
Fat) is an example of a semicontinuous extraction method for fat analysis. In this type of
method, the solvent builds up in the extraction chamber for a number of minutes, completely
surrounding the sample, and then siphons back to the boiling flask. The fat content is
measured by either the weight loss of the sample or by the weight of the fat extracted from the

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sample. If the sample contains more than 10% water, it should normally be dried to constant
weight at 95–100ºC under vacuum for about 5 h (AOAC Method 934.01).

Assessment (6%)
Assessment for Lipids in Foods consists of the following:
• Answers to the Quiz written individually (12 Questions). Do the Quiz on Canvas
(6%). Some of the questions are on the practical class. Other questions are on
polysaccharides in foods more generally and include some calculation questions.

Deadline for submission of the Food Polysaccharides Quiz:

Odd Group: 30 March 2023
Even Group: 06 April 2023

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