wk3

Lec 3
HS4322
Halal Food Analysis
Sem 1, 2023-2024

Instructors
Hamzah Mohd. Salleh (Prof. Dr.)
hamzah.salleh@unissa.edu.bn

Siti Nur Idayu Haji Matusin (Dr.)

idayu.matusin@unissa.edu.bn
 ‫ع ِلِّ ْمنِي َمب يَ ْنفَعُنِي َو ِز ْدنِي ِع ْل امب‬
َ ‫علَّ ْمتَنِي َو‬
َ ‫اللَّ ُه َّم ا ْنفَ ْعنِي ِب َمب‬
“O Allah! Grant me benefit in what you have taught me, and teach me useful
knowledge and increase me in knowledge.”
[Sunan at-Tirmizi]

ُ ‫ش ُع َو ِم ْن نَ ْف ٍس ََل ت َ ْشبَ ُع َو ِم ْن َدع َْوةٍ ََل يُ ْست َ ََج‬

‫بب لَ َهب‬ ٍ ‫عوذُ ِب َك ِم ْن ِع ْل ٍم ََل يَ ْنفَ ُع َو ِم ْن قَ ْل‬
َ ‫ب ََل يَ ْخ‬ ُ َ ‫اللَّ ُه َّم ِإنِِّي أ‬
“O Allah, I seek refuge in You from knowledge which does not benefit, from a heart that does not entertain the fear
(of Allah), from a soul that is not satisfied and the supplication that is not answered.”
[Sahih Muslim]
 General Steps Performed in (Halal) Food Analysis
1. 2. 3.
perform the test Calculate/analyse
select & prepare and interpret
sample / assay
results

COMPOSITIONAL
FOOD ANALYSIS:
Analysis of
After this lecture, you should know:
LIPIDS (Fats and Oils)
• General introduction about lipids • How to determine the chemical
• What is the importance of lipids analysis? composition of lipids?
• What are the general steps for making lipid • How to analyse lipid oxidation?
analysis • Analysis of physicochemical properties
of lipids
General Introduction about Lipids
Lipids definition:
• No one definition accepted by all
examples of (free) fatty acids
• Most textbooks definition of lipids:
“A group of naturally-occurring organic ❑ waxes ❑ phospholipids ❑ free fatty acids
compounds that are generally soluble in
❑ fat-soluble vitamins (A & D) ❑ glycolipids
non-polar organic solvents (e.g. hexane,

Examples:
acetone, chloroform, etc.) and sparingly ❑ mono- & diacylglycerols
in water”
❑ fats and oils
• in other words, lipid molecules (the term lipids, fats and oils are
are lipophilic (fat loving) often used interchangeably)
and FATS
hydrophobic (water fearing) ➢ triglycerides
(triacylglycerols) that are
solid or semi-solid at
RT, and occur mainly in
animals
❑ steroids like
cholesterol
❑ carotenoids, etc.
OILS
➢ triglycerides (triacylglycerols)
that are liquid at RT, and
occur mainly in plants,
although they also occur in fish
 Classification of Lipids
1. 2. 3.
Simple Compound Derived
Lipids Lipids Lipids
esters of FAs with alcohol
esters of FAs with substances derived
fats & oils waxes alcohol conjugated for simple lipids or
esters of FAs esters of FAs with with other groups compound lipids by
with glycerol long-chain alcohol e.g. phospholipids hydrolysis
other than glycerol (to phosphoric acid) (they have the general
properties of lipids)
e.g. glycolipids
glyceride e.g. FAs
(to carbohydrate)
e.g. long chain alcohols
e.g. sterols

therefore, the lipid fraction of any food contains a complex mixture of different types of molecules
Oils & Fats
❑ Oils and fats are the major component of
lipid fraction of food (typically, 95-99% of
total lipids present)

❑ Both oils and fats consists of triesters

of FAs with glycerol, and so known as
triglycerides (= triacylglycerols) as shown

❑ The FA present in triglycerides differ in

chain length, degree of unsaturation
and position on the glycerol molecule.
(hence, by itself triglycerides consist of a
➢ monoacylglycerols & diacylglycerols come from triacylglycerols
complex mixture of different types of
molecules.)
 Why lipids are important
constituents of food?
➢ Lipids represent a major source of energy
➢ In many foods, the lipid component plays a
major role in determining the overall physical
characteristics such as flavour, texture, mouth
feel and appearance
❑ over consumption of certain lipids can be
harmful to our health (e.g. cholesterol,
saturated fats)
❑ many lipids are prone to lipid oxidation,
which leads to the formation of potentially
harmful product
Why the need to perform
lipid food analysis?
➢ Nutrition labelling:
fat content is one of the mandatory items in
standard format of the “Nutrition Facts” label
➢ Legal requirements:
to confirm that the food meets the standards of
identity and the manufacturing specifications
(i.e. comform to standards of identity)
➢ Food quality:
the texture, taste and appearance is affected by
its lipid content
➢ Health reasons (e.g. development of low fat foods)
➢ Processing
processing conditions depend on the total lipid content)
 COMPOSITIONAL
FOOD ANALYSIS:
Analysis of LIPIDS (Fats and Oils)
analysis of lipids include:

(A) (B) (C) (D)

Determination of Determination of Analysis of lipid Analysis of
physicochemical
total lipid content lipid composition oxidation properties
 COMPOSITIONAL
FOOD ANALYSIS:
Analysis of LIPIDS (Fats and Oils)
analysis of lipids include:

(A)
Determination of
total lipid content
 COMPOSITIONAL
FOOD ANALYSIS:
Analysis of LIPIDS (Fats and Oils)
analysis of lipids include:

(A)
Determination of
total lipid content

Sampling and sample preservation store highly-unsaturated lipid contents under nitrogen
precaution or some other inert gas, in dark rooms or covered
amber bottles, and in refrigerated temp.
Lipids can be oxidised by exposure to:
▪ Light in addition, antioxidants may be added to retard
▪ Elevated temp. oxidation if they do not interfere with the analysis
▪ Oxygen
(1) SOLVENT EXTRACTION METHODS Solvent Selection for Lipid Extraction
(ideal characteristics)
❑ organic solvent extraction step is necessary to
separate lipids from food water-soluble components ❑ inexpensive
❑ the lipid content in extract is measured by either: ❑ able to completely extract all lipid
components while leaving non-lipid
(i) Measuring the weight loss of sample after extraction of lipids components (e.g. proteins,
OR carbohydrates…etc.)
(ii) Measuring the weight of the extracted lipids after evaporation
of organic solvent ❑ relatively low boiling point (can easily be
removed by evaporation)
To facilitate the extraction process the following steps are ❑ non-toxic
performed in sample preparation: ❑ non-flammable
❑ drying the sample
➢ ethyl ether and petroleum ether are the
❑ particle size reduction most commonly used solvents, but
❑ acid hydrolysis (in case of compound lipids) hexane and pentane are also used for
some foods.
➢ a combination of solvents is also used
 Methods of Solvent Extraction
There are several methods for solvent extraction of lipids
including:
❑ continuous solvent extraction method
❑ semi-continuous solvent extraction method or
Soxhlet method
(official AOAC method for cereal fat and meat fat)
❑ discontinuous solvent extraction method……and
others

https://www.youtube.com/watch?v=Nrm4F_P8pVQ
[5.45 min]
Extraction Unit E 812 816 SOX, HE, ECE
(2) NON-SOLVENT WET EXTRACTION METHODS

❑ do not rely on organic solvents, but use other chemicals to separate lipids from the rest of the food
❑ these methods are mainly used for lipid determination in milk and some dairy products

Babcock Method (official AOAC method for determination of milk fat)

❑ H2SO4 is added to known amount of milk in a Babcock bottle
(the acid digests the milk sample and releases the fat)

❑ then centrifuged, followed by addition of hot water

(the liquefied fat will rise into the graduated neck of Babcock bottle)
❑ the fat is measured volumetrically, but the fat content in milk is
expressed as wt%
(3) INSTRUMENTAL METHODS (I) Gas Chromatography (GC)
(official AOAC method for determination of total fat for Nutrition Labelling)
Advantages food
sample
❑ rapid
❑ non-destructive
hydrolysis
❑ require minimal sample preparation (acidic hydrolysis, alkaline hydrolysis, or both)
❑ require minimal chemical consumption
extraction
of fat content with ether
Disadvantages
❑ expensive instrumentation derivatization
❑ measurements often require the into a volatile fatty acid methyl esters
(FAMEs)
establishment of calibration curves
Triacylglycerol Fatty acid methyl esters (FAMEs) + methylated glycerol
specific to various compositions

analysis by GC
 (II) Infrared Method (IR)

❑the IR method is based on adsorption of

IR energy by fat at specific wavelength
(5.73 µm). The more the energy
absorption, the higher is the fat content
of the sample.
 analysis of lipids include:
(A) (B) (C) (D)
Determination of Determination of Analysis of lipid Analysis of
physicochemical
total lipid content lipid composition oxidation properties
❑ Lipids are an extremely diverse group of compounds consisting of tri-, di- and monoacylglycercols, free FAs,
phospholipids, sterols, carotenoids and vitamins A and D.
❑ In addition, most of these sub-groups are themselves chemically complex.
e.g. All triacylglycerols are esters of glycerol and three FA molecules, nevertheless, the FAs can have different chain
lengths, branching, unsaturation, and positions on the glycerol molecule.
SOME OF THE MOST IMPORTANT REASONS FOR DETERMINING THE TYPE OF LIPIDS present in foods:
• Legal: Government regulations often demand that the amounts of saturated, unsaturated and polyunsaturated lipids, as
well as the amount of cholesterol, be specified on food labels.
• Food Quality: Desirable physical characteristics of foods, such as appearance, flavour, mouthfeel and texture, depend on
the type of lipids present.
• Lipid oxidation: Unsaturated lipids are particularly susceptible to lipid oxidation, which can lead to the formation of
undesirable off-flavours and aromas, as well as potentially toxic compounds e.g., cholesterol oxides.
• Adulteration: Adulteration of fats and oils can be detected by measuring the type of lipids present, and comparing them
with the profile expected for an unadulterated sample.
• Food Processing: The manufacture of many foods relies on a knowledge of the type of lipids present in order to adjust the
processing conditions to their optimum values, e.g. temperatures, flow rates etc.
 analysis of lipids include:

(A) (B) (C) (D)

Determination of Determination of Analysis of lipid Analysis of
physicochemical
total lipid content lipid composition oxidation properties

Analytical methods used to “specify” and “determine the concentration” of the different types of
lipid molecules present in the sample (i.e. compositional analysis of lipids). These methods are
classified into:

(1) Chemical Methods (2) Instrumental Methods

 (1) Chemical Methods (I) Iodine Value

Examples of chemical methods used for Definition: it is the number of grams of iodine
determination of lipid composition: absorbed per 100 g of lipid

(I) Iodine value Significance:

▪ It is a measure of degree of unsaturation (i.e.
(II) Acid value number of double bonds) of lipid.
(III) Saponification value ▪ Higher iodine value indicates greater number of
double bonds (C=C) in the lipid
(IV) Specific colour tests for some oils
Application of iodine value:
▪ To characterise (identify) some oils.
H H
I_I ▪ To monitor hydrogenation process of oil, which is
C=C C C
associated with a decrease in iodine value
O- O- ▪ To indicate lipid oxidation (form of spoilage), which
I I
C=C C _C is associated with a decrease in iodine value
 (I) Iodine Value (cont’d)
Most commonly used methods involved the back titration:
(i) Wijs method (pronounced, Ves)
➢ Iodine itself has a reddish brown colour, but this is
▪ Dissolve a known amount of lipid in a suitable organic often not intense enough to be used as a good
solvent before adding excess iodine monochloride. indication of the end-point of the reaction.
H H HH ➢ For this reason, starch is usually used as an
I I I I indicator because it forms a molecular complex
R-C=C-R’ + ICl R-C-C-R’
known I I with the iodine that has a deep blue colour.
excess I Cl ➢ Initially, starch is added to the solution that
▪ Then the remaining unreacted ICl is determined by contains the iodine and the solution goes a dark
addition of excess KI which reacts with ICl to produce I2 blue. Then, the solution is titrated with a
(reddish brown colour) which is then titrated against sodium thiosulfate solution of known molarity.
Na2S2O3 using starch indicator.
➢ While there is any I2 remaining in the solution it
(IClreacted = IClexcess - IClremaining) stays blue, but once all of the I2 has been
converted to I- it turns colourless.
ICl + KI KI + I2
remaining ➢ Thus, a change in solution appearance from blue
I2 + 2 S2O32- 2 I- + S4O62- to colourless can be used as the end-point of
(blue) (colorless) the titration.
 (I) Iodine Value (cont’d)
Most commonly used methods involved the back titration:
(i) Wijs method (pronounced, Ves) (ii) Hanus method
▪ Principle is the same as Wijs method but uses a
different reagent.
▪ Dissolve a known amount of lipid in a suitable organic
solvent before adding excess iodine monobromide
(IBr).
▪ Wijs method is preferred over Hanus method
because ICl reacts faster than IBr with C=C double
bonds.
https://www.youtube.com/watch?v=fQ1hSNGnXYY
Iodine value determination for 2.7234 g sample
using Wijs method [13.23 min]

https://www.youtube.com/watch?v=1IXvcf7rDXM
Determination of Iodine value of an oil sample
[7.43 min]
 (II) Acid Value Principle:

Definition: it is the number of milligrams of KOH Dissolve lipid sample in ethanol

required to neutralise free FAs present containing an indicator (phenolphthalein)
in 1 g of lipid
Titrate with standard KOH
Significance:
▪ It is a measure of the amount of free FAs present Calculate the acid value from the titration
in lipid. end point
Application of acid value: potassium salt of
free fatty acid
the fatty acid
▪ To measure the degree of hydrolysis of lipid. A OH K+ O-
high acid value means poor quality (or bad I I
R-C=O + KOH R-C=O + H2O
storage conditions) of lipids. R-COOH R-COOK
▪ To test the efficiency of fat refining steps
designed to remove FAs. A high acid value means
a poorly refined fat.
NOTE: Acid value may be over-estimated if any acidic additives are
present in the sample (e.g. citric acid used as preservative)
 Principle: (back titration)
(III) Saponification Value
Dissolve lipid sample in excess ethanoic
Definition: it is the number of milligrams of KOH KOH solution of high concentration
required to saponify 1 g of lipid (at least 0.5 N)

Significance: Heat to saponify the lipid

▪ It is a measure of the mean molecular weight of the
triglycerides (triacylglycerols) in the lipid sample. Back titrate the remaining unreacted KOH with
standard HCl using an indicator (phenolphthalein)
Application of saponification value:
▪ To detect adulteration of lipid. Calculate the saponification value from
the titration end point
▪ Example: adulteration with mineral oils such as
liquid paraffin (very cheap) leads to a decrease in
saponification value because mineral oils do not
heat soap
contain esters, and so are not saponifiable.
Trigylceride + 3 KOH glycerol + 3 potassium salt of
NOTE: fatty acid
Calculation of saponification value is not applicable if sample has high The higher the number of combined fatty acids per 1 g
amounts of free FAs (>0.1%) because FAs will also react with KOH.
of lipid sample, the higher the saponification value.
https://www.youtube.com/watch?v=DpgmHx-dl1A
[16.18 min]
Determination of Saponification Value of Oil or Fat
Sample_A Complete Procedure (AOAC 920.160)
https://www.youtube.com/watch?v=ersHDsiAVko
[3.03 min]
Estimation of Saponification Value of Oils & Fats
QUESTION:
The higher the saponification value, the ________ will be
the mean molecular weight of the triglycerides present.
The higher the saponification value, the lower will be
the mean molecular weight of the triglycerides present.
WHY?
Because high saponification value means the presence of
high number of triglyceride molecules per 1 g of liquid
sample, which means lower molecular weight.
e.g.
If 100 molecules of triglycerides (i.e. 300 RCOO-) are present in 1 g
lipid, that means that the molecular weight in this case is lower than
the sample containing 20 molecules (60 RCOO-) per 1 g lipid.
 (IV) Specific Colour Tests for Selected Oils
In addition to the chemical methods mentioned in
CHEMICAL METHODS SUMMARY TABLE previous slides, there are also several specific
Iodine Acid Saponification colour tests for some oils/fats.
value value value
Definition Examples:
Significance
Application
▪ Halphen’s test for cotton-seed oil
Principle ▪ Halphen’s insoluble bromide test for fish oil
▪ Baudouin;s test for sesame oil
▪ etc.
 analysis of lipids include:

(A) (B) (C) (D)

Determination of Determination of Analysis of lipid Analysis of
physicochemical
total lipid content lipid composition oxidation properties
Analytical methods used to “specify” and “determine the concentration” of the different types of
lipid molecules present in the sample (i.e. compositional analysis of lipids). These methods are
classified into:

(1) Chemical Methods (2) Instrumental Methods

A variety of instrumental methods can be used
to provide information about lipid composition:
▪ Chromatography (TLC, GC, HPLC)
▪ Nuclear magnetic resonance (NMR)
spectroscopy
 analysis of lipids include:
(A) (B) (C) (D)
Determination of Determination of Analysis of lipid Analysis of
physicochemical
total lipid content lipid composition oxidation properties
▪ Lipids are especially unsaturated ones are susceptible to oxidation.
▪ Lipid oxidation is a complex process involving numerous reactions that produces a variety of chemical
and physical changes in lipids.
▪ Lipid oxidation is one major form of spoilage, leading to bad flavours and potentially toxic cmpds.
▪ Rancidity is the condition at which the lipid has unpleasant odour and flavour as a result of lipid
hydrolysis (hydrolytic rancidity) or lipid oxidation (oxidative rancidity).

METHODS OF ANALYSING LIPID OXIDATION

Several analytical methods used for detection/determination of lipid oxidation include:
(1) Peroxide Value (2) Kreis Test (3) Thiobarbituric Acid (TBA) Test etc.
 (1) Peroxide Value (2) Kreis Test
Definition: it is the number of milliequivalents of ▪ It is a test for oxidative rancidity
peroxide per kg of sample
Significance: sample + + HCl
concentrated
▪ It is a measure of peroxides (R-O-O-H) which are phloroglucinol
transient products of lipid oxidation (i.e. formed
during lipid oxidation)
shake for 30 seconds
Principle: Iodometry
▪ The method is based on the ability of peroxides to pink colour indicate rancidity
liberate iodine from potassium iodide.
▪ Lipid is dissolved in a suitable organic solvent and (3) Thiobarbituric Acid (TBA) Test
an excess potassium iodide is added.
▪ This is one of the most widely used tests for determining
R-O-O-H + KI R-O-H + KOH + I2 the extent of lipid oxidation.
▪ The liberated iodine is titrated with sodium thiosulfate ▪ It measures secondary products of lipid oxidation,
mainly malonaldehyde.
and starch indicator.
▪ TBA reacts with malonaldehyde to yield coloured
I2 + 2 S2O3 2-
2 I + S4O6
- 2-
cmpd. That is measured spectrophotometrically.
 analysis of lipids include:

(A) (B) (C) (D)

Determination of Determination of Analysis of lipid Analysis of
physicochemical
total lipid content lipid composition oxidation properties

▪ Different analytical methods can be used to characterise

the physicochemical properties of lipids.
▪ Examples of these properties:
1. Melting point
2. Specific gravity
3. Refractive index
4. Viscosity
5. Colour
6. Odour (flavour) etc.
 SUMMARY: Analysis of Lipids (Oils and Fats)
▪ Introduction (B) Detmn. of Total Lipid Composition
▪ Oils & Fats (definition & difference)
(1) Chemical Methods
▪ Classification of Lipids (i) Iodine Value (ii) Acid Value
▪ Why lipids are important constituents of food? (iii) Saponification Value (iv) Specific Colour Tests
▪ Importance of lipid analysis (2) Instrumental Methods
▪ Analysis of lipids include (A) Detmn. of Total Lipid Content, ❑ Chromatography
(B) Detmn. of Lipid Composition, (C) Analysis of Lipid ❑ NMR spectroscopy
Oxidation, (D) Analysis of Physicochemical Properties
(C) Analysis of Lipid Oxidation
(A) Detmn. of Total Lipid Content
❑ Peroxide value
(1) Solvent Extraction Methods ❑ Kreis test
▪ Introduction ▪ Sample preparation ▪ Solvent selection ❑ Thiobarbituric Acid (TBA) test
▪ Methods of solvent extractions (continuous, semi-continuous, discontinuous)
(2) Non-solvent Wet Extraction Methods (Babcock Method) (D) Analysis of Physicochemical Properties
(3) Instrumental Methods (GC & IR) ❑ Melting point ❑ Refractive index ❑ Colour
❑ Specific gravity ❑ Viscosity ❑ Odour/flavour
etc.
What Are Emulsifiers?

Role of an emulsifier:
stabilises an emulsion
mayonnaise mayonnaise with
without emulsifier
emulsifier
Margarine, spread
Ice-cream
Cake

Emulsifiers: Example of
Applications in Food
 Example of an Emulsifier
E322, LECITHIN = phosphatidylcholine (a phospholipid)

❑ Egg yolk, one of the best sources of

lecithin in food. Other animal
sources include liver, milk, fish, fish
Phosphatidylcholine: eggs, chicken, and meat.
red – choline residue and phosphate group; ❑ Good plant sources of lecithin
black glycerol residue; include seeds, peanuts, wheat
green – monounsaturated FA residue; germ, olives, avocado, and
blue – saturated FA residue cabbage.

❑ Lecithin in food additives often

derived from soybean oil (soy
lecithin) & sunflower oil.
Example of an Emulsifier
E322, LECITHIN = phosphatidylcholine (a phospholipid)

Muslims are not forbidden from eating lecithin

However, since lecithin may be derived from

animals as well as plant sources, care needs to be
taken to ensure the source is halal

Lecithin derived from plant and egg yolk is

permissible, as well as that derived from animals
slaughtered according to the Islamic Shari’ah.
Example of an Emulsifier
E322, LECITHIN = phosphatidylcholine (a phospholipid)

Non-food applications include:

In the pharmaceutical industry, it acts as a wetting, stabilising agent

and chlorine enrichment carrier. It helps in emulsifications and
encapsulation, and is a good dispersing agent. It can be used in the
manufacturing of intervenors fat infusions

In animal feed, it enriches

fat and protein an
improves palletisation
In the paint industry, it forms
protective coating for
surfaces with painting and
printing ink.
 Example of an Emulsifier
E 471 mono & diglycerides of fatty acid
E 472 esters of mono & diglycerides of fatty acid
Examples:
Commercial source may be either :

❑ Animal (cow- or pig-derived), or fish

or
❑ Vegetable, derived primarily from soy 1,3-diglyceride
bean and canola oil

❑ May also be synthetically produced

1-monoglyceride

❖ E471 & E472 also can function as stabiliser & thickener

 Example of an Emulsifier
E 471 mono & diglycerides of fatty acid
E 472 esters of mono & diglycerides of fatty acid

1,3-diglyceride
hydrolysis

FAT or OIL = triglycerides (triacylglycerols)

fatty acids 1-monoglyceride

glycerol or glycerin
Example of an Emulsifier
E 471 mono & diglycerides of fatty acid SAMPLE
E 472 esters of mono & diglycerides of fatty acid APPLICATIONS

Macaroni & pasta Ice-cream Bakery

Pharmaceuticals & Cosmetics

Example of an Emulsifier
E 471 mono & diglycerides of fatty acid SAMPLE
E 472 esters of mono & diglycerides of fatty acid APPLICATIONS
E 472e (diacetyl tartaric esters of monoglycerides)
without E 472e with E 472e better texture

E472a E472d
Acetic acid esters of mono- and diglycerides of fatty acids Tartaric acid esters of mono- and diglycerides of
E472b fatty acids
Lactic acid esters of mono- and diglycerides of fatty acids
E472f
E472c Mixed acetic and tartaric acid esters of mono- and
Citric acid esters of mono- and diglycerides of fatty acids diglycerides of fatty acids
End of
Wk3
Lec 3