APPLICATION OF GAS CHROMATOGRAPHY MASS SPECTROMETRY

AND ELEMENTAL ANALYZER-ISOTOPE RATIO

MASS SPECTROMETRY TECHNIQUES
TO DISTINGUISH LARD FROM SELECTED ANIMAL FATS
BEFORE AND AFTER CHEMICAL GLYCEROLYSIS

By

NINA NAQUIAH BINTI AHMAD NIZAR

Thesis Submitted to the School of Graduate Studies,

Universiti Putra Malaysia,
In Fulfillment of the Requirements for the Degree of Master of Science

July 2013
 2

Appendix B3

COPYRIGHT

All material contained within the thesis, including without limitation text, logos,
icons, photographs and all other artwork, is copyright material of Universiti
Putra Malaysia unless otherwise stated. Use may be made of any material
contained within the thesis for non-commercial purposes from the copyright
holder. Commercial use of material may only be made with the express, prior
written permission from Universiti Putra Malaysia.

Copyright © Universiti Putra Malaysia

2
 DEDICATION

In the Name of Allah

Especially for:

Abah and Ibu,

Shahid, Hanis, Syahirah, Sharaf,

All my friends and relatives,

For the never ending love and support…

I love you Lillahitaala!

ii
 Abstract of thesis presented to the Senate of Universiti Putra Malaysia in
fulfillment of the requirement for the degree of Master of Science

APPLICATION OF GAS CHROMATOGRAPHY MASS SPECTROMETRY

AND ELEMENTAL ANALYZER-ISOTOPE RATIO
MASS SPECTROMETRY TECHNIQUES
TO DISTINGUISH LARD FROM SELECTED ANIMAL FATS
BEFORE AND AFTER CHEMICAL GLYCEROLYSIS

By

NINA NAQUIAH BINTI AHMAD NIZAR

July 2013

Chair: Ir. Dzulkifly Bin Mat Hashim, MSc

Institute: Halal Products Research Institute

A study was conducted to differentiate lard from selected animal fats

namely chicken fat, beef fat and mutton fat, before and after chemical

glycerolysis. It was carried out using Gas Chromatography Mass

Spectrometry (GCMS) and Elemental Analyzer–Isotope Ratio Mass

Spectrometry (EA-IRMS) techniques. The comparison of overall fatty acid

data obtained by Gas Chromatography analysis before and after chemical

glycerolysis showed that lard and chicken fats shared common

characteristics by having palmitic, oleic and linoleic acids as major fatty

acids. On the other hand, beef and mutton fats shared common

characteristics by possessing palmitic, stearic and oleic acid as major fatty

acids. Direct comparisons among the fatty acid data therefore may not be

iii
suitable for differentiation of animal fats. When the fatty acid distributional

data of the animal fats was subjected to Principle Component Analysis

(PCA), it was demonstrated that stearic, oleic and linoleic acids were the

most discriminating parameters in the clustering of animal fats to four

subclasses. The stable isotope analysis of lard and selected animal fats before

chemical glycerolysis using EA-IRMS showed significant difference in the

carbon isotope ratios (δ 13C). The same finding was observed after chemical

glycerolysis. This would be a good indicator in discrimination of lard,

chicken, beef and mutton fats. The current finding leads to a more efficient

method, to screen and ascertain the source of origin of fats used in food

products.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk Ijazah Master Sains

iv
APLIKASI TEKNIK KROMATOGRAFI GAS SPEKTROMETRI JISIM DAN
ANALISIS ELEMEN NISBAH ISOTOP SPEKTROMETRI JISIM BAGI
PEMBEZAAN LEMAK BABI DARIPADA LELEMAK HAIWAN LAIN
SEBELUM DAN SELEPAS GLISEROLISIS KIMIA

Oleh

NINA NAQUIAH BINTI AHMAD NIZAR

Julai 2013

Pengerusi: Ir. Dzulkifly Bin Mat Hashim, MSc

Institut: Institut Penyelidikan Produk Halal

Satu kajian telah dijalankan untuk membezakan lelemak babi daripada lelemak

haiwan lain iaitu lelemak ayam, lelemak lembu dan lelemak kambing, sebelum

dan selepas gliserolisis kimia. Kajian ini telah dijalankan dengan menggunakan

Teknik Kromatografi Gas Spektrometri Jisim (GCMS) dan Analisis Elemen

Nisbah Isotop Spektrometri Jisim (EA-IRMS). Perbandingan keseluruhan data

asid lemak yang diperolehi daripada analisis Kromatografi Gas sebelum dan

selepas gliserolisis kimia menunjukkan lelemak babi dan lelemak ayam

mempunyai ciri-ciri yang sama iaitu mempunyai asid palmitik, asid oleic dan

asid linoleik sebagai komponen asid lemak yang utama. Sementara itu, lelemak

lembu dan kambing pula berkongsi ciri-ciri yang sama dengan memiliki asid

palmitik, asid stearik dan asid oleik sebagai asid lemak utama. Walau

bagaimanapun, perbandingan taburan data asid lemak sahaja tidak dapat

membezakan lelemak haiwan. Oleh itu, taburan data asid lemak tersebut

v
diproses menggunakan Analisis Prinsip Komponen (PCA). Analisis tersebut

telah menunjukkan bahawa asid stearik, oleic dan linoleik adalah parameter

yang paling utama dalam membezakan lelemak haiwan kepada empat

kumpulan berasingan. Analisis isotop stabil lelemak babi dan lelemak haiwan

sebelum gliserolisis kimia menggunakan EA-IRMS menunjukkan perbezaan

yang signifikan dalam nilai isotop karbon (δ13C). Pemerhatian yang sama turut

didapati selepas proses gliserolisis kimia dijalankan. Ini dapat dijadikan asas

dalam pembezaan lelemak babi, ayam, lembu dan kambing. Penemuan ini akan

membawa kepada kaedah yang lebih cekap untuk tujuan saringan (screening)

makanan, selain dapat memastikan sumber lelemak yang digunakan dalam

produk makanan.

ACKNOWLEDGEMENTS

Thank you Allah the Most Gracious; for His mercy granted me wisdom, faith

vi
and courage to initiate and successfully complete this endeavor. Alhamdulillah!

Thank you to my supervisors, Ir. Dzulkifly Mat Hashim and Dr. Nazrim

Marikkar, for their sincere and knowledgeable guidance.

My family, especially Abah and Ibu for their love and encouragement. You saw

me through.

To all my friends, near and far, your support means the world to me. Thank

you, for being there.

To all staffs and colleagues of Halal Products Research Institute, UPM, your

contributions, are deeply appreciated. Last but not least, I want to thank UPM

for providing the funds and opportunity to study here.

Thank you so much!

APPROVAL SHEET 1

I certify that a Thesis Examination Committee has met on 11 th July 2013 to

conduct the final examination of Nina Naquiah binti Ahmad Nizar on her thesis

vii
entitled “Application Of Gas Chromatography Mass Spectrometry and
Elemental Analyzer-Isotope Ratio Mass Spectrometry Techniques to
Distinguish Lard from Selected Animal Fats Before and After Chemical
Glycerolysis” in accordance with the Universities and University Colleges Act
1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15
March 1998. The Committee recommends that the student be awarded the
Masters of Science degree.
Members of the Thesis Examination Committee were as follows:

Hasanah, PhD
Prof. Dr.
Faculty of Food Technology
Universiti Putra Malaysia
(Chairman)

Badlishah, PhD
Prof. MadyaDr.
Faculty of Food technology
Universiti Putra Malaysia
(Internal Examiner)

Amin Ismail, PhD

Prof. Dr.
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Internal Examiner)

______________________
Bujang Kim Huat
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia

Date:

This thesis was submitted to the Senate of Universiti Putra Malaysia and has
been accepted as fulfillment of the requirement for the degree of Master of
Science. The members of the Supervisory Committee were as follows:

viii
Dzulkifly bin Mat Hashim, MSc, Ir.
Lecturer
Halal Products Research Institute
Universiti Putra Malaysia
(Chairman)

Mohammed NazrimMarikkar, PhD

Senior Lecturer
Faculty of Biotechnology & Biomolecular Sciences
Universiti Putra Malaysia
(Member)

_____________________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia

Date:

DECLARATION

I declare that the thesis is my original work except for quotations and citations
which have been duly acknowledged. I also declare that it has not been
previously, and is not concurrently, submitted for any other degree at Universiti
Putra Malaysia or at any other institution.

ix
 _____________________________________

NINA NAQUIAH BINTI AHMAD NIZAR

Date: 11th July 2013

TABLE OF CONTENTS
Page
ABSTRACT iii
ABSTRAK v
ACKNOWLEDGEMENT vii
APPROVAL viii
DECLARATION x
LIST OF TABLES xiii
LIST OF FIGURES xiv
x
LIST OF ABBREVIATIONS xv

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW
2.1 Animal Fats 5
2.1.1 Lard, beef fat, mutton fat and chicken fat 5
2.2 Animal fats in different forms 9
2.2.1 Chemical Glycerolysis: 10
Mono- (MAG) and Di-acylglycerols (DAG)
2.2.2 Fractionation 13
2.3 Analytical Tools for Detection of Animal Fats 15
2.3.1 Isotope Ratio Mass Spectrometry (IRMS) 15
2.3.2 Gas Chromatography Mass Spectrometry (GCMS) 21
2.3.3Principle Component Analysis (PCA) 23

3 DIFFERENTIATION OF LARD FROM SELECTED

ANIMAL FATS BY EA-IRMS AND GCMS
3.1 Introduction 25
3.2 Materials and Methods 28
3.2.1 Materials 28
3.2.2 Determination of δ13C for Animal Fats 29
3.2.3 Preparation of fatty acid methyl esters (FAME) 30
3.2.4 GCMS analysis of FAME 30
3.2.5 Fractional Crystallization of Lard 31
3.2.6 Statistical Analysis 31
3.3 Results and Discussions 32
3.3.1 δ13C Values of Lard and Selected Animal Fats 32
3.3.2 Fatty Acid Distributional Pattern of Animal Fats 35
3.4 Conclusion 42

4 DIFFERENTIATION OF LARD FROM SELECTED

ANIMAL FATS AFTER CHEMICAL GLYCEROLYSIS BY
EA-IRMS AND GCMS
4.1 Introduction 44
4.2 Materials and Methods 45
4.2.1 Materials 45
4.2.2 Chemical Glycerolysis of Selected Animal Fats 46
4.2.3 Separation of MAG and DAG by column 46
chromatography
4.2.4 TLC Verification 47
4.2.5 GCMS analysis of FAME 47
xi
 4.2.6 Determination of δ13C for MAG and DAG derived
from animal fats 47
4.2.7 Statistical Analysis 47
4.3 Results and Discussion 48
4.3.1 Quantitative recovery of MAG and DAG 48
4.3.2δ13C Values of Selected Animal Fats After Chemical
Glycerolysis 49
4.3.3 Fatty Acid Composition of MAG 52
4.3.4 Fatty Acid Composition of DAG 57
4.4Conclusion 60

5 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATION FOR FUTURE RESEARCH
5.1 Summary and general conclusion 61
5.2 Recommendation for future research 63

REFERENCES/ BIBLIOGRAPHY 65
APPENDICES 74
BIODATA OF STUDENT 80
PUBLICATIONS 81
CONFERENCES 81
AWARD 81

LIST OF TABLES

Table
Page
1 δ13C values of different animal fats 32

2 Pearson’s Correlation Coefficient (r)between δ13C

values and unsaturated fatty acid contents in lard, 33
chicken fat, beef fat and mutton fat

xii
3 Fatty acid compositions (%, peak area) of lard,
35
chicken fat, beef fat and mutton fat

4 δ13C values of lard, LO and LS 41

5 Fatty acid composition (%, peak area) of lard, LO

and LS 42

6 Recovery of partial acylglycerols of lard, chicken

49
fat, beef fat and mutton fat

7 δ13C values of MAG and DAG of chicken fat, lard,

50
beef fat and mutton fat

8 Pearson’s correlation coefficient between δ13C

values and unsaturated fatty acid contents of MAG
54
and DAG derived from lard, chicken fat, beef fat
and mutton fat

9 Fatty acid composition (%, peak area) of MAG and

DAG of lard, chicken fat, beef fat and mutton fat 68

LIST OF FIGURES

Figure Page

1 Stereo isomeric sn-1- and sn-2- and sn-3- 11

monoacylglycerols

A racemic mixture of sn-1,2- and 2,3- 12

2
diacylglycerols

3 Simplified chemical equation for fat 12

glycerolysis (R=alkyl chain of fatty acid)

xiii
4 Schematic diagram of Isotope Ratio Mass 18
Spectrometer

5 Schematic diagram of Elemental Analyzer 19

6 Flowchart of EA-IRMS analysis 19

7 Schematic diagram for a GC system 22

Loading plot of PCA of animal fats based on

8
fatty acid composition 38

Loading plot of PCA of animal fats based on

9
fatty acid composition 38

10 Score plot of PCA of MAG derived from 40

animal fats based on fatty acid composition

11 Loading plot of PCA of MAG derived from 40

animal fats based on fatty acid composition

12 Score plot of PCA of DAG derived from 56

animal fats based on fatty acid composition

13 Loading plot of PCA of DAG derived from 56

animal fats based on fatty acid composition

14 DSC cooling curve for (a) lard, (b) lard stearin 59

and (c) lard olein

LIST OF ABBREVIATIONS

AOAC Association of Analytical Chemist

AOCS American oil Chemist Society

BF Beef fat

ANOVA Analysis of Variance

δ13C Carbon Isotope Value

DSC Differential Scanning Calorimetry

xiv
FA Fatty Acid

FAME Fatty Acid Methyl Esters

GC Gas Chromatography

GCxGC-TOF-MS Gas Chromatography x Gas Chromatography- Time of Flight-

Mass Spectrometer

MF Mutton fat

IUPAC International Union of Pure and Applied Chemistry

LD Lard

MAG Monocylglycerol

DAG Diacylglycerol

EA-IRMS Elemental Analyzer-Isotope Ratio Mass Spectrometer

PCA Principal Component Analysis

TAG Triacylglycerol

xv
 CHAPTER 1

GENERAL INTRODUCTION

Lard is one of the important products traded worldwide. In 2010, the annual

production of lard is 109.9 million tonnes (Food Outlook, 2012), wherein the

highest production was from China (51.7 mt), followed by European Union

(22.5 mt) and USA (9.9 mt) (FAO, 2010). For certain segments of the society,

consumption of lard is not desirable due to religious restriction (Al-Taher, 2004)

and various health reasons (Wang and Lin, 1995). According to past studies,

lard is reported to be mixed with other fat species such as beef, mutton, and

chicken in different food products (Anna, 2006; Saeed et al., 1989;

Saeed et al., 1986). Hence, various efforts have been made to develop analytical

approaches to differentiate lard from other animal and plant species. A

considerable amount of literature on lipid-based methodologies for such

purpose have been published (Sawaya et al., 1990; Marikkar et al., 2005a;

Marikkar et al., 2005b; Rohman and Che Man, 2010; Rohman et al., 2011;

Che Man et al., 2011; Nurjuliana et al., 2010).

In the last 40 years, extensive works on modification of fat, cholesterol content

and fatty acid composition of animal products were done, in requirement of

producing high quality food products, that meet the optimum dietary

recommendations for human diet as well as enhancing the versatility of fats and

oils in different industrial applications (Jakobsen, 1999). The two most common

1
methods used to modify the physico-chemical properties of the original fats and

oil arechemical glycerolysis and fractionation. The products of the former are

partial acylglycerols (MAG and DAG). In this research, fractionation of lardthat

yields lard stearin and lard olein would be investigated. Since modifications

affect the composition and physical-chemical properties of the original oil,

therefore, a proper basis for differentiation of lard and selected animal fats in its

modified forms is timely.

Another new and interesting field of research that could be explored for

differentiation or detectionof this kind is the carbon isotope ratio analysis by

using Isotope Ratio Mass Spectrometry (IRMS). Already there are some reports

to illustrate the use of stable isotope ratio analysis of light elements such as

carbon, hydrogen, nitrogen, and oxygen to verify authenticity and geographical

origin of some food samples (Jochmann, 2009). Most of the past researches,

however, were mainly focused on honey (Chesson et al., 2011; Simsek et al.,

2012), fish oil (Aursand et al., 2000), vegetable oils such as olive oil, sunflower

oil, groundnut oil, palm oil, rapeseed oil and corn oil (Angerosa et al., 1999;

Kelly et al., 1997; Bianchi et al., 1993), and essential oils (Schipilliti et al., 2010). As

of date, very few studies have been reported on the use of IRMS to investigate

animal fats. It was reported by Hamilton, (1998) that non-maize oils (animal fats

included) are clearly differentiated from maize oils. To the best of our

knowledge, there is hardly any studies to show the potential application of

IRMS in Halal authentication purposes, thus establishing their isotope ratios for
2
detection purposes has become important. The information of this kind would

be greatly helpful as a basis for food control authorities who are required to

carry out routine tests on commercial products that are suspected to contain lard

(Yantyet al., 2011) other than to ensure food safety and to protect the consumers

from fraud and deception.

The application of chromatographic analysis in the earlier studies on

characterization and comparison of edible oils have shown to give accurate and

consistent results (Aparicio and Aparicio-Ruíz, 2000). Animal oils from different

species, brands and grades can be discriminated conveniently using Gas

Chromatography (GC) (Araujo et al., 2010).However, there may be difficulties in

sorting or differentiating the animal fats solely based on fatty acid distributional

pattern. Thus, Principle Component Analysis (PCA) may be effectively applied

in chromatography for the purpose of measuring similarity and dissimilarity

among calculated data. Owing to the high occurrence of components in oils, the

evaluation and comparison of the chromatographic profiles by visual methods

may be enhanced by the usage of PCA (Cserháti, 2010). Therefore, in this work,

a study to evaluate the potential of GC combined with PCA for the

differentiation of lard and selected animal fats and its derivatives is important.

As the fatty acid distributional data from gas chromatography techniques are

well established, it would be interesting to see its correlation with carbon

isotope values acquired from IRMS.

3
Hence, the objectives of the present study are:

1. To distinguish lard from selected animal fats namely chicken, beef and

mutton fats in terms of fatty acid components using Elemental Analyzer-

Isotope Ratio Mass Spectrometry (EA-IRMS) andGas Chromatography

Mass Spectrometry (GCMS)

2. To determine whether chemical glycerolysis of lard and the selected

animal fats namely chicken, beef and mutton fatswould affect the ability

to distinguish them using Elemental Analyzer-Isotope Ratio Mass

Spectrometry (EA-IRMS) and Gas Chromatography Mass Spectrometry

(GCMS)

4
 CHAPTER 2

LITERATURE REVIEW

2.1 Animal Fats

2.1.1 Lard, beef fat, mutton fat and chicken fat

Animal fats have long been utilized in human diets despite a remarkable

diversification to include other lipid types over the years. The depot fats of

animals are readily available during the butchering of slaughtered animals and

are easily extracted. The major animal fats (also termed meat fats) traded world-

wide are produced from pigs (Sus scrofa), termed as lard or rendered pork fat,

from cattle (Bos taurus) or sheep (Ovis aries) termed tallow, and from poultry

(primarily chickens, Gallus gallus) termed as poultry fat. Tallow from domestic

cattle is known as beef tallow, whereas that from sheep is termed mutton tallow.

‘‘Acylglycerol,’’ is the primary component of animal lipids which may exist as

either fats or oils. At room temperature (240C), the former are primarily solid

and the latter are liquid (Haas, 2005). The major classes of lipids of interest in

feedstuffs and foods include fatty acids, triacylglycerols and phospholipids

(Carrasco et al., 2009).

Lard is the fat rendered from fresh, uncontaminated, healthy fatty tissues from

the meat of sound swine (Sus scrofa) at the time of slaughter, and hence, fit for

human consumption. It is also imperative that the fat is pure and without the

presence of bones, isolated skin, blood or any other viscera. Edible tallow
5
(dripping), on the other hand, is the product obtained by rendering the clean,

sound, fatty tissues (including trimming and cutting fats), attendant muscles

and bones of bovine and/or sheep (Ovis aries) in good health at the time of

slaughter and fit for human consumption (Codex Alimentarius Commission,

2001).

In the food industry, animal fats were used immensely for domestic frying due

to the desirable flavors they impart on some foods, as in the flavor added to

french-fried potatoes by tallow and to pie crusts by lard (Haas, 2005). Besides,

they are used as embedded ingredients in the formulation of food products such

as breads and cakes (Fadzlillah et al., 2011) because animal fats exhibit good

stability and have generally been economical to use (Haas, 2005). Likewise, both

genuine and industrially-modified randomized lard (shortening) are used

commercially (Al-Rashood and Abou-Shaaban, 1996) to blend with vegetable

oils as they are capable to be mixed efficiently with vegetable oils to produce

cost-effective margarines, shortenings, and other oil-based foods

(Mansor et al., 2011). However, concerns regarding the relationship of dietary

cholesterol and saturated fatty acid to coronary heart disease made the expert

committees to recommend reduction of animal fat consumption by switching to

plant oils and fish oils (Jakobsen, 1999). From a religious perspective, the

presence of lard in any food products precludes consumption by Muslims

(Che Man et al., 2010).

6
In the recent past, many investigations on differentiation and detection of

animal fatsin food samples have been conducted due to these reasons. One of

the earliest works is the detection of lard in animal body fats extracted from

cow, lamb, and chicken, reported by Che Man and Mirghani, (2001). They

investigated the potential application of FTIR spectroscopy method to detect the

presence of lard in lamb fat, chicken fat and beef fat. In subsequent years, the

same work was re-investigated by Jaswir et al., (2003) by using advanced

statistical technique such as multivariate analyses. A while later,

GCxGC-ToF-MS was utilized to differentiate lard from other animal fats namely

beef fat, goat fat and chicken fat by identifying lipid markers and adoption of

chemometric methods (Chin et al., 2009; Indrasti et al., 2010).

Apart from differentiation of animal fats, studies on adulteration of lard in

vegetable oil were also instigated. Differential Scanning Calorimetry (DSC) has

been employed to monitor the adulteration of animal fats in sunflower oil

(Naysrah et al., 2012), canola oil (Marikkar et al., 2002), and palm oil

(Marikkar et al., 2001). Then, application of liquid chromatography (HPLC)

coupled with multivariate data analysis to distinguish lard from other animal

fat, as well as lard and other animal fats from some refined vegetable oils were

implemented (Marikkar et al., 2005a). In another separate research, the

pancreatic lipolysis technique was adopted to monitor the compositional

variations in sn-2 positional fatty acids in some vegetable oils after adulteration

with animal depot fats using a multivariate data analysis approach

7
(Marikkar et al., 2005b). The technique of combining FTIR with multivariate

analyses have also been used to quantify the presence of lard in food products

such as cakes (Syahariza et al., 2005a) and chocolates (Syahariza et al., 2005b).

Study on animal fat derivatives is another area which has drawn considerable

interest among researchers. Kallio et al., (2001) investigated the use of tandem

mass spectrometry to determine the regioisomerism of fatty acids in lard,

tallow, yolk, chicken skin, palm oil, palm olein, palm stearin, and a

transesterified blend of palm stearin and coconut oil. Later, Indrasti and

co-researchers (2010) used GC×GC-TOF-MS in characterization of regiospecific

isomers of mono- and diglycerides (MAG, DAG) in the glycerolysis products

derived from lard, sunflower seed oil, corn oil, butter and palm oil. In a recently

concluded study, Nasyrah et al., (2012) attempted to characterize animal and

plant derived MAG and DAG using fatty acid composition and thermal profiles.

The application of principle component analysis (PCA) to fatty acid composition

and thermal profile data has been shown to differentiate MAG and DAG from

plant oils from those derived from animal sources. Apart from MAG and DAG

discrimination, common characteristic of native lard and its fractions were also

identified (Yantyet al., 2011). From the aforementioned examples, it shows that

authentication of food products is vital for consumers and industries, in every

production process, from raw materials to finished products.

8
From the economic perspective, product authentication is crucial to evade unfair

competition that can lead to market catastrophe since most targeted food

products for adulteration are monetary-value products and/or produced in

abundant around the globe (Cordella, 2003). From the religious view, Muslims

stress on the importance of the permissibility of sources of food to be consumed.

The Orthodox Jewish religion also prohibits the consumption of both pork and

lard derived from pigs in any products (Al-Rashood and Abou-Shaaban, 1996).

This is because food intake will affect the development of human wellness and

behavior. Hence, harmonization of modern science and Islamic law is very

important especially with regard to halal authentication to protect from fraud

and deception (Fadzlillah et al., 2011).

2.2 Animal Fats in Different Forms

Animal fats consist primarily of triacylglycerol (TAG), these being esters

of glycerol with three fatty acids. As fatty acids (both saturated and

unsaturated) occur in large numbers in oils, the number of different TAG

in an oil may vary considerably. The versatility of fatty oils could be

enhanced significantly with the availability of processes to modify the

solid-liquid balance of the TAG molecules in the temperature range of

interest in a given application. Fractionation (fractional crystallization),

hydrogenation and esterification (Hamm, 1995) are the three major

modification processes used extensively by the industry. As modification

brings considerable changes in the chemical composition and physico-

9
 chemical properties of the original fats, it may pose a challenge to the

detection of adulteration practices taking place in food systems.

2.2.1 Chemical Glycerolysis: Mono- (MAG) and Di-acylglycerols

(DAG)

Chemical glycerolysis is the process of breaking a chemical bond with the use of

glycerine, in the presence of an alkaline catalyst. It is a process whereby fats and

oils of the desired hardness are mixed with an excess of glycerine at elevated

temperatures in the presence of (usually) either sodium or calcium hydroxide.

The reaction mixture is kept at an elevated temperature until the fatty acid

radicals of the triglycerides are redistributed at random among the available

hydroxyl groups of the glycerine. The reaction mixture is cooled after

equilibrium has been attained. The excess glycerine will separate as a lower

layer upon cooling and can be partially removed by decanting (O’Brien, 2009).

The products of chemical glycerolysis are MAGs and DAGs.They are classified

as the food additives class E471 in the EuropeanUnion (EU), whereas, hold a

GRAS (generally recognized as safe) status in the US. In the food industry,

MAGs and DAGs are widely used in bakery products, margarines, dairy

products, and confectionary because of their emulsifying, stabilizing, and

conditioning properties. In cosmetic and pharmaceutical industries, they

function as drug carriers, whereas in creams and lotionsthey are required for

consistency improvements. Their lubricating and plasticizing properties allow

MAGs to be used in textile and fiber processing, as well as in the manufacturing

10
of plastics (Damstrupet al., 2005). MAGs accounts for more than 70% of the total

world usage of food emulsifiers. They are esters of the trihydric alcohol glycerol

in which only one of the hydroxyl groups is esterified with a long-chain fatty

acid. They can exist in three stereochemical forms as shown in Figure 1. MAGs

consist of single fatty acyl chain esterified (-COO-) to a glycerol backbone

(Damstrup, 2008).The chain length of the acyl group in MAG would determine

its application in various formulations.

Figure 1. Stereo isomeric sn-1- and sn-2- and sn-3-monoacylglycerols

(Source: Lipidlibrary, 2012)

On the other hand, DAGs are esters of the trihydric alcohol glycerol in

which two of the hydroxyl groups are esterified with long-chain fatty

acids. They also can exist in three stereochemical forms as shown in

Figure 2. It is generally noted that thecommercial products often contain

DAG with the positional isomers of sn-1,2(2,3)- and sn-1,3-DAG (Yanget

al., 2004).A simplified equation representing chemicalglycerolysis is

shown in Figure 3.

11
Figure 2. A racemic mixture of sn-1,2- and 2,3-diacylglycerols
(Source: Lipidlibrary, 2012)

Figure 3. Simplified chemical equation for fat glycerolysis (R=alkyl chain of

fatty acid)
(Source: Negi, 2006)

As of date, various analyses on MAGs and DAGs have been carried out.

Nasyrahet al., (2012) and Indrastiet al., (2010) characterized MAG and DAG by

means of thermal properties and fatty acid distributional pattern. The former

uses GCFID and DSC, while the latter uses GCxGC-ToF-MS in their respective

research. Although some amounts of data on the fatty acid composition of MAG

and DAG have already been determined, the carbon isotope ratios for MAG and

DAG have not been documented before.

12
 2.2.2 Fractionation

Besides chemical glycerolysis, fractionation is also one of the common methods

in modification of fats. Fractionation is one of the activities carried out by the

fats and oils industry to expand the application range of products. Fractionation

of edible fats and oils offer new materials that are more functional than the

original products. Edible fats are complex multi component mixtures of various

triglycerides with different melting points. The melting behavior and the clear

point of fats are important properties for functionality in the various prepared

food products (Gunstone, 2008).

Lard too, as an edible fat could be utilized in diversified forms in the food

systems. It maybe fractionated into its subcomponents namely lard stearin and

lard olein. This can be achieved through fractional crystallization via either by

dry process or solvent assisted process. In dry fractionation, the separation of

TAG molecules occurs on the basis of their melting point differences.

Schematically, single step fractionation yields a hard fraction called stearin and

a soft or liquid fraction known as olein. Stearin can be used as hard-stock for

ghee production and replacer of hard butters in hot climate countries or as a

component for spreadable butter. Besides, it is also used as a hard-stock in place

of hydrogenated oil when blended with liquid oil. Olein, on the other hand, is

used as lipid component to be incorporated into milk fat giving certain mouth

feel for cakes or cookies. In addition, olein is also used as lipid component in

13
creaming applications and spreads as well as for softening butter

(Deffense, 1993).

As of date, various studies have been conducted for fractionation, including lard

(Yanty et al., 2011a), chicken fat (Lee and Foglia, 2000), palm oil (Che Man, 1999)

and beef fat (Grompone, 1989). For example, characterization and thermal

behavior of lard and its fractions (Yanty et al., 2011a) and avocado butter at

different crystallization temperatures (Yanty et al., 2011b) were conducted using

Nuclear Magnetic Resonance (NMR), Differential Scanning Calorimetry (DSC),

Gas Liquid Chromatography (GLC) and High Performance Liquid

Chromatography (HPLC) techniques. Besides, Lee and Foglia (2000) conducted

analysis on fractionated chicken fats using Gas Chromatography (GC).

However, the effect of fractionation on carbon isotope values obtained by IRMS

has not been established before. Hence this study would observe the potential of

the IRMS application in determination of lard and its fractionated forms as a

basis for halal authentication.

2.3 Analytical Tools for Differentiation of Animal Fats

2.3.1 Isotope Ratio Mass Spectrometry (IRMS)

In addition to the common methods in distinguishing characteristics of

animal oils, an interesting field of research that could be tapped is the

determination of stable isotopes in the common animal fats. Isotopic

14
 variations are found in a wide variety of materials occurring in the

nature. The isotopic profile is unique to the origin and history of the

substance. As the isotopic abundances of these elements were fixed at the

Earth was formed, they have not changed since then on a global scale.

IRMS is an analytical technique used to measure the relative abundance

of isotopes in different materials (Carter and Barwick, 2011).

Stable isotope ratio analysis of light elements such as carbon, hydrogen,

nitrogen, and oxygen is widely used for food authentication and geographical

origin tracking (Jochmann et al., 2009). In order to establish an isotopic “profile”

or “signature” for a material, the ratios of the stable isotopes of a number of

elements such as 2H/1H, 13

C/12C, N/14N and
15
O/16O are required to be
18

determined. For example, IRMS is capable of determination in δ 13C values of oils

arising through biosynthesis differences in the mode of carbon fixation in C4

and C3 plants. Differences in δ13C values of individual fatty acids components of

the vegetable oil determined by GC-C-IRMS raises important questions

concerning isotopic discrimination of chain elongation and desaturase enzymes

involved in fatty acid’s biosyntheses (Woodbury, 1995). From a fundamental

point of view, differences in the 13

C contents of fatty acids are expected as a

result of fractionation effects accompanying the different steps of the

biosynthesis (Kelly et al., 1997). Lipids are the major form of carbon storage in

seeds of several plant species, and carbon discrimination during their synthesis

might differ between individual fatty acids (Richter et al., 2010).

15
The principle of IRMS consists of measuring the isotope ratio of an

analyte converted into a simple gas, isotopically representative of the

original sample before entering the ion source of an IRMS system. The

system performs isotope ratio measurements (2H/1H, N/14N,

15
C/12C,
13

O/16O, and 34S/32S) continuously on gases of H2, N2, CO2, CO, and SO2,
18

respectively. The IRMS measures the ratio of ions that correspond to

these gases. For example, in the analysis of carbon isotope ratios, the

mass spectrometer monitors ions with mass to charge ratios (m/z) of 44,

45 and 46 which correspond to the ions produced from CO 2 molecules

containing 12C, 13C, 16O, 17O and 18O in various combinations. The 13C/12C

ratio is naturally constant in all biological products but can fluctuate

from one carbon source to another (atmospheric CO 2, C4 plants, C3

plants, animal carbon, etc.). Therefore, it is easy to detect whether these

different carbon sources were mixed because the C/12C ratio of the
13

product will have shifted compared to that of the natural product

(Cordella et al., 2002).

Ratio (R) = abundance of heavy isotope/ abundance of light isotope

(1)

δ = (Rsample/Rstandard) -1

(2)

Rsample ratio of the sample

16
 Rstandard ratio of the international standard (defined by the IAEA)

δ -values are commonly multiplied by 1000 so that they are reported in

parts per thousand (‰ or per mil) or by 1000 000, to give results in parts

per million (ppm).

There are five main sections of an IRMS instrument: a sample

introduction system, an electron ionization source, a magnetic sector

analyzer, a Faraday-collector detector array, and a computer-controlled

data acquisition system as shown in Figure 4. One of the most common

interfaces used to introduce samples into the IRMS, is the elemental

analyzers (EA-IRMS) as would be discussed in this work (Muccio and

Jackson, 2009).

Figure 4: Schematic diagram of Isotope Ratio Mass Spectrometer

(Source: Muccio and Jackson, 2009)

17
In the case of Elemental Analyzer IRMS (EA-IRMS), to measure the

average isotope ratios for non-volatile liquids or solids, the bulk sample

can simply be weighed and placed in a tin or silver capsule. Figure 5

shows the schematic diagram of an elemental analyzer. The sample

capsule is lowered into a combustion furnace through an auto-sampler

carousel, during which time the sample is combusted at elevated

temperatures under a flow of oxygen into CO 2. Depending on the

isotopes of interest, the combustion products may need to be specifically

treated to reduce interferences. The analyte is next carried through a

chemical trap to remove water which is produced from combustion, and

then into the gas chromatograph where separation of CO2 is performed.

The effluent from the elemental analyzer is then sent to the IRMS

(Muccio and Jackson, 2009). The flow diagram provided in Figure 6

shows that the sequence of analysis can be divided in four stages.

18
 Figure 5: Schematic diagram of Elemental Analyzer
(Source: Muccio and Jackson, 2009)

Combustion of the sample material using the elemental analyzer

Introduction of the evolved gases into the ion source of

the mass spectrometer via the interface
↓

Ionisation of the gas molecules followed by separation

and detection of the ions in the mass spectrometer
↓

Evaluation of the raw data

Figure 6. Flowchart of EA-IRMS analysis

(Source: Muccio and Jackson, 2009)

Stable isotope analysis has become a reliable tool for distinguishing

biological substances, such as food (Baskaran, 2011). This is based on the

fact that C3 and C4 plants possess distinctly different 13C/12C ratios (δ13C

value) due to a different isotopic fractionation during photosynthetic

carbon fixation. Therefore, IRMS has been used extensively for detection

of adulteration. Olive oil and wine are the most studied food items with

regards to commercial frauds (Ghidini et al., 2006).Test on young beef

bulls has shown that stable carbon analysis from tissues can be used as a

method to trace diets containing variable proportions of C3 and C4

plants (De Smet et al., 2004). Isotopic variations are found in a wide

variety of materials occurring in the nature. The isotopic profile is unique

to the origin and history of the substance. Because of this, IRMS has a

19
wide range of applications. Over the years, it has been employed to

detect adulteration honey (Chesson et al., 2011; Simsek et al., 2012),

differentiate farm and wild salmon species (Aursand et al., 2000) and to

test authenticity of vegetable oils namely sunflower oil, corn oil,

groundnut oil, palm oil, rapeseed oil and olive oil (Kelly et al., 1997;

Bianchi et al., 1993).There were studies on animal fats done separately to

investigate the relationship between different diets of the animals with

the changes of carbon isotope values (Rhodes et al., (2010), Bojlul et al.,

(2007), De Smet et al., (2004) and Piasentier et al., 2003). However, two

issues that were not addressed in these studies were whether these fats

could be distinguished from each other based on their carbon isotope

values, in relation to halal in particularand secondly, how modification

of the selected animal fats such as chemical glycerolysis and fractionation

would affect the carbon isotope values.The present study would provide

additional evidence with respect tothese two matters.

2.3.2 Gas Chromatography Mass Spectrometry (GCMS)

As of date, GC technique has been widely used in analysis of fatty acid

composition for various fats and oils (Che Man et al., 2010; Cheong et al.,

2010; Mustafa and Karakaya 2009; Mustafa et al., 2010; Lee and Foglia,

2000; Grompone, 1990). It is used for separating volatile organic

compounds among different varieties of the same product for detection

of adulteration or for authentication of the genuineness of products.

20
Lipids consist of building blocks of the triacylglycerol esters of fatty

acids. Naturally, fatty acids are synthesized via condensation of malonyl

coenzyme A units by a fatty acid synthase complex (Xiao et al., 2010).

As intact TAG molecules and free fatty acids are not readily volatile,

lipids are usually derivitized prior to analysis. The process of

saponification breaks the TAG molecules into glycerol and fatty acids,

which are thenmethylated. Saponification process reduces the molecular

weight while methylation reduces the polarity, both of which increase

the volatility of the lipids. FAME analysis demands high

chromatographic resolution especially to provide evidence of positional

and geometrical isomers of unsaturated fatty acids in complex mixtures.

In order to meet these requirements, polar stationary phases are

normally used for the separation of complex FAME mixtures, and they

enable separation of fatty acids according to their degree of unsaturation

and carbon number (Ruiz-Rodriguez et al., 2010). The FAMEs are

dissolved in a suitable organic solvent which are then injected into a GC

injection chamber. Upon injection, the sample is heated in the injection

chamber to volatilize and then carried into the separating column by a

heated carrier gas. As FAMEs pass through the column, they undergo

separation into a number of peaks based on the differences in their

molecular weights and polarities. Determination of the total fatty acid

profile allows one to calculate the type and concentration of fatty acids
21
 present in the original lipid sample. Figure 7 shows a schematic diagram

for a GC system.

Figure 7. Schematic diagram for a GC system

(Source: Wikipedia, 2012)
GCxGC-ToF-MS was capable of illustrating the glyceride profile within 20 min

which is beneficial for product development and quality monitoring in various

sectors (Indrasti et al., 2010). Although it is based on more advanced technology,

this 2D technique is a less than ideal solution because of the limitation of the

analysis to a few discrete target regions of the chromatogram, besides it requires

sophisticated instrumentation and experienced analysts (Adahchour et al., 2003).

Gas Chromatography- Flame Ionization Detector (GC-FID) on the other hand, is

relatively inexpensive to acquire, operate and requires low maintenance.

Nonetheless it cannot differentiate between multiple molecules that have the

same retention time, resulting in two or more molecules that co-elute. GC can

separate volatile and semi volatile compounds with great resolution, but it

cannot identify them. MS can provide detailed structural information on most

compounds such that they can be exactly identified, but it cannot readily

22
separate them.Hence alternatively, GCMS reduces the possibility of errorwhen

two different molecules have a similar pattern of ionized fragments in a mass

spectrometer, as it is very unlikely that two different molecules will behave

similarly in both a gas chromatograph and a mass spectrometer

(Wikipedia, 2013).

2.3.3 Principle Component Analysis

Principle component analysis (PCA) is a pattern classification procedurethat is

increasingly applied to the data set to compare similarities or differences of

sample data with original data. PCA allows the number of variables to be

reduced while maintaining most of the information by simultaneously studying

all of the variable relationships. Because of its simplicity, PCA has frequently

been used in food science and technology to classify foodstuffs according to

their chemical composition and to group samples with similar features.

Theoretically, after a multivariate analysis taking into account many analytical

factors, authentic samples may form singular group that can be easily

distinguishable from adulterated samples (Cordella et al., 2002).

For instance, application of PCA to fatty acid data has been studied for

authentication of commercial edible oils (Brodnjak et al., 2005), coffee varieties

(Alves et al., 2003) and peanut cultivars (Shin et al., 2007). Recently, the fatty acid

profile of 119 oil samples was measured by GC and the correlation among

23
peanut oil, soybean oil, palm oil and rapeseed oil was elucidated by PCA

whereby computations proved that the samples form clusters according to the

type of oil (Cserháti, 2010). By using PCA, Nasyrah et al., (2012) has successfully

discriminated plant and animal derived MAG and DAG using GCFID, while

Indrastiet al., (2010) applied the same multivariate technique to differentiate

animal fats using GCxGC-ToF-MS. In this research, the fatty acid distributional

data of animal fats and the animal derived MAGs and DAGs obtained from

GCMS would later be subjected to PCA for further classification, as it might be

hard to differentiate them using mere comparison of the general fatty acid data.

CHAPTER 3

DIFFERENTIATION OF LARD FROM SELECTED ANIMAL FATS BY

EA-IRMS AND GCMS

3.1 Introduction

Authenticity is an important issue for the food industry due to legal compliance,

economic reasons, guarantee of a constant quality, use of safe ingredients, and

religious regulations (Kamm et al., 2007). Over the past 10 years, isotope ratio

mass spectrometry (IRMS) emerged as newer analytical tool to cross-check food

authenticity. IRMS is a specialized technique used to provide information about

the geographic, chemical, and biological origins of substances. The ability to

determine the source of an organic substance stems from the relative isotopic

abundances of the elements which comprise the material. Because the isotope

24
ratios of elements such as carbon, hydrogen, oxygen, sulfur, and nitrogen can

become locally enriched or depleted through a variety of kinetic and

thermodynamic factors, measurement of the isotope ratios can be used to

differentiate between samples which otherwise share identical chemical

compositions. Variations in the abundance of isotopic ratios of light element

namely C/12C, was measured in this experiment (Muccio & Jackson, 2009;
13

Kelly and Heaton 2005). Over the years, IRMS has been used successfully to

obtain specific information about the authenticity and source of origins of

different biological substances such as beef (Bojlul et al., 2007), poultry

(Rhodes et al., 2010) and lamb (Piasentier et al., 2003). The ability to determine

the source of an organic substance stems from the relative isotopic abundances

of the elements which comprise the material. As elements such as carbon,

hydrogen, oxygen, sulfur, and nitrogen can become locally enriched or depleted

through a variety of kinetic and thermodynamic factors, measurement of the

isotope ratios can be used to differentiate between samples which otherwise

share identical chemical compositions. Many past reports highlighted the use of

stable isotope ratio in the detection of adulteration in edible oils. For instance,

the potential use of IRMS has been investigated for detection of sesame oil

adulteration with corn oil (Seo et al., 2010), adulteration of ganoderma spore

lipid with cheaper vegetable oils (Liu et al., 2007) as well as adulteration of

maize oil with other vegetable oils (Mottram et al., 2003). According to our

literature search, the carbon isotope ratios determination has been scantily used

25
for differentiation of animal fats such as lard, chicken fat, mutton fat and beef

fat.

In this connection, adulteration and misbranding of oils from cow, lamb,

chicken, and swine is a particular concern for followers of certain religious

practices. In the food industry, lard particularly is fractionated to lard olein (LO)

and lard stearin (LS) to be used in different applications. The high-melting

fraction (LS) is important with regard to its sensorial and functionality

properties at room temperature. Lower melting fraction (LO) promotes

pourability and ease of handling at low temperatures (Meng et al., 2011). While

olein isolates was found useful as frying medium or bread pan lubricating

agent, stearin has been used to improve the firmness of lard (Yanty et al., 2011).

Because of this, there has been a great deal of interest among researchers to

develop analytical methodologies to authenticate different oil species. For

instance, Marikkar et al., (2002) investigated the differentiation of lard, chicken

fat, beef fat, and mutton fat using differential scanning calorimetric analysis.

Che Man and Mirghani (2001) used Fourier transform infrared spectroscopy to

study the possibility of discriminating these four animal fats. Chin et al. (2009),

followed by Dias et al. (2010) employed GCxGC-Tof-MS to differentiate lard

from other animal fats by using long branched fatty acids. Although

GCxGC-ToF-MS is based on more advanced technology and requires short time

of analysis, this 2D technique limits the analysis to a few discrete target regions

of the chromatogram and is not favored for screening purposes. Moreover, it

26
requires sophisticated instrumentation and experienced analysts

(Adahchour et al., 2003). GC-FID on the other hand, offers analysis time of

37 minutes. Alternatively, GC-MS could offer a convenientscreening method of

analysis in less than 17 minutes without risking the credibility of the data. This

makes it an attractive technique, as the industry and analytical laboratories

demand for high throughput and fast turnaround analytical approaches.

If problems are encountered in the classification, the fatty acid data could be

processed using multivariate data analysis techniques such as Principal

component analysis (PCA) (Christy and Egeberg, 2006). PCA has been well-

known for its capability to identify patterns in data, and express the data in such

a way as to emphasize their similarities and differences (Shin et al., 2007).

Eventually, it helps to establish the authenticity of various kinds of foodstuffs

and other agricultural produces. Hence, the objective of this study is to

distinguish lard from selected animal fats namely chicken fat, mutton fat, and

beef fat by IRMS and GCMS approach. As an addition, it may be of a great

interest to know whether lard and its fractions such as LS and LO are able to be

distinguish from the unfractionated lard based on their δ13C values.

3.2 Materials and Methods

3.2.1 Materials

Three batches of animal fats namely lard, beef fat, mutton fat, and chicken fat

were extracted by rendering of adipose tissues of animals collected from local

27
wet markets in Sri Serdang, Selangor, Malaysia. Melted animal fats were filtered

through double-folded muslin cloth to remove impurities. After adding the

samples with small proportions of anhydrous sodium sulfate to remove residual

moisture, they were filtered through Whatman No. 2 filter paper and stored at

4°C (Marikkar et al., 2001).All chemicals used in this experiment were of

analytical or HPLC grade (boron trifluoride in 25% methanol, hexane, sodium

hydroxide). A standard mixture containing 37 FAME standards was purchased

from Sigma-Aldrich (St. Louise, MO, USA). NBS-22 standard was purchased

from International Atomic Energy Agency, Vienna, Austria.

3.2.2 Determination of δ13C for Animal Fats

About 0.2 µg of each animal fat was weighed and loaded into a clean tin capsule

to determine their δ13C values. The capsules containing samples were placed in

auto-sampler system of elemental analyzer (Europa Scientific, UK) for burning

in an O2 atmosphere of the combustion CuO tube with its temperature set at 960

°C. Combustion gases were eluted through a reduction column by a stream of

He gas and passed into the gas chromatograph where CO 2, still in the He

stream, was separated from the other gases. The gas stream was then entered

into the IRMS system (Sercon Ltd., Crewe, U.K.) where the CO 2 gas was

analyzed by comparison with NBS-22 reference material (with a δ13C value of

−30.03‰). During every batch of analyses, an empty tin capsule was analyzed

as the blank to check the background (Liu et al., 2007). Results are referenced to

Vienna Pee Dee Belemnite (V-PDB). The isotopic values were calculated against

28
the international isotope reference standards: NBS-22 (International Atomic

Energy Agency, Vienna, Austria) for 13 C/12C measurements (Osorio et al.,

2011).Calibration to an internationally recognized scale is achieved through

comparison of closely spaced sample and standard peaks. Such systems are

capable of measuring 13C/12C ratios with a precision approaching 0.1‰ (for

values reported in the standard delta notation). Detection limits to achieve this

level of precision are typically <1 nmol Carbon (roughly 10 ng of a typical

hydrocarbon) injected on-column. The time of processing per sample is less than

400 seconds.

3.2.3 Preparation of Fatty Acid Methyl Esters (FAME)

A 50-mg portion of animal fat was weighed into a 20-ml test tube (with screw

cap). After adding 2ml portion of 2N sodium hydroxide in methanol, the sample

tube was closed and heated at 800C for 1 hour. After allowing the tube and its

content for a few minutes, a 2 ml portion of 25% boron trifluoride solution in

methanol was added. The tube was closed and heated again for 1 hour at 80 0C.

Subsequently, 5 ml portions of water and hexane were added into this. The

contents of the tube were shaken well and allowed to undergo phase separation.

The clear supernatant of the solution were transferred into a 2-ml auto-sampler

vial (AOAC, 2007).

3.2.4 GCMS Analysis of FAME

29
The top hexane layer FAME solution (1 µL) was injected on an Agilent 6890N

GC (Agilent Technologies, Singapore) equipped with a polar capillary column

DB-23 (0.15mm internal diameter, 60 m length and 0.25 µm film thickness;

Agilent Technologies, Singapore) and an Agilent 5973 MS. Split injection was

conducted with a split ratio of 100:1, using helium as a carrier gas at a flow rate

of 1.00mL/min. The temperature of the column was 500C (for 1 min) and

programmed to increase to 1750C at the rate of 25 0C/min. Then, it was increased

at the rate of 40C/min to 2300C for 5 min. The temperature of the injector and

detector was maintained at 2500C (David et al., 2003). Individual peaks of

FAMEs were identified by comparing their retention time with those of 37 fatty

acids standard (Supelco Bellefonte, PA). The percentage of fatty acids was

calculated as the ratio of the partial area to the total area (Yanty et al., 2011).The

samples to be injected need to contain 0.1 to 100 ng of each fatty acid

components.

3.2.5 Fractional crystallization of lard

Fractional crystallization was carried out using acetone as solvent medium. Lard

was melted at 60℃ and mixed with acetone in 1:2 (w/v) ratios. The solution was

boiled at 60℃ until become uniformly dissolved and left at 5±1℃ for 24 h to

crystallize. The precipitated fat was filtered off to give a high melting fat fraction

(LS). After removing the precipitate, the mother-liquor was evaporated under

reduced pressure to yield a liquid called low-melting fraction (Yanty et al., 2011).

30
3.2.6 Statistical Analysis

Data were statistically analyzed by one-way analysis of variance

(ANOVA) using MINITAB (version 14) statistical package at 0.05

probability level.The relationships between individual unsaturated fatty

acid and δ13C values of the animal fats were determined by Pearson’s

Correlation analysis.For grouping and classificationmodels, PCA was

carried out using Unscrambler 9.7 (Camo, USA) software.

3.3 Results and Discussions

3.3.1 δ13C Values of Lard and Selected Animal Fats

The data presented in Table 3 compares the δ 13C values of lard, chicken fat, beef

fat and mutton fat. According to Table 3, the highest δ 13C value is found with

chicken fat (-20.3‰) while the lowest value of the same is found for mutton fat

(-33.2‰). The δ13C values of lard (-23.2‰) and beef fat (-29.5‰) are within the

range of these two extremes of the mean δ13C values.

Table 1.δ13C values of different animal fats

Sample N δ13C (‰)
Chicken fat 3 -20.3d±0.93
Lard 3 -23.2c±1.17
Beef fat 3 -29.5b±0.87
Mutton fat 3 -33.2a±0.32
abcd
Means within column with different superscript are significantly (p < 0.05) different

Interestingly, the δ13C values of these fats are found to show good correlation

(+0.997; p<0.003) with the increasing degree of unsaturation as shown in Table

4. When chicken fat with highest proportion of unsaturated fatty acids (67.52 %)

31
having the highest δ13C value, mutton fat with the lowest proportion of the same

(31.92%) shows the lowest δ13C value. Among the three unsaturated fatty acids

enlisted in Table 2, linoleic acid shows the best positive correlation (+0.988;

p<0.012) with the δ13C values of animal fats (Table 1).

Table 2. Pearson’s correlation coefficient (r) between δ13C values and

unsaturated fatty acid contents in lard, chicken fat, beef fat and mutton fat

Fatty acid r p value

Palmitoleic (C16:1) +0.683 p<0.317

Oleic (C18:1) +0.987 p<0.013

Linoleic (C18:2) +0.988 p<0.012

∑USFA +0.997 p<0.003

The δ13C values of beef fat, chicken fat and mutton fat obtained in this study are

comparably similar to those reported in some previous investigations. For

instance, Rhodes et al. (2010) found that δ13C values of chicken fat obtained from

Hubbard and Ross breeds were around -21‰, which is closely comparable to

the δ13C value of chicken fat obtained in the present study. Likewise,

Bojlul et al. (2007) found that the δ13C value of beef fat was -29.2‰, which is

closely similar to the δ13C value of beef fat obtained in the present study.

Bojlul et al. (2007) further pointed out that the δ 13C value obtained for beef fat

was in accordance with the previous findings reported by De Smet et al. (2004).

In an attempt to authenticate lamb meat, Piasentier et al. (2003) observed that

δ13C values of mutton fat extracted from the lamb meat originated from Great

32
Britain, France and Iceland were within a narrow range from -31.3 to -32.5‰.

Interestingly, the δ13C value of mutton fat obtained in the present study is

roughly closer to the range of values reported by them. In the case of lard, there

is hardly any report to compare the δ 13C value, except the report of

Gonzalez et al. (1999), which indicated that the δ 13C values of pigs’ adipose

tissues were within a narrow range from -22.14 and -23.87‰. The δ 13C values of

adipose tissue of pig may still be considered for comparison purpose as it is the

part of the animal where roughly about 80% of animal fats are deposited

(Wikipedia, 2012).

The statistical analysis of the data from the present study suggests that the

determination of δ13C value for bulk animal fats can be a useful tool since the

δ13C value of lard (-23.2‰) is significantly (p<0.05) different from those of beef

fat (-29.5‰), chicken fat (-20.3‰), and mutton fat (-33.2‰) (Table 3). The

observed variation in the δ13C values of animal fats could be attributed to their

species difference (Osorio et al., 2011), genetic factors (Wood et al., 2008) as well

as the diet fed to the animals (Bojlul et al., 2007). According to previous

investigators, the variation in the δ13C values of oils and fats originated from

different plant sources are due to isotopic fractionation during physical,

chemical and biological processes in plants (Kelly and Rhodes, 2002). This could

also be the factors that affect the values of carbon isotope in animal fats. To

understand the chemical components of the selected animal fats, they will be

33
subjected to gas chromatography analysis to identify the components that

contributes to the carbon isotope values.

3.3.2 Fatty Acid Distributional Pattern of Animal Fats

Fatty acid distributional patterns of lard, chicken fat, mutton fat and beef fat are

compared as shown in Table 2. Lard and chicken fat are found to have more

unsaturated fatty acids (60.98 to 67.52%) than saturated fatty acids (32.48 to

67.52%).

Table 3. Fatty acid compositions (%, peak area) of lard, chicken fat, beef fat
and mutton fat
Fatty acids Lard Chicken fat Beef fat Mutton fat
C10:0 0.08±0.01a - - 0.29±0.06b
C12:0 0.19±0.11a 0.64±0.93a 0.11±0.05a 0.46±0.06 a
C14:0 2.28±1.10 a 1.62±0.65a 6.15±0.31b 6.40±0.49b
C15:0 0.05±0.04a - 0.46±0.24b 0.76±0.02c
C16:0 24.64±1.90a 25.39±1.01a 31.07±0.78b 27.38±1.22ab
C16:1 1.07±0.46a 5.32±0.48c 2.56±0.07b 0.52±0.18a
C17:0 0.25±0.23a - 0.82±0.62a 1.85±0.13b
C18:0 11.53±1.67b 4.84±0.18a 16.53±1.25c 30.90±0.50d
C18:1 42.62±0.74c 43.94±1.77c 35.70±1.71b 29.82±1.04a
C18:2 17.29±3.11b 18.26±1.64b 6.59±0.61a 1.61±0.06a
∑SFA 39.02 32.48 55.15 68.05
∑USFA 60.98 67.52 44.85 31.95
Each fatty acid value in the table represents the mean ± standard deviation of three replicates.
Means within each row with different superscripts are significantly (p<0.05) different.

34
On the other hand, beef and mutton fats are found to possess more saturated

fatty acids (55.15 to 68.05%) than unsaturated fatty acids (44.85 to 31.95%). The

basic difference in the degree of unsaturation of these animal fats could be due

to the diverse pattern of distribution of individual fatty acids. The most

predominant fatty acid of lard is oleic acid (42.62%), followed by palmitic

(24.66%) and linolenic (17.29%), which is in accordance with the findings

reported by previous workers (Che Man et al., 2010; Nurjuliana et al., 2010;

Cheong et al., 2009; Marikkar et al., 2002). Chicken fat is also found to have oleic

acid (43.94%) as the most dominant fatty acid, followed by palmitic (25.39%)

and linolenic (18.26%) acids. In addition, lard and chicken fat having lower

levels of myristic acid was a common characteristic feature, when compared to

the proportion of myristic in mutton and beef fats.These values are comparable

to the values in Codex Alimentarius Comission, (2001).

These similarities in fatty acid distribution would cause adifficulty in making a

clear distinction between them.The proportional distributions of individual fatty

acids of mutton and beef fats in this study are somewhat comparable to those

reported previously by other research workers (Yilmaz and Karakaya 2009;

Yilmaz et al., 2010). Although these two animal fats are also found to have oleic

and palmitic as their most prominent fatty acids, they may tend to differ from

lard and chicken fat with regard to the third and the fourth most abundant fatty

acids. While linoleic is the third most fatty acid in lard and chicken fat, stearic is

the third most fatty acid in beef and mutton fats, resulting firmer fats in the
35
latter (Haas, 2005). This difference could be attributed to the differences in

metabolic process taking place in ruminant and non-ruminant animals. For

instance, Wood et al. (2008) emphasized that linoleic acid in animal fats is

derived entirely from the diet, which is passed through the pig’s stomach

unchanged, absorbed into the blood stream through the small intestine, and

incorporated from there into tissue lipids, resulting in higher levels of linoleic

acid in lard. In contrast to this, in ruminants, linoleic acid is degraded into

saturated and monounsaturated fatty acids through a process known as

microbial bio-hydrogenation. As a result, only a small proportion of dietary

linoleic acid (around 10%) is incorporated into their tissue lipids.However, the

stearic acid contents in beef and mutton fats may vary slightly due to the

influence of various factors namely breed, sex, age and nutritional conditions

(Yilmaz and Karakaya, 2009).

As beef and mutton fat share common characteristics in the way lard and

chicken fat share common characteristic with regard to major fatty acids, it

might be difficult to find differentiation among them using mere comparison of

the overall fatty acid data. In such situations, it would be more appropriate to

use multivariate data analysis techniques such as principle component

analysis.As shown in Table 1, fatty acids namely lauric (C12:0), myristic (C14:0),

palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1) and linoleic

(C18:2) acids are found to occur in variable amounts in all animal fats and could

be used as independent variables in PCA procedure.

36
Figure 8. Score plot of PCA of animal fats based on fatty acid composition
Abbreviations:L, Lard; C, Chicken fat; B, Beef fat; M, Mutton fat.

Figure 9.Loading plot of PCA of animal fats based on fatty acid composition

(PC1: , PC2: )

The score plot of fatty acids derived from the four animal fats as shown in

Figure 13 represents the projection of samples defined by principle component 1

37
(PC1) and principle component 2 (PC2). PC1 is the linear combination of

variables that explain the highest variation among the samples, while PC2 is

orthogonal to PC1 and exhibited the second largest variation

(Cordella et al., 2003). The score plot projected on PC1 described 86% of the

variation while PC2 accounted for 8% of the variation, making up of 94% of

variance for PC1 and PC2. According to the group separation illustrated in

Figure 13, lard is located in upper left quadrant, chicken fat in lower left

quadrant, mutton fats in upper right quadrant, and beef fat in lower right

quadrant. Fatty acid variables giving high influence on the group separation of

the samples in the score plot could be traced from the analysis of the loading

plot. As explained by Cordella et al. (2003), a variable which is farther from the

origin of axis contributes to the most variation in the statistical model generated

by the PCA. According to the loading plot in Figure 14, out of the seven fatty

acid variables stearic, oleic and linoleic, palmitic and palmitoleic acids were the

most discriminating variables that influence the group separation into four

different clusters.

In a separate study, lard fractions namely lard olein (LO) and lard stearin (LS)

were obtained to see whether fractional crystallization would impose any

changes to the δ13C values of lard. The cooling curve of Lard, LO and LSwere

exhibited in Figure 19. It is evident that both LO and LS possessed cooling

profiles, which are dissimilar from that of the native sample. The existence of

two major exothermic thermal transitions at broadly distanced temperature

38
regions signify the occurrence of two distinguishable TAG groups with differing

melting ranges in the cooling curve of LD. This is in agreement with the

previous findings of (Marikkar et al., 2001; Yanty et al., 2011).

Figure 10. DSC cooling curve for (a) lard, (b) lard stearin and (c) lard olein

Table 4 shows the δ13C values of lard, LO and LS. In the case of LS and LO, there

is hardly any report on their δ 13C values. It is observed that no significant

difference is observed among all samples. Gonzalez et al., (1999) reported that

the δ13C value of pigs’ adipose tissue is -22.14 and -23.87‰, which are

comparable to the values observed in this experiment. Even though

fractionation is done, only slight deviations in the δ13C values were observed,

due to the same sample of lard taken for fractionation. The same sample of lard

portrays the same type of diet consumed as highlighted by Ulfert, (2004) in

39
which the δ13C values of the animal tissues may vary due to diet as per the

inclusion of different proportions of C3 and C4 plant species in the fodder.

Table 4. δ13C values of lard, LO and LS

Samples N δ13C(‰)
Mean
Lard 3 -23.20a ±1.17
LO 3 -23.41a ±0.41
LS 3 -22.64a ±0.89
Means within column with different superscript are significantly (p < 0.05) different
abcd

Abbreviation: LO (lard olein), LS (lard stearin)

The data presented in Table 5 compares the fatty acid profiles of LS and LO with

that of lard. LO possessed 65.33% unsaturated fatty acid, followed by lard with

59.91% while LS with 39.71%. As a common feature, both LO and lard were

found to possess higher percentage of unsaturated fatty acids than saturated

fatty acids. The basic difference in the degree of unsaturation of these animal

fats is due to the increase of linoleicacid in LO with simultaneous decrease of

stearic acid. This is the opposite the LS in which higher stearic acid is observed,

with a decrease in linoleic acid content. The major fatty acids of LS are palmitic

followed by oleic, stearic and linoleic, comprising about 95% of the total.

Firmness of lard and all the measured traits related to melting behaviour were

quite strongly affected by the fatty acid composition (Glaser et al., 2004). With

respect to the native sample, great increases in the proportions of stearic and

palmitic could be noticed in LS, with simultaneous reductions in oleic and

40
linoleic acid. In LO, oleic (43.57%) is the most dominant fatty acid followed by

linoleic (21.76%).

Table 5.Fatty acid composition (%, peak area) of lard, LO and LS

FA C12:0 C14:0 C16:0 C18:0 C18:1 C18:2

LS 0.1±0.00a 1.00±0.17a 29.15±1.06a 26.33±0.77a 27.69±1.54b 12.02±0.10b
LO 0.22±0.00a 1.26±0.40a 21.16±0.60b 7.13±0.68b 43.57±0.54a 21.76±2.66a
LAR
D 0.19±0.11a 2.28±1.10a 24.64±1.90b 11.53±1.67b 42.62±0.74a 17.29±3.11a
Each fatty acid value in the table represents the mean ± standard deviation of three replicates.
1

Means within each column with different superscripts are significantly (p<0.05) different.

Naturally, with the migration of more palmitic and stearic acids into the solid

phase, the liquid phase becomes more enriched with oleic and linoleic acids. The

data on fatty acid composition obtained in this study is in accordance with

previous work by Yanty et al., (2011). The correlation is observed here, whereby

LO with the highest proportion of unsaturated fatty acids (65.33%) recorded the

highest δ13C value (-23.41‰). On the other hand, LS with the lowest proportion

of the same (39.71%) shows the lowest δ13C value (-22.64‰).

3.4 Conclusion

This study investigated the application EA-IRMS and GCMS techniques for

differentiation of lard, chicken fat, beef fat, and mutton fat (before chemical

glycerolysis). The significant differences in the values of carbon isotope ratios

(δ13C) of all animal fats shown to be good indicators for differentiation of lard,

chicken fat, beef fat and mutton fat.However, there are no significant difference

41
observed in carbon isotope values of lard, LO and LS. This showed that

fractionation of samples would not affect the carbon isotope values. Comparison

of the overall fatty acid data showed that use of single fatty acid as parameter

may not be suitable to classify animal fats into distinct subclasses. Hence, the

application of multivariate statistical techniques such as PCA would be required

to classify them to determine the source of origin. According to the outcome of

PCA, stearic, oleic and linoleic acids were found to be the most discriminating

parameters in the clustering of animal fats. Hence, this study showed that PCA

of fatty acid data allowed separation of lard from the selected animal fats.

CHAPTER 4
42
 DIFFERENTIATION OF LARD FROM SELECTED ANIMAL FATS AFTER
CHEMICAL GLYCEROLYSIS BY EA-IRMS AND GCMS

4.1 Introduction

In the industry, chemical glycerolysisis a common method used to synthesize

and manufacture partial acylglycerols (MAGs and DAGs)

(Damstrup et al., 2005). The excellent self-emulsifying and surface-active

properties of partial acylglycerols have made them popular emulsifying agents

(Cheng et al., 2005). MAGs and DAGs can be obtained from both animal fats and

plant fats. The animal fat that is highly used in the production of MAG and

DAG is lard. However, some religions like Islam, Judaism and Hinduism forbid

their followers to consume any foods containing porcine or lard, and its

derivatives (Regenstein et al., 2003). Hence, the use of lard may not be desirable

as raw material for production of MAG and DAG due to this reason (Riaz and

Chaudhary 2004). Therefore, there has been a great deal of interest among

researchers to develop analytical methodologies to differentiate animal fats

species. Previously, several methods have been used to characterize MAGs and

DAGs, including differential scanning calorimetric and gas chromatographic

methods (Yang et al., 2004). Analysis of partial acylglycerols or emulsifiers in

food systems were previously performed by several other chromatographic

techniques, namely high performance liquid chromatography (HPLC),

supercritical fluid chromatography (SFC), and planar chromatography (paper

and thin layer chromatography) (Rohman et al., 2012). Multivariate data analysis

43
techniques such as Principal component analysis (PCA) could be used to

processthe fatty acid data if problems arise in the classification of data (Christy

and Egeberg, 2006). PCA is capable to identify patterns in data, and express the

data by emphasizing their similarities and differences (Shin et al., 2007).

Although some amounts of data on the fatty acid composition of MAGs and

DAGs have already been determined, the carbon isotope ratios for MAGs and

DAGs have not been documented before. Hence, this study would determine

whether chemical glycerolysis of lard and the selected animal fats would affect

the ability to distinguish those using EA-IRMS and GCMS. MAG, DAG and

TAG were first separated by chemical glycerolysis method with sodium

hydroxide (NaOH) as catalyst. Then, isolation of the fractions was done by

column chromatography. Next, the fatty acid composition and carbon isotope

ratio of MAG and DAG for animal fats were determined by GCMS and EA-

IRMS in order to establish their characteristics.

4.2 Materials and Method

4.2.1 Materials

Three batches of lard, beef fat, chicken fat and mutton fat werepurchased

from different suppliers and extracted from the adipose tissue of animals

using oven extraction method (Marikkaret al., 2001). 25cm x 2cm glass

column with Teflon™ stopcock plug (Favorit), TLC plate (Merck), silica

gel (Davison grade 923) (Sigma-Aldrich) and a mixture of MAG, DAG

and TAG standards (Sigma-Aldrich). All the reagents and chemicals used
44
 in this study were either analytical or HPLC grade unless otherwise

specified (glycerol, chloroform, petroleum ether, diethyl ether and

iodine).

4.2.2 Chemical Glycerolysis of Selected Animal Fats

Chemical glycerolysis of each animal fat was performed according to the

method reported previously by Indrastiet al., (2010). For glycerolysis reaction,

35-g oil sample was mixed with 15-g of glycerol (99% purity) and 0.2-g sodium

hydroxide, the mixture was then heated at 2500C with vigorous mixing for 60

min.

4.2.3 Separation of MAG and DAG byColumn Chromatography

Separation of MAG and DAG was carried out according to AOCS method Cd

11c-93 (AOCS 2007) using a glass column filled with silica gel of Davison 923

type (Sigma-Aldrich). TAGs are eluted with 250ml of 10% diethyl ether in

petroleum ether, DAG with 250ml of 25% diethyl ether in petroleum ether, and

MAG with 250ml of 100% diethyl ether.

4.2.4 TLC verification

TLC was carried out to verify the purity of the fractions collected through

column chromatography. The TLC plate was activated by heating at 110 0C-
45
1200C in oven. Spot around 30-60 µg of each TAG, DAG and MAG fraction from

a 30-40 mg/mL chloroform onto the TLC plate and place it in the developing

chamber after saturating it for 15 min with 92% petroleum ether and 8%

acetone. After the solvent front has moved to the top of the plate, plate is

removed from chamber, air dried in a suitable operating fume hood and placed

in a chamber saturated with iodine (AOCS, 2007).

4.2.5 GCMS Analysis of FAME

Analysis of FAME was carried out as described before in Chapter 3

(Section 3.2.4).

4.2.6 Determination of Carbon Isotope Ratiosof Selected Animal Fats After

Chemical Glycerolysis

Analysis of bulk δ13C in MAG and DAG samples was carried out as described in

Chapter 3 (Section 3.2.2).

4.2.7 Statistical analysis

Data were statistically analyzed by one-way analysis of variance (ANOVA)

using MINITAB (version 14) statistical package at 0.05 probability level. The

relationships between individual unsaturated fatty acids and δ13C values of

selected animal fats after chemical glycerolysis were determined by Pearson’s

Correlation analysis using MINITAB (version 14). For grouping and

46
classificationmodels, PCA was carried out using Unscrambler 9.7 (Camo, USA)

software.

4.3 Results and Discussions

4.3.1 Quantitative recovery of MAG and DAG

The recoveries of MAG, DAG and TAG by silica gel column chromatography

are as in Table 6. Based on the data, the chemical glycerolysis reaction of lard,

chicken fats, beef fats and mutton fats produced around 38–48% MAG and 31–

35% DAG. The amounts of TAG remaining in the four fats were noticeably less

compared to the overall total of the proportions of MAG and DAG in individual

cases, ranging from 13-25%. While the highest MAG yield was obtained for LD

(48%), the lowest MAG yield was recorded for BF (38%). In the case of DAG, the

highest DAG yield was for CF (35.36%) while the lowest DAG yield was for MF

(31.99%). To maximize the contact between the reactants namely glycerol and

the animal fats in the glycerolysis reaction, the liquid phases are generally

stirred at high speeds. This leads to one phase being dispersed into another as

long as the agitation is continued (Negi, 2006). According to

Damstrup et al., (2005), MAG, DAG, and TAG distribution under a

commercially synthesized glycerolysis process usually amount between 45–55%,

38–45%, and 8–12%, respectively. This is comparable to the values obtained for

chicken fat and lard. Nevertheless, as the proportions of TAGs in all samples are

very much lower than the sum total of the proportions of MAG and DAG, it is

concluded that the chemical glycerolysis process is successful. Verification of

47
MAG and DAG by column chromatography using TLC Plates are as shown in

Appendices.

Table 6. Recovery of partial acylglycerols of lard, chicken fat, beef fat and
mutton fat

Compound(%) MAG DAG TAG

LD 48.81 ± 0.57 35.04 ± 1.11 13.12 ± 1.06

CF 47.03 ± 0.87 35.36 ± 1.09 14.01 ± 1.80
BF 38.62 ± 0.48 32.34 ± 0.98 23.99 ± 0.71
MF 39.89 ± 0.61 31.99 ± 1.02 25.01 ± 0.42

a
Each value represents the mean ± standard deviation of three replicates
b
Abbreviations: MAG, monoacylglycerol: DAG, diacylglycerol; TAG, triacylglycerol; LD, lard; CF, Chicken fat; BF, beef
fat; MF, mutton fat.

4.3.2 δ13C Values of Selected Animal Fats After Chemical Glycerolysis

The data presented in Table 7 compares the δ 13C values of lard, chicken fat, beef

fat and mutton fat after chemical glycerolysis (MAG and DAG). According to

Table 2, the highest δ13C value of MAG is found with chicken fat (-20.3‰) while

the lowest value of the same is found for mutton fat (-31.9‰). The δ 13C values

of lard(-22.3‰) and beef fat (-29.0‰) are within the range of these two extremes

of the mean δ13C values lard, chicken fat, beef fat and mutton fat. On the other

hand, the highest δ13C value of DAG is found with chicken fat (-19.2‰) while

the lowest value of the same is found for mutton fat (-32.2‰). The δ 13C values

of lard (-22.2‰) and beef fat (-29.3‰) are within the range of these two

extremes of the mean δ13C values. In comparison to the animal fats, the δ 13C

48
values of these animal fats before and after chemical glycerolysis showed no

significant difference.

Fat Types n δ13C (‰)

Mean
Chi
cken 3 -20.32a ±0.38
Lar
M d 3 -22.29b ±0.59
AG Bee
f fat 3 -29.01c ±0.92
Mu
tton fat 3 -31.93d±0.81
Chi
cken 3 -19.17a ±0.51

Lar
D d 3 -22.16b±0.75
AG Bee
f fat 3 -29.34c ±0.41
Mu
tton fat 3 -32.17d±0.33

Table 7. δ13C values of chicken fat, lard, beef fat and mutton fat after chemical
glycerolysis
1
Each value represents the means and standard deviation of triplicates
2
Abbreviations: MAG, monoacylglycerol; DAG, diacylglycerol; LD, Lard; CF, Chicken
fat; BF, Beef fat; MF, Mutton fat

The statistical analysis of the data from the present study suggests that the

determination of δ13C value for bulk animal fats can be a useful tool since the

49
δ13C value of lard is significantly (p<0.05) different from those of beef fat,

chicken fat and mutton fat (Table 7). The observed variation in the δ 13C values

of animal fats could be attributed to their species difference (Osorio et al., 2011),

genetic factors (Wood et al., 2008) as well as the diet fed to the animals

(Bojlul et al., 2007). According to previous investigators, the variation in the δ 13C

values of oils and fats originated from different plant sources are due to isotopic

fractionation during physical, chemical and biological processes in plants (Kelly

and Rhodes 2002).

Table 8. Pearson’s correlation coefficient (r) between δ13C values and

unsaturated fatty acid contents of MAG and DAG derived from lard, chicken
fat, beef fat and mutton fat
Sa Fatty acid R p value
mples

M Palmitoleic (C16:1) +0.713 <0.287

AG

Oleic (C18:1) +0.538 <0.462

Linoleic (C18:2) +0.695 <0.305

∑USFA +0.875 <0.125

D Palmitoleic (C16:1) +0.777 <0.233

AG

Oleic (C18:1) +0.803 <0.197

Linoleic (C18:2) +0.779 <0.221

∑USFA +0. <0.06

938 2
USFA: Unsaturated fatty acids

50
Interestingly, the δ13C values of MAG derived these fats are found to show a

correlation (+0.875; p<0.12) with the increasing degree of unsaturation, and

(+0.938; p<0.062) for DAG as shown in Table 8. The δ 13C values of MAG and

DAG of lard, beef fat, chicken fat and mutton fat have not been investigated

before; therefore there is hardly any report to compare the δ 13C values.

However, the δ13C values of MAG and DAG obtained are comparable with the

values obtained before chemical glycerolysis. Among the three unsaturated fatty

acids of MAG enlisted in Table 8, palmitoleic acid shows the best positive

correlation (+0.713; p<0.287) with the δ13C values of animal fats, whereas for

DAG, oleic acid has the best positive correlation (+0.803; p<0.197).

4.3.3 Fatty Acid Composition of MAG

Fatty acid distributional pattern of MAG derived from lard, chicken fat, beef fat

and mutton fat are compared as shown in Table 9. Lard and chicken fat are

distinguishable from beef fat and mutton fat due to the high content of

unsaturated fatty acids (60.3% to 60.92%) than saturated fatty acids (39.08% to

39.7%). On the other hand, high saturated fatty acid content is observed in beef

fat (69.39%) and mutton fat (60.27%) compared to unsaturated fatty acids

(30.61% to 39.72%). This basic difference could be due to the diverse pattern of

distribution of individual fatty acids among these animal fats. Although these

animal fats are found to have high amount of oleic and palmitic acids, they may

tend to differ with regard to the third most abundant fatty acids.

51
While high occurrence of linoleic acid is observed in MAG derived from lard,

high occurrence of stearic acid is observed in beef fat and mutton fat. The most

predominant fatty acid of lard is oleic acid (38.75%), followed by palmitic

(28.16%) and linoleic (21.55%). Chicken fat is also found to have oleic acid

(45.94%) as the most dominant fatty acid, followed by palmitic (30.99%) and

linoleic (8.97%) acids. These values are in accordance with the findings reported

by (Nasyrah et al., 2012). MAG derived from beef and mutton fat share a

common characteristic, whereby palmitic, stearic and oleic acids are major fatty

acids.

52
 Table 9. Fatty acid composition (%, peak area) of lard, chicken fat, beef fat and mutton fat
after chemical glycerolysis (MAG and DAG)

Fatty acid (methyl esters) (%)

∑SF ∑US

Fat types C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 A FA

28.16±0. 11.54±0. 38.75±0. 21.55±0.

LD - 00a - 03b 02a 04a 39.7 60.30

30.99±0. 6.01±0. 8.09±0.0 45.94±0. 8.97±0.0 39.0

CF - 02a 01a 0b 03a 0b 8 60.92

MAG

5.90±0. 28.16±0. 35.34±0. 24.83±0. 5.77±0.0 69.3

BF 01a 04a - 05a 04c 4b 9 30.61

28.96±0. 31.31±0. 39.72±0. 60.2

MF - 05a - 10a 14a - 7 39.72

0.79±0. 31.35±1. 0.86±1. 14.92±0. 40.29±0. 11.79±0. 47.0

DAG

LD 91b 38a 00b 62b 79b 32a 6 52.94

CF 0.80±0. 31.36±2. 5.71±0. 7.85±0.2 47.24±1. 5.92±3.5 41.1 58.87

53
 10b 63a 33a 6c 55a 8b 3

4.89±0. 25.56±0. 1.14±0. 38.57±0. 28.28±0. 1.57±0.2 69.0

BF 09a 08b 12b 53a 33c 0c 2 30.99

27.38±0. 36.49±0. 36.13±0. 63.8

MF - 08b - 28a 36b - 7 36.13

1
Each fatty acid value in the table represents the mean ± standard deviation of three replicates. Means within each column with different
superscripts are significantly (p<0.05) different.
2
Abbreviations: MAG, monoacylglycerol; DAG, diacylglycerol; LD, Lard; CF, Chicken fat; BF, Beef fat; MF, Mutton fat; SFA, saturated
fatty acid; USFA, unsaturated fatty acid

54
As beef and mutton fat share common characteristics in the way lard and

chicken fat share common characteristic with regard to major fatty acids, it

might be difficult to find differentiation among them using mere comparison of

the overall fatty acid data. In such situations, it would be more appropriate to

use multivariate data analysis techniques such as principle component analysis.

As shown in Table 9, fatty acids namely myristic (C14:0), palmitic (C16:0),

palmitoleic (C16:1), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) acids are

found to occur in variable amounts in all animal fats and could be used as

independent variables in PCA procedure. The score plot of fatty acids derived

from the four animal fats as shown in Figure 11 represents the projection of

samples defined by principle component 1 (PC1) and principle component 2

(PC2). PC1 is the linear combination of variables that explain the highest

variation among the samples, while PC2 is orthogonal to PC1 and exhibited the

second largest variation (Cordellaet al., 2003). The score plot projected on PC1

described 89% of the variation while PC2 accounted for 8% of the variation,

making up of 97% of variance for PC1 and PC2.

55
Figure 11. Score plot of PCA of MAG derived from animal fats based on fatty
acid composition

Figure 12. Loading plot of PCA of MAG derived from animal fats based on
fatty acid composition (PC1: , PC2: )

According to the group separation illustrated in Figure 11, lard is located in

lower left quadrant, chicken fat in upper left quadrant, mutton fats in upper

right quadrant, and beef fat in lower right quadrant. Fatty acid variables giving

56
high influence on the group separation of the samples in the score plot could be

traced from the analysis of the loading plot (Figure 12). As explained by

Cordellaet al. (2003), a variable which is farther from the origin of axis

contributes to the most variation in the statistical model generated by the PCA.

According to the loading plot in Figure 16, out of the six fatty acid variables

palmitic, stearic, oleic and linoleic acids were the most discriminating variables

that influence the group separation into four different clusters.

4.3.4 Fatty Acid Composition of DAG

Fatty acid distributional pattern of DAG derived from lard, chicken fat, beef fat

and mutton fat are compared as shown in Table 9. As for DAG, the pattern

distribution is similar with MAGs, except that DAG derived from mutton fat

possessed higher amount of stearic acid compared to oleic acid. All samples

show oleic and palmitic acid as the most predominant fatty acid respectively.

Lard and chicken fat are distinguishable from beef fat and mutton fat due to the

high content of unsaturated fatty acids (52.94% to 58.87%) than saturated fatty

acids (47.16% to 41.13%). On the other hand, high saturated fatty acid content is

observed in beef fat (69.02%) and mutton fat (63.87%) compared to unsaturated

fatty acids (30.99% to 36.13%). It is also observed that these amounts are

comparable to those of raw animal fats.

As previously seen in fatty acid composition of MAG, DAG of lard and chicken

fat were shown to contain palmitic, oleic and linoleic acid as the most dominant

fatty acids as well. Akin to MAG, beef and mutton fat share a common
57
characteristic, whereby palmitic, stearic and oleic acid are the major fatty acids

(Table 9). The high content of linoleic acid in DAG of lard; resembling MAG

may discriminate lard from other animal fats; however differentiation of chicken

fat, beef fat and mutton fat is hard. As listed in Table 9, fatty acids namely

myristic (14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1)

and linoleic (C18:2) acid were found to occur in variable amounts in all DAG

derived from animal fats. PCA was performed using these fatty acids as

variables to establish classification between DAG originating from animal fats.

The score plot of fatty acids derived from the animal fats as shown in Figure 13

represent the projection of samples defined by principle component 1 (PC1)

describing 87% of the variation while PC2 accounted for 9% of the variation,

making up of 96% of variance explained for the PC1 and PC2. According to the

group separation illustrated in Figure 13, lard is located in lower left quadrant,

chicken fat in upper left quadrant, mutton fats in upper right quadrant, and beef

fat in lower right quadrant. Fatty acid variables giving high influence on the

group separation of the samples in the score plot could be traced from the

analysis of the loading plot. As explained by Cordella et al. (2003), a variable

which is farther from the origin of axis contributes to the most variation in the

statistical model generated by the PCA. According to the loading plot in

Figure 14, out of the six fatty acids, stearic, oleic and linoleic acids were the most

discriminating variables that influence the group separation into four different

58
clusters.

Figure 13.Score plot of PCA of DAG derived from animal fats based on fatty
acid composition

Figure 14. Loading plot of PCA of DAG derived from animal fats based on
fatty acid composition(PC1: , PC2: )

4.4 Conclusion

This study concluded that chemical glycerolysis of lard and the selected animal

fats namely chicken fat, beef fat, and mutton fat do not affect the values of

59
carbon isotope values.The significant differences in the values of carbon isotope

(δ13C) of all MAGs (Chicken fat: -20.3‰, Lard: -22.29‰; Beef fat: -29.01‰; and

Mutton fat: -31.93‰) and DAGs (Chicken fat: -19.17‰, Lard: -22.16‰; Beef fat: -

29.34‰; and Mutton fat: -32.17‰) derived from animal fats are shown to be

good indicators for discriminating lard, chicken fat, beef fat and mutton fat.

Comparison of the overall fatty acid data showed that use of single fatty acid as

parameter is insufficient to classify animal fats into distinct subclasses. Hence,

the application of multivariate statistical techniques such as PCA is used to

classify them. According to the outcome of PCA, stearic, oleic and linoleic acids

were found to be the most discriminating parameters in the clustering of animal

fats. Hence, this study showed that PCA of fatty acid data allowed separation of

lard from other selected animal fats after chemical glycerolysis.

CHAPTER 5

SUMMARY, CONCLUSION AND

RECOMMENDATIONS FOR FUTURE RESEARCH

60
5.1 Summary and general conclusions

The present work highlighted the potential use of GCMS with PCA and EA-

IRMS techniques to distinguish lard from other animal fats before and after

glycerolysis, as well as after fractionation. Owing to the significant implication

of lard adulteration cases to Muslims, developing rapid test methods for animal

fats discrimination has become essential.The use of fatty acid composition, PCA,

and carbon isotope ratio data has been made possible to identify the source of

origin of common animal fats.

The investigation on the application of EA-IRMS techniques for differentiation

of lard, chicken fat, beef fat, and mutton fat before undergoing chemical

glycerolysis was carried out. The significant differences in the values of carbon

isotope ratios (δ13C) of all animal fats have shown to be able to distinguish lard

from chicken fat, beef fat and mutton fat. Surprisingly, it was found that there

are no significant difference observed in carbon isotope values of lard and lard

fractionates, namely LO and LS.This established that fractionation of samples

would not affect the carbon isotope values. Interestingly too, the chemical

glycerolysis of lard and the selected animal fats do not affect the carbon isotope

values, as ascertained in Chapter 4.

In the determination of fatty acid distributional data using GCMS, it was found

that the use of single fatty acid as parameter may not be suitable to classify

animal fats into distinct subclasses. Hence, the use of multivariate statistical

techniques, in this respect, Principal Component Analysis (PCA) - would be

61
necessary to classify those animal fats to determine their source of origin by

comparing the overall fatty acid data. Based on the outcome of PCA, stearic,

oleic and linoleic acids were found to be the most discriminating parameters in

the clustering of lard, chicken fats, beef fats and mutton fatsbefore and after

chemical glycerolysis. Hence, this study showed that PCA of fatty acid data

helped in separation of lard from the selected animal fats.

The results of this investigation showed that GCMS and EA-IRMS could be

applied to discriminate lard as well as lard in other forms from other animal

fats before and after chemical glycerolysis, as well as after fractionation. The

findings indicated that EA-IRMS analysis is potential as a rapid and reliable test

for lard detection, which could serve as routine analysis and screening in food.

EA-IRMS analysis requires short processing time (less than 400 seconds), no

sample preparation (melted fats to be directly measured into the tin capsule),

little amount of sample (0.2µg per tin capsule) and inexpensive compared to gas

chromatography analysis (RM100.00 compared to RM300.00, Cost Analysis as

in the Appendices). However, the samples would need to be sent to the

laboratory for screening, whereas, nowadays a handier tool is preferred to test

samples on site, for instance the portable Hafys which is based on DNA

detection. Furthermore, further profiling analysis for identification and

confirmation purposes contributing to the carbon isotope values would be

required. GCMS on the other hand, needs laborious preparation method and

clean-up procedures.
62
5.2 Recommendations for future research

This study determines the carbon isotope valuesof selected animal fats before

and after chemical glycerolysis, as well as lard fractionates using EA-IRMS.

Then, GCMS analysis for determination of fatty acid composition with the

application of PCA to discriminate lard from other animal fats was carried

out.From the research, it was established that EA-IRMS and GCMS are

compatible combination, and chemical glycerolysis and fractionation did not

affect the carbon isotope values of the raw materials.However, more research

needs to be undertaken before the association between carbon isotope values

and fatty acid distributional data, with the selected animal fatsto be clearly

understood. Hence, in future, it would be interesting to recommended that

those commercial samples or the products on the shelves to be tested using the

same techniques. Future trials on raw samples should assess age of the animal,

different parts of the body, sex and species of the animals that samples are being

taken from. Furthermore, tests can be carried out when the selected animal fats

were mixed. The adulterators may be of more than three types of animal fats or

even vegetable oils. This could determine whether thesemethods can distinguish

lard from selected animal fats and other edible oils in mixed form, besides it

allows the detection on the amount of adulteration.

63
 REFERENCES

Al-Rashood, K. A. and Abou-Shaaban, R. R. A. (1996). Compositional and

Thermal Characterization of Genuine and Randomized Lard : A
Comparative Study. JAOCS 73(3): 303–309.

Al-Taher, F. (2004). Halal food production: Mian N. Riaz and Muhammad M.

Chaudry (Eds.); CRC Press, Boca Raton, FL, 2004, 379pp, Hardcover. Food
Microbiology 21(4): 490-490.

64
Alves, M.R., Casal, S., Oliveira, M.B.P.P and Ferreira, M.A. (2003). Contribution
of FA profile obtained by high-resolution GC/chemometric techniques to
the authenticity of green and roasted coffee varieties. AOCS Press, 80:
511-517.

Angerosa, F.B.O., Contento, S., Guillou, C., Reniero, F. and Sada, E. (1999).
Application of stable isotope ratio analysis to the characterization of the
geographical origin of olive oils. Journal of agricultural and food chemistry
47(3): 1013–1017.

Anna, J. (2006). Species specific detection of meat by polymerase chain reaction

techniques.PhD Thesis, Corvinus University of Budapest.

AOAC (2007).Official methods of analysis of AOAC International.18th ed.

Association of Official Analytical Chemists, Washington, DC.

AOCS (1993). Official method and recommended practices of the American Oil
Chemists’ Society, 6th ed, IL.

Aparicio, R. and Aparicio-Ruíz, R. (2000). Authentication of vegetable oils by

chromatographic techniques. Journal of chromatography A 881(1-2): 93–104.

Araujo, P., Zeng, Y., Du, Z.-Y., Nguyen, T.-T., Froyland, L. and Grung, B. (2010).
Discrimination of n-3 Rich Oils by Gas Chromatography. Lipids 45: 1147–
1158.

Aursand, M., Mabon, F. and Martin, G. J. (2000). Characterization of Farmed and

Wild Salmon ( Salmo salar ) by a Combined Use of Compositional and
Isotopic Analyses. JAOCS 77(6): 659-666.

Baskaran, M. (2011).Handbook of Environmental Isotope Geochemistry,

Advances in Isotope Geochemistry, Berlin Heidelberg: Springer-Verlag.
Page 707.
Bianchi, G., Angerosa, F., Camera, L., Reneiro, F. and Anglani, C. (1993). Stable
Carbon Isotope Ratios ( 13C / 12C ) of Olive Oil Components, J Agri. Food
Chem. 41:1936–1940.

Bojlul, B., Monahan, F. J., Moloney, A. P., Kiely, P. O., Scrimgeour, C. M. and
Schmidt, O. (2007).Alteration of the carbon and nitrogen stable isotope
composition of beef by substitution of grass silage with maize
silage.Rapid Commun.Mass Spectrom. 19: 1937-1942.

Brodnjak-Voncina D., Kodba, Z.C. and Novic, M. (2005).Multivariate data

analysis in classification of vegetable oils characterized by the content of
fatty acids.Chemometric and Intelligence Laboratory Systems.75: 31- 43.
65
Carrasco-Pancorbo, A., Navas-Iglesias, N. and Cuadros-Rodríguez, L. (2009).
From lipid analysis towards lipidomics, a new challenge for the analytical
chemistry of the 21st century. Part I: Modern lipid analysis. Trends in
Analytical Chemistry, 28(3): 263–278.

Carter, J. F. and Barwick, V. J. (2011). Good Practice Guide for Isotope Ratio Mass
Spectrometry. FIRMS.ISBN 978-0-948926-31-0.

Che Man, Rohman, A. and Mansor, T. S. T. (2011). Differentiation of Lard From

Other Edible Fats and Oils by Means of Fourier Transform Infrared
Spectroscopy and Chemometrics. JAOCS 88: 187–192.

Che Man, Y. B. and Mirghani, M. E. S. (2001). Detection of Lard Mixed with

Body Fats of Chicken , Lamb , and Cow by Fourier Transform Infrared
Spectroscopy. JAOCS 78(7): 753-761.

Che Man, Y. B., Sazili, A. Q. and Regenstein, J. M. (2003). Chapter 3 Food

Production From The Halal Perspective. pp: 1–59.

Cheng, S.F., Choo, Y.M., Ma, A.N. and Chuah, C.H. (2005). Rapid synthesis of
palm-based monoacylglycerols. Journal of the American Oil Chemists’
Society 82(11): 791–795.

Cheong, L.Z., Hong, Z., Lise, N., Kirsten J., Haagensen, J.A.J. and Xuebing, X.
(2010). Physical and sensory characteristics of pork sausages from
enzymatically modified blends of lard and rapeseed oil during storage.
Meat science 85(4): 691–9.

Chesson, L., Tipple, B.J., Erkkila, B.R., Cerling, T.E. and Ehleringer, J. R. (2011).
B-HIVE: Beeswax hydrogen isotopes as validation of environment. Part I:
Bulk honey and honeycomb stable isotope analysis. Food Chemistry 125(2):
576–581.

Chin, S.T., Yaakob, C.M, Tan, C.P. and Dzulkifly, M.H. (2009) Rapid Profiling of
Animal-Derived Fatty Acids Using Fast GCxGC Coupled to Time-of-
Flight Mass Spectrometry. J Am Oil ChemSoc 86:949–958.

Christy, A.A. and Egeberg, P.K. (2006).Quantitative determination of saturated

and unsaturated fatty acids in edible oils by infrared spectroscopy and
chemometrics.Chemometric Intelligence Laboratory Systems 82: 130 – 136.

66
Codex Alimentarius Commission.(2001). Codex Standard for Named Animal
Fats, Codex-Stan 211-1999.Volume 8. Rome: Food and Agriculture
Organization of the United Nations, World Health organization (WHO).

Cordella, C., Moussa, I., Martel, A.C., Sbirrazzuoli, N. and Lizzani-Cuvelier, L.

(2002). Recent Developments in Food Characterization and Adulteration
Detection : Technique-Oriented Perspectives. Journal of Agricultural and
Food Chemistry 50: 1751-1764.

Cordella, C.B.Y, Militão, J.S.L.T, Clément, M.C. and Cabrol-Bass, D. (2003).

Honey characterization and adulteration detection by pattern recognition
applied on HPAEC-PAD profiles. 1. Honey floral species
characterization. Journal of agricultural and food chemistry 51(11): 3234–42.

Cserháti, T. (2010). Data evaluation in chromatography by principal component

analysis. Biomedical chromatography : BMC 24(1): 20–28.

David, F., Sandra, P. and Wylie, P.L. (2003) Improving the Analysis of fatty Acid
Methyl Esters Using Retention Time Locked Methods and Retention
Time Databases. Agilent Technologies, Inc.

Damstrup, M.L., Jensen, T., Sparsøc, F.V., Kiil, S.Z., Jensen, A.D. and Xu X.
(2005). Solvent Optimization for Efficient Enzymatic
MonoacylglycerolProduction Based on a Glycerolysis Reaction JAOCS
82(8): 559-564.

Damstrup, M. L. (2008). Process Development of Enzymatic Glycerolysis for

Industrial Monoacylglycerol Production. PhD Theses. Technical
University of Denmark.
Deffense, E. (1993). Milk fat fractionation today: A review. Journal of the American
Oil Chemists’ Society 70(12): 1193–1201.

De Smet, S., Balcaen, A., Claeys, E., Boeckx, P. and Van Cleemput, O.
(2004).Stable carbon isotope analysis of different tissues of beef animals in
relation to their diet.Rapid Commun.Mass Spectrom. 18: 1227–1232.

Ghidini, S., Ianieri, A., Zanardi, E., Conter, M., Boschetti, T., Iacumin, P.
andBracchi, P.G.(2006). Stable isotopes determination in food
authentication: A review. Ann. Fac. Medic. Vet. di Parma 27: 193-204

Indrasti, D., Yaakob, C.M., Shuhaimi, M. and Dzulkifly, M.H. (2010).Lard

detection based on fatty acids profile using comprehensive gas
chromatography hyphenated with time-of-flight mass spectrometry. Food
Chemistry 122: 1273–1277.

67
Fadzlillah, N. A., Che Man, Y. B., Jamaludin, M. A., Rahman, S. A., and Al-
Kahtani, H. A. (2011). Halal Food Issues from Islamic and Modern
Science Perspectives. 2nd International Conference on Humanities, Historical
and Social Sciences IPEDR 17: 159–163.

Food Agriculture Organization.(2010). Top Production- Indigenous Pigmeat

2010. Retrieved May 2012 from
http://faostat.fao.org/site/339/default.aspx.

Food Outlook.(2012). Meat and Meat Products. Retrieved May 2012 from
http://www.fao.org/docrep/012/ak341e/ak341e09.htm

Gan, H.L., Tan, C.P., Che Man, Y.B., NorAini, I. and Nazimah, S.A.H. (2005).
Monitoring the storage stability of RBD palm olein using the electronic
nose. Food Chemistry 89: 271–282.

Glaser K.R., Wenk C. and Scheeder, M. R. (2004). Evaluation of pork backfat

firmness and lard consistency. J Sci Food Agric 84: 853-862.

Gonzalez, I., Gonzalez, C., Hernandez, J., Marques, E. and Sanz, P.F. (1999).Use
of isotope analysis to characterize meat from Iberian-breed swine.Meat
Science 52: 437–441.

Grompone, M. A. (1990). Characteristics of Uruguayan Mutton Tallow. Journal of

American Oil and Chemist Society. 67(12): 1990.

Hamilton, R.J. (1998). Purity Criteria for Edible Vegetable Oils in Lipid Analysis
of Oils and Fats.pp.284. Blackie Academic and Professional.

Hamm, W. (1995). Trends in edible oil fractionation. Trends in Food Science and
Technology 6: 121–126.

Jafari, M., Kadivar, M. and Keramat, J. (2009). Detection of Adulteration in

Iranian Olive Oils Using Instrumental (GC, NMR, DSC) Methods. J Am
Oil Chem Soc 86:103–110.

Jakobsen, K. (1999). Dietary modifications of animal fats: status and future

perspectives. Lipid – Fett. 101(12): 475–483.

Jaswir, I., Mirghani, M.E.S., Hassan, T.H. and Said, M.Z.M. (2003).
Determination of Lard in Mixture of Body Fats of Mutton and Cow Using
Fourier Transforms Infrared Spectroscopy. Journal of Oleo Science 52(12):
633–638.

68
Jochmann, M., Steinmann, D., Stephan, M. and Schmidt, T. C. (2009). Flow
injection analysis-isotope ratio mass spectrometry for bulk carbon stable
isotope analysis of alcoholic beverages. Journal of agricultural and food
chemistry 57(22): 10489–10496.

Kallio, H., Yli-Jokipii, K., Kurvinen, J.P., Sjo¨vall, O and Tahvonen, R.

(2001).Regioisomerism of Triacylglycerols in Lard, Tallow, Yolk, Chicken
Skin, Palm Oil, Palm Olein, Palm Stearin, and a Transesterified Blend of
Palm Stearin and Coconut Oil Analyzed by Tandem Mass Spectrometry.
J. Agric. Food Chem. 49: 3363−3369.

Kamm, W., Dionisi, F. and Hischenhuber, C. (2007). Authenticity Assessment of

Fats and Oils. Food Reviews International 17(3): 37–41.

Kelly, S. and Heaton, K. (2005).Tracing the geographical origin of food: The

application of multi-element and analysis.Trends in Food Science &
Technology 16: 555–567.

Lee, D., Noh, B., Bae, S. and Kim, K. (1998). Characterization of fatty acids
composition in vegetable oils by gas chromatography and chemometrics.
Analytica Chemica Acta 358:163-175.

Lee, K. and Foglia, T. A. (2000). Synthesis, Purification and Characterization of

Structured Lipids Produced from Chicken Fat. JAOCS 77: 1027–1034.

Lipidlibrary (2012). A racemic mixture of sn-1,2- and 2,3-diacylglycerols

Retrieved May 2012 from
http://lipidlibrary.aocs.org/lipids/mg/index.htm

Lipidlibrary (2012).Stereo isomeric sn-1- and sn-2- and sn-3-monoacylglycerols.

Retrieved May 2012 from
http://lipidlibrary.aocs.org/lipids/mg/index.htm

Liu, X., Xu, S.P., Wang, J.H., Yuan, J.P., Guo, L.X., Xin, L. and Huang, X.N.
(2007). Characterization of ganoderma spore lipid by stable carbon
isotope analysis: implications for authentication. Analytical and
bioanalytical chemistry 388(3): 723–31.

Liu, Y., Meng, Z., Shan, L., Jin, Q. and Wang, X. (2009). Preparation of specialty
fats from beef tallow and canola oil by chemical interesterification:
physico-chemical properties and bread applications of the products.
European Food Research and Technology 230(3): 457-466.

Marikkar, J.M.N., Ghazali, H. M., Che Man, Y. B., Peiris, T. S. G. and Lai, O. M.
(2005a). Distinguishing lard from other animal fats in admixtures of some
69
 vegetable oils using liquid chromatographic data coupled with
multivariate data analysis. Food Chemistry 91(1): 5–14.

Marikkar, J.M.N., Ghazali, H. M., Che Man, Y. B., Peiris, T. S. G. and Lai, O. M.
(2005b). Use of gas liquid chromatography in combination with
pancreatic lipolysis and multivariate data analysis techniques for
identification of lard contamination in some vegetable oils. Food
Chemistry90(1-2): 23–30.

Marikkar, J.M.N., Jayasundera, J.M.M.A., Kumari, A.G.O. and Waidyarathana,

K.P. (2008). A Predictive Model for Determination of the Iodine Value of
Coconut Oil by GLC Analysis of the Component Fatty Acids.Cord 24(2):
21.

Marikkar, J.M.N., Lai, O. M., Ghazali, H. M., and Man, Y. B. C. (2001). Detection
of Lard and Randomized Lard as Adulterants in Refined-Bleached-
Deodorized Palm Oil by Differential Scanning Calorimetry. JAOCS
78(11): 1113–1119.

Marikkar, J.M.N., Lai, O.M., Ghazali, H.M. and Che Man, Y.B. (2002).
Compositional and thermal analysis of RBD palm oil adulterated with
lipase-catalyzed interesterified lard. Food Chemistry 76(2): 249–258.

Marikkar, J.M.N., Ghazali, H.M., Long, K. and Lai O.M. (2003). Lard uptake and
its detection in selected food products deep-fried in lard. Food Research
International 36: 1047–1060.

Michael J. Haas (2005). Animal Fats in Bailey’s Industrial Oil and Fat Products,
6th. Ed. Edited by FereidoonShahidi. pp: 161-212. New York: John Wiley
and Sons, Inc.

Mottram, H. R., Woodbury, S. E., Rossell, J. B. and Evershed, R. P. (2003). High-

resolution detection of adulteration of maize oil using multi-component
compound-specific δ13C values of major and minor components and
discriminant analysis. Rapid communications in mass spectrometry : RCM,
17(7): 706–12.

Muccio, Z. and Jackson, G. P. (2009). Isotope Ratio Mass Spectrometry. The

Analyst 134(2): 213–22.

Nasyrah, A. R., Marikkar, J. M. N. and Dzulkifly, M. H. (2012). Discrimination of

plant and animal derived MAG and DAG by principal component
analysis of fatty acid composition and thermal profile data. IFRJ 19(4):
1497–1501.

70
Negi, D. S. (2006). Base Catalyzed Glycerolysis of Fatty Acid Methyl Esters:
Investigations towards the Development of a Continuous Process. PhD
Theses. Technical University of Berlin

Nurjuliana, M., Che Man, Y.B. and Dzulkifly, M.H. (2011).Analysis of lard's
aroma by an electronic nose for rapid halal authentication.Journal of the
American Oil Chemists' Society 88(1): 75–82.

O’ Brien, R. 2009.Fats and Oils. Formulating and Processing for Applications.

pp. 54-55, 265-275, 15-57 3rd Ed. Boca Raton: CRC Press Taylor & Francis
Group.

Osorio, M. T., Moloney, A. P., Schmidt, O., and Monahan, F. J.

(2011).Multielement Isotope Analysis of Bovine Muscle for Determination
of International Geographical Origin of Meat, Journal of Agricultural and
Food Chemistry 59: 3285-3294.

Perini, M., Camin, F., Bontempo, L., Rossmann, A., and Piasentier, E. (2009).
Multielement (H, C, N, O, S) stable isotope characteristics of lamb meat
from different Italian regions Rapid Commun. Mass Spectrom. 23: 2573–
2585
Piasentier, E.R.,Valusso, F.,Camin, and Versini,G. (2003). Stable isotope ratio
analysis for authentication of lamb meat.Meat science 64(3): 239–47.

Rand R. Wilcox. (2003). Chapter 6 - Least squares regression and Pearson's

correlation.Applying Contemporary Statistical Techniques.pp. 173-206.
California: Elsevier Science (USA) Academic Press.

Rhodes, C. N., Lofthouse, J. H., Hird, S., Rose, P., Reece, P., Christy, J. and
Macarthur, R. (2010).The use of stable carbon isotopes to authenticate
claims that poultry have been corn-fed.Food Chemistry, 118(4), 927-932.

Riaz, M.N. and Chaudary, M.M. (2004).Halal Food Production, pp. 1-379. Florida:
CRC press.

Richter, E.K., Spangenberg, J.E., Michael, K. and Florian, L.

(2010).Characterization of Rapeseed (Brassica napus) Oils by Bulk C, O, H,
and Fatty Acid C Stable Isotope Analyses J. Agric. Food Chem 58: 8048–
8055.

Rohman, A. and Che Man, Y.B. (2010). FTIR spectroscopy combined with
chemometrics for analysis of lard in the mixtures with body fats of lamb,
cow, and chicken. International Food Research Journal 17: 519–526.

71
Rohman, A., Che Man, Y.B. and Noviana, E. (2012).Analysis of Emulsifier in
Food Using Chromatographic Techniques.In Press.

Rohman, A., Sismindari, Erwanto, Y. and Che Man, Y.B. (2011).Analysis of pork
adulteration in beef meatball using Fourier transform infrared (FTIR)
spectroscopy. Meat Science 88: 91–95.

Ruiz-Rodrigueza ,A., Reglero, G. and Iba˜nez, E. (2010). Recent trends in the

advanced analysis of bioactive fatty acids. Journal of Pharmaceutical and
Biomedical Analysis 51: 305–326.

Saeed T, Ali S.G., Rahman H.A.A and Sawaya WN. (1989). Detection of Pork
and Lard as adulterants in Processed Meat: Liquid Chromatography
analysis of derivatized triglycerides. J Ass of Off Anal Chem 72:921-925.

Saeed, T., Abu-Dagga, F. and Rahman, H.A. (1986).Detection of Pork and lard as
Adulterants in Beef and mixtures.J Ass of Off Anal Chem 69: 999-1002.

Sawaya, W.N., Saeed T., Mameesh, M., El-Rayes, E., Husain, A., Ali, A. and
Rahman, H.A. (1990). Detection of Pork in Processed Meat: Experimental
comparison of methodology. Food Chem 37:201-219.

Schipilliti, L., Tranchida, P. Q., Sciarrone, D., Russo, M., Dugo, P., Dugo, G. and
Mondello, L. (2010). Genuineness assessment of mandarin essential oils
employing gas chromatography-combustion-isotope ratio MS (GC-C-
IRMS). Journal of separation science 33: 617–25.

Seo, H.Y., Ha, J., Shin, D.B., Shim, S.L., No, K.M., Kim, K.S. and Lee, K.B. (2010).
Detection of Corn Oil in Adulterated Sesame Oil by Chromatography and
Carbon Isotope Analysis. Journal of the American Oil Chemists’ Society
87(6): 621–626.

Shin, E.C., Craft, B.D., Pegg, R.B., Phillips, R.D. and Eitenmiller, R.R. (2011)
Chemometric approach to fatty acid profiles in Runner-type peanut
cultivars by principal component analysis (PCA).Food Chemistry119(3):
1262-1270.

Simsek, A., Bilsel, M. and Goren, A. C. (2012). 13C/12C pattern of honey from
Turkey and determination of adulteration in commercially available
honey samples using EA-IRMS. Food Chemistry 130(4): 1115–1121.

Ulfert Focken (2004).Feeding fish with diets of different ratios of C3- and C4-
plant-derived ingredients: a laboratory analysis with implications for the
back-calculation of diet from stable isotope data.Rapid Commun. Mass
Spectrom18: 2087–2092.
72
Wang, F.S. and Lin, C.W. (1995).Turbidimetry for Crystalline Fractionation of
Lard.JAOCS 72(5): 585-589.

Wikipedia 2013.[GCMS].Retrieved from http://en.wikipedia.org/wiki/GCMS.

Wikipedia 2012.[Schematic diagram for a GC system].Retrieved from

http://en.wikipedia.org/wiki/GC system.

Wikipedia. 2012. Adipose tissue. en.wikipedia.org/wiki/Adipose_tissue.

Accessed on December 2012.

Wood, J.D., Enser, M., Fisher, A.V., Nute, G.R., Sheard, P.R., Richardson, R.R.,
Hughes, S.I. and Whittington F.M. (2008).Fat deposition, fatty acid
composition and meat quality: A review. Meat Science 78: 343–358.

Woodbury, S.E., Evershed, R.P. and Rossell, J.B. (1998).Purity assessments of

major vegetable oils based on δ13C values of individual fatty acids. JAOCS
75(3): 371–379.

Yang, T., Zhang, H., Mu, H., Sinclair, A. J., and Xu, X. (2004). Diacylglycerols
from butterfat: Production by glycerolysis and short-path distillation and
analysis of physical properties. Journal of the American Oil Chemists’
Society, 81(10): 979–987.

Yanty, N. A. M., Marikkar, J. M. N. and Che Man, Y.B. (2011b). Effect of

fractional crystallization on composition and thermal characteristics of
avocado (Perseaamericana) butter. J Therm Anal Calorim 1-7.

Yanty, N. A. M., Marikkar, J. M. N., Che Man, Y. B. and Long, K. (2011).

Composition and thermal analysis of lard stearin and lard olein. Journal of
oleo science 60(7): 333–338.

Yılmaz, M.T. and Karakaya, M. (2009). Differential Scanning Calorimetry

Analysis of Goat Fats: Comparison of Chemical Composition and
Thermal Properties. Journal of the American Oil Chemists’ Society 86(9): 877–
883.

Yılmaz, M.T., Mustafa, K. and Aktaş, N. (2010).Composition and thermal

properties of cattle fats.European Journal of Lipid Science and Technology
112(3): 410–416.

73
APPENDICES

Appendix 1

74
Figure. MAG, DAG and TAG Standard

75
 (a) (b) (c) (d)

Appendix 2

Figure. TLC separation of (a) Lard, (b) Chicken fat, (c) Beef fat and (d) Mutton fat

76
 Appendix 3

Cost Analysis: Determination of Carbon Isotope Values of Edible Oils by

Total Cost of
Cost per Hour
A Electricity kWh (unit) Electricity
(RM)
(RM0.37/unit)
1 EA-IRMS 0.0425 0.02 0.02
Depreciation Cost Cost per hour
B Equipments Depreciation Cost
(RM/hour) (RM)
1 (20% Equipment Cost)/365*24 1300000.00 29.68 29.68
Cost Cost per sample
C Preventive Maintenance Per Year Cost
(RM/hour) (RM)
1 Cost/ (312*8) 39000 15.63 15.63
D Direct Cost Cost (RM)
A+B+C 45.32
E Admin Cost Cost (RM)
20% Direct Cost 9.06
GRAND TOTAL COST per hour
54.39
(RM)
GRAND TOTAL COST per minute (RM) 0.906430306

Direct
Summary 10% 20% 30%
Cost (RM)
EA-IRMS Edible oil 54.39 59.82 65.26 70.70

Direct
Summary 40% 50% 60%
Cost (RM)
EA-IRMS Edible oil 54.39 76.14 81.58 87.02

Direct
Summary 70% 80% 90%
Cost (RM)
EA-IRMS Edible oil 54.39 92.46 97.89 103.33
EA-IRMS

Total Cost per analysis: RM 100.00

Appendix 4

77
Figure: Chromatograms of EA-IRMS for selected animal fats, lard olein and
lard stearin

78
 Appendix 5

Figure : Chromatograms of EA-IRMS for MAG and DAG of selected animal

fats

79
 BIODATA OF STUDENT

Nina Naquiah binti Ahmad Nizar was born in Kuala Lumpur on the

28th October 1987. She is the daughter of Ahmad Nizar Zolfakar and Aishah

Hashim, and the eldest of five siblings. After completing SPM in 2004 with 7A’s

from Sekolah Menengah Sains Seri Puteri KL, she was called for the National

Service Programme in Kem Bakau Resort Kerteh, Terengganu. Then, she

pursued her Foundation in Life Science in Kedah Matriculation College,

Changloon. She was awarded the Bachelor of Science in Food Science and

Nutrition from Universiti Kebangsaan Malaysia in 2010. She is optimistic with

the idea of integrating science and Islam. With her high interest in Halal and

Food Science, she pursued Masters of Halal Products Science from Halal

Products Research Institute (HPRI), UPM directly after completing her first

degree to nourish her insights in this blooming field. While waiting for her viva

voce, she works at the Laboratory of Halal Services, HPRI UPM for six months.

Currently, she works in Wisma Putra, Ministry of Foreign Affairs.

80
 LIST OF PUBLICATIONS

Nina Naquiah Ahmad Nizar, Jalaldeen Mohamed Nazrim Marikkar and

Dzulkifly Mat Hashim, (2013). Differentiation of lard, chicken fat, beef fat
and mutton fat by GCMS and EA-IRMS techniques. Journal of Oleoscience.
62(7): 459-464.

Nina Naquiah, A.N., Marikkar, J.M.N. and Dzulkifly, M.H. (2013). Bulk carbon
isotope ratio determination for MAG and DAG derived from animal fats
by GCMS and IRMS techniques. Journal of Oleoscience (Awaiting
submission)

Conferences

Marikkar, J.M.N. and Nina Naquiah A.N. Differentiation of pork from other
meat species by lipid analysis methodologies.15th Food Innovation Asia
Conference, Bangkok , Thailand, 13th-14th June 2013

Nina Naquiah, A.N., Marikkar, J.M.N. and Dzulkifly, M.H. Differentiation of

Animal Oils by Means of Gas Chromatography Mass Spectrometry and
Principle Component Analysis for Halal Authentication. World Halal
Research, Kuala Lumpur Convention Centre, Kuala Lumpur, 4 th-5th
April 2012.

Award

Nina Naquiah, A.N., Marikkar, J.M.N. and Dzulkifly, M.H. Differentiation of

Animal Oils by Means of Gas Chromatography Mass Spectrometry and
Principle Component Analysis for Halal Authentication. Research,
Innovation, Invention & Design, UiTM 2012 (SILVER MEDAL).

81