Meat Science 96 (2014) 9498

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Meat Science
journal homepage: www.elsevier.com/locate/meatsci

Analysis of lard in meatball broth using Fourier transform infrared

spectroscopy and chemometrics
Endah Kurniawati a,b, Abdul Rohman a,b,c,, Kuwat Triyana a,d
a

Research Center of Halal Products, Gadjah Mada University, Yogyakarta 55281, Indonesia
Faculty of Pharmacy, Gadjah Mada University, Yogyakarta 55281, Indonesia
Centre of Research for Fiqh Science and Technology (CFiRST), Universiti Teknologi Malaysia, Malaysia
d
Department of Physics, Faculty of Mathematics and Natural Sciences, Gadjah Mada University, Yogyakarta 55281, Indonesia
b
c

a r t i c l e

i n f o

Article history:
Received 16 December 2012
Received in revised form 28 June 2013
Accepted 1 July 2013
Keywords:
Lard
Meatball broth
FTIR spectroscopy
Chemometrics

a b s t r a c t
Meatball is one of the favorite foods in Indonesia. For the economic reason (due to the price difference), the substitution of beef meat with pork can occur. In this study, FTIR spectroscopy in combination with chemometrics of partial least square (PLS) and principal component analysis (PCA) was used for analysis of pork fat (lard) in meatball
broth. Lard in meatball broth was quantitatively determined at wavenumber region of 10181284 cm1. The coefcient of determination (R2) and root mean square error of calibration (RMSEC) values obtained were 0.9975 and
1.34% (v/v), respectively. Furthermore, the classication of lard and beef fat in meatball broth as well as in commercial samples was performed at wavenumber region of 12001000 cm1. The results showed that FTIR spectroscopy
coupled with chemometrics can be used for quantitative analysis and classication of lard in meatball broth for
Halal verication studies. The developed method is simple in operation, rapid and not involving extensive sample
preparation.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Transparency in meat speciation used in food products is an ever
increasing demand and is essential for the protection of consumers'
right, religious credence and hard-earned fortunes (Ali, Hashim,
Mustafa, Che Man, Dhadi et al., 2012; Doosti, Ghasemi Dehkordi, &
Rahimi, 2011; Fajardo, Gonzlez, Rojas, Garca, & Martn, 2010). Verication of declared components in food products is also necessary
for the prevention of adulteration practice. For this purpose, some countries make regulation for assuring that food products available are safe
and authentic. Therefore, detection of species fraud in meat products including meatball is important for consumer protection and food industries (Doosti et al., 2011).
In Indonesia, one of the favorite foods consumed is meatball, or
known as bakso (Rohman, Sismindary, Erwanto, & Che Man, 2011).
Currently, in Indonesia, due to the high different prices between pork
and beef, the adulteration practice of beef meatball with pork meatball
is occurring. Meatball is a processed comminuted meat which can be
classied as restructured meat. It can be prepared using beef, chicken,
pork, or sh meat, and the one that is very popular and widely found
in the Indonesian market is beef meatball (Purnomo & Rahardiyan,
2008). Sometimes, unscrupulous seller replaces beef meatball with
pork meatball in order to gain economical prots.
Corresponding author at: Faculty of Pharmacy, Gadjah Mada University, Yogyakarta
55281, Indonesia. Tel.: + 60 3 89430405; fax: + 60 3 89439745.
E-mail address: abdulkimfar@gmail.com (A. Rohman).
0309-1740/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.meatsci.2013.07.003

The substitution of beef with pork is a serious problem not only for
economic reason but also for religious point of view. Muslim and Jew
communities are not allowed to consume food products containing
pig derivatives like pork. In Islamic and Jews, pork is not halal and
not kosher (Regenstein, Chaudry, & Regenstein, 2003; Rohman &
Che Man, 2012). In the Middle East and other Islamic countries, especially in East Asia, halal certication has been made mandatory for all
meat and meat based imported food products like meatball. Halal verication needs a reliable method assuring that non-halal items like pork
are absent in food products (Nakyinsige, Che Man, & Sazili, 2012). As a
consequence, some analytical methods have been developed, proposed
and used for analysis of pork in food products (Mursyidi, 2013).
Analysis of pork in meatball and other food products can be performed
by DNA amplication present in pork using polymerase chain reaction
using different targets of amplication (Ali, Hashim, Dhahi, et al., 2012;
Ali, Hashim, Mustafa, & Che Man, 2012; Ali, Hashim, Mustafa, Che Man,
Dhahi, et al., 2012), electronic nose and gas chromatographymass spectrometry by detection of the aroma and volatile compounds of lard
present in meatball (Nurjuliana, Che Man, Mat Hashim, & Mohamed,
2011), and Fourier transform infrared (FTIR) spectroscopy by analyzing
pork fat or lard as a whole of matter extracted from pork (Rohman,
Sismindary, et al., 2011).
FTIR spectroscopy coupled with chemometrics is promising analytical techniques to be used in the halal verication studies. It is fast, not
destructive and not involving laborious sample preparation. In halal authentication, FTIR spectroscopy has been exploited for analysis of lard in
the binary mixture with other animal fats with the aid of multivariate

E. Kurniawati et al. / Meat Science 96 (2014) 9498

calibration in combination with discriminant analysis (Che Man &

Mirghani, 2001; Jaswir, Mirghani, Hassan, & Mohd Said, 2003), as well
as for analysis of lard in cake and chocolate formulation using partial
least square calibration (Che Man, Syahariza, Mirghani, Jinap, & Bakar,
2005; Syahariza, Che Man, Selamat, & Bakar, 2005). Previously, we
have analyzed pork fat in meatball products (Rohman, Sismindary,
et al., 2011). In that research, we extracted lard from pork fat contained
in meatball in which some other components may be present in
extracted lard. In this study, we develop FTIR spectroscopy in combination with partial least square and principal component analysis for quantication and classication of lard in broth of beef meatball. Due to the
different components that may be present in lard extracted from meatball broth and lard extracted from pork fat in meatball, indeed, the calibration model should be developed.
2. Materials and methods
2.1. Preparation of animal fats
The pork fat (lard) and beef fat were obtained by rendering process
of corresponding animal. The procedure of rendering follows with that
reported in Rohman and Che Man (2009). The adipose tissues of pig and
cattle were cut into small pieces using commercial cutter in order to effectively extract lard and beef fat. Using Beaker glass, the cut tissue was
introduced into conventional oven during 3 h at 100 C. The melted fat
was strained with lter paper. The residue of water was removed using
anhydrous sodium sulfate. The fat obtained was further used for analysis of fatty acid composition and FTIR spectra analysis.
2.2. Fatty acid composition
The composition of fatty acids composed of lard and beef fat was
carried out using gas chromatography with ame ionization detector
(GC-FID). The GC condition was as reported in Rohman et al. (2012).
As standard of fatty acid methyl esters (FAMEs), we used 37 compounds (C4 to C24) from Sigma Chemicals (St. Louis, MO, USA) to
identify the retention times of FAME in lard and beef fat. Quantication analysis of FAMEs was performed using internal normalization
technique.
2.3. Preparation of calibration samples
A set of standards consisting of lard in beef fat was prepared by
mixing of both at concentration ranges of 0100% (v/v) of lard in beef
fat. Selected samples, which were different from calibration samples,
were used as validation samples. Lard, beef fat along with their blends
were measured using FTIR spectrophotometer. The spectral regions in
which the variations among analytes were observed were selected for
developing multivariate calibration and principal component analysis.
2.4. Analysis of lard in meatball broth
The meatball broth was taken from several markets in Yogyakarta.
An approximately 100 mL of meatball broth was taken, added with
100 mL hexane, and subjected to liquidliquid extraction in a separatory
funnel. The mixture was shaken vigorously and the hexane phase containing fats was taken. The water phase was further extracted using
50 mL of hexane. The hexane phases were collected and evaporated
under vacuum rotary evaporator at 60 C, and the residue of water was
dried with anhydrous sodium sulfate. The fats obtained were subjected
to FTIR measurements.
2.5. FTIR spectral acquisition
FTIR spectra of all samples was scanned in the mid infrared region of
4004000 cm1 using ABB MB3000 FTIR spectrophotometer (Clairet

95

Scientic, Northampton, UK) equipped with deuterated triglycine sulfate

(DTGS) detector and KBR as beam splitter, with a resolution of 8 cm1
and 32 scanning. Spectra were processed using Horizon MB FTIR software version 3.0.13.1 (ABB, Canada). The samples were placed in good
contact with horizontal attenuated total reectance (HATR) element
(ZnSe crystal) at controlled ambient temperature (20 C). All spectra
were rationed against a background of air spectrum. After every scan, a
new reference air background spectrum was taken. These spectra were
recorded as absorbance values at each data point in triplicate.

2.6. Statistical analysis

Quantitative analysis of lard was performed using partial least
square calibration with the aid of Horizon MB software (Canada) included in FTIR spectrophotometer. While, the classication among
meatball broth samples was carried out using principal component
analysis with the software of Minitab (version 16, USA).

3. Results and discussion

3.1. Fatty acid analysis
Fatty acid composition is one of the parameter used in the quality
control of edible fats and oils. The fatty acid proles can be used for
differentiation of edible fats and oils due to its capability to provide
the ngerprint proles of studied fats and oils.
Table 1 revealed the fatty acid composition of lard and beef fat.
Beef fat contained more palmitic and oleic acids, while lard contained
more oleic and stearic acids, respectively. The composition of these
fatty acids in lard and beef fat was in agreement with those specied
in Codex Allimentarius (2003).

3.2. FTIR spectra of lard and beef fat

The importance of IR spectroscopy in identication of samples
originates from the much information content obtained, and the possibility to assign certain absorption bands related to functional groups
(Bendini et al., 2007). Fig. 1 is FTIR spectra of lard and beef fat. Each
peaks and shoulder indicated the functional groups responsible for
infrared absorption at wavenumbers of 4000400 cm1, corresponding to stretching and bending vibrations of functional groups. Peak
assigned with (a) at 3007 cm1 attributed from the stretching vibration of cis C_CH. Table 2 described the assignment peaks and shoulders present in lard and beef fat (Guillen & Cabo, 1997; Lerma-Garcia,
Ramis-Ramos, Herrero-Martinez, & Simo-Alfonso, 2010).
FTIR spectra of beef fat and lard are difcult to be differentiated,
however, due to the capability of FTIR spectra as ngerprint tools,
both spectra revealed bit differences in peak/shoulder intensities and
the exact wavenumbers at which the maximum absorbance was observed for each fat and oil, due to the different nature and composition
of the both lard and beef fat, especially at wavenumber regions of 3007
(a), 1117 (l) and 1098 cm1 (m). The wavenumber of 3008 cm1 originates from cis-olenic C_H, which can be used as an indicative of
unsaturation degree. The more unsaturation degree of fats and oils,
the higher the peak intensities in that wavenumber. Meanwhile,
wavenumbers of 1117 and 1098 cm 1 originate from the stretching
vibrations of C\O in triacylglycerols. As shown in Table 1, lard has
more unsaturated fatty acid than that in beef fat. As a consequence,
lard revealed higher peak intensity than beef fat at wavenumber of
3007 cm 1 (Che Man, Rohman, & Mansor, 2011). This wavenumber
region in which lard and beef fat showed some differences was further optimized to be selected as wavenumber used for quantitative
analysis of lard in meatball broth.

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E. Kurniawati et al. / Meat Science 96 (2014) 9498

Fig. 1. FTIR spectra of lard and beef fat at mid infrared region (4000650 cm1).

3.3. Quantitative analysis of lard in meatball broth

Analysis of lard in meatball broth was performed with the aid of
multivariate calibration of partial least square (PLS) at wavenumber
region of 10181284 cm1. The selection of this wavenumber was
based on the optimization process, i.e. the used wavenumber offered
the best prediction model for the relationship between actual value of
lard and FTIR predicted values. Besides, this wavenumber also gave
the highest coefcient of determination (R2) and the lowest values
of errors in calibration and prediction, expressed with root mean
square error of calibration (RMSEC) and prediction (RMSEP).
Fig. 2 exhibited the relationship between actual values of lard (x-axis)
and FTIR calculated values (y-axis) having R2 and RMSEC values of 0.9975
and 1.34% (v/v), respectively. Furthermore, this calibration model was
used for prediction of independent samples called with prediction/
validation samples. The R2 and root mean square error of prediction
(RMSEP) obtained were 0.9944 and 2.89% (v/v), respectively. From
this result, it is imperative that FTIR spectroscopy combined with
multivariate calibration of PLS provide the accurate results with

low errors (from R2, RMSEC dan RMSEP values) for analysis of lard
in meatball broth. Some authors have reported that as low as 1% of
lard in the mixture with other fats and oils could be detected using
FTIR spectroscopy in combination with multivariate calibration of
PLS (Che Man & Mirghani, 2001; Jaswir et al., 2003; Rohman, Che
Man, Ismail, & Puziah, 2011).
3.4. Classication of meatball broth containing lard and beef fat
The meatball broth with lard or beef fat was classied using
chemometrics of principal component analysis (PCA). The wavenumber
regions for PCA were also optimized. Finally, the wavenumbers of 1200
1000 cm1 were chosen due to its capability to provide good separation
among evaluated samples. Fig. 3 demonstrates the score plot of PCA of
lard and beef fat in meatball broth, representing the projection of samples dened by the rst principle component (PC 1) and the second principle component (PC 2). Using this projection, meatball broth containing

Table 2
The functional groups responsible for infrared absorption of lard and beef fat.
Table 1
Fatty acid composition composed of lard and beef fat.
Fatty acids

Kaproic acid (C10:0)

Lauric acid (C12:0)
Miristoleic acid (C14:1)
Miristic acid (C14:0)
Pentadecanoic acid (C15:0)
Palmitoleic acid (C16:1)
Palmitic acid (C16:0)
Heptadecanoic acid (C17:0)
Oleic acid (C18:1)
Stearic acid (C18:0)
Linoleic acid (C18:2)
Gamma linolenic acid (C18:3)
Linolenic acid (C18:3)
Arachidonic acid (C20:4)

Fatty acid composition (%)

Beef fat

Lard

nd
0.16 0.01
0.34 0.03
5.53 0.11
2.14 0.08
2.16 0.09
45.67 0.24
3.61 0.05
31.20 0.17
7.17 0.06
1.05 0.02
0.65 0.02
0.16 0.01
nd

0.004 0.00
0.10 0.01
nd
1.34 0.04
0.04 0.00
0.90 0.00
21.92 0.12
nd
38.64 0.41
26.85 0.25
10.08 0.10
nd
0.039 0.00
0.001 0.00

Assignment

Wavenumbers
(cm1)

Functional group responsible for IR absorption

a
b
c
d
e
f
g
h

3007
2970
2925
2875
1715
1650
1462
1418

i
j
k
l
m
n
o
p

1375
1226
1160
1117
1098
1031
962

cis-olenic C_H
CH3 stretching asymmetric
CH2 stretching asymmetric
CH3 stretching asymmetric
C_O carbonyl stretching
cis C_C
CH2 bending
CH rocking (bending) from cis-disubstituted
alkenes
CH3 bending
C\O (ether) stretching
C\O (ether) stretching
C\O (ether) stretching
C\O (ether) stretching
C\O (ether) stretching
_CH from isolated trans-olen
\CH2 rocking vibration

E. Kurniawati et al. / Meat Science 96 (2014) 9498

97

Fig. 2. The relationship between actual value (x-axis) and FTIR predicted value (y-axis) of lard in binary mixture with beef fat in meatball broth.

Acknowledgment
The authors thank the Directorate of Higher Education, Ministry of
Education and Culture, Republic of Indonesia for nancial support during this research via project grant of Riset Unggulan Strategis Nasional
with contract number: LPPM-UGM/0004/2011.

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Fig. 3. The Score plot of rst principal components (PC 1) and second principal components (PC 2) of lard (A) beef fat (C), and selected samples (B, D, E).

lard, beef fat and commercial meatball is well separated meaning that
PCA can accomplish the classication among them. Based on this prole,
it can be stated that commercial samples (region B) do not contain lard in
the products.

4. Conclusions
It can be concluded that FTIR spectroscopy in combination with
partial least square (PLS) and principal component analysis (PCA)
can be successfully used for analysis of lard in meatball broth. The
coefcient of determination (R2) and root mean square error of calibration (RMSEC) values obtained for quantication were 0.9975
and 1.34% (v/v), respectively. PCA for classication of lard and beef
fat was performed at wavenumber region of 12001000 cm 1. The
developed method was rapid, ease in sample presentation and reliable enough.

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