Analysis of lard in meatball broth using Fourier transform infrared spectroscopy

and chemometrics
Analisis lemak babi dalam kuah bakso menggunakan spektroskopi Fourier
transform infrared (FTIR) dan kemometrika
Endah Kurniawati a,b, Abdul Rohman a,b,c,d , Kuwat Triyana a,d
a
Research Center of Halal Products, Gadjah Mada University, Yogyakarta 55281, Indonesia
b
Faculty of Pharmacy, Gadjah Mada University, Yogyakarta 55281, Indonesia
c
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
abstract
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 cm-1. The coefficient of
determination (R2) and root mean square error of calibration (RMSEC) values obtained were
0.9975 and 1.34% (v/v), respectively. Furthermore, the classification of lard and beef fat in
meatball broth as well as in commercial samples was performed at wavenumber region of
12001000 cm-1. The results showed that FTIR spectroscopy coupled with chemometrics can
be used for quantitative analysis and classification of lard in meatball broth for Halal
verification studies. The developed method is simple in operation, rapid and not involving
extensive sample preparation.
Bakso merupakan salah satu makanan favorit di Indonesia. Untuk alasan ekonomi (karena
perbedaan harga), substitusi daging sapi dengan daging babi dapat terjadi. Dalam penelitian
ini, spektroskopi FTIR dikombinasikan dengan kemometrika dari kuadrat terkecil parsial atau
partial least square (PLS) dan analisis komponen utama atau principal component analysis
(PCA) digunakan untuk analisis lemak babi (lard) dalam kuah bakso. Lemak babi dalam kuah
bakso secara kuantitatif ditentukan pada daerah bilangan gelombang 1018-1284 cm -1.
Koefisien determinasi (R2) dan nilai akar kuadrat tengah atau root mean square error kalibrasi
(RMSE) yang diperoleh masing-masing adalah 0,9975 dan 1,34% (v / v). Selanjutnya,
klasifikasi lemak babi dan lemak sapi dalam kuah bakso serta dalam sampel komersial
dilakukan pada daerah bilangan gelombang 1200-1000 cm -1. Hasil penelitian menunjukkan
bahwa spektroskopi FTIR dipasangkan dengan kemometrika dapat digunakan untuk analisis
kuantitatif dan klasifikasi lemak babi dalam kuah bakso untuk studi verifikasi Halal. Metode
yang dikembangkan sederhana dalam pengoperasian, cepat dan tidak memerlukan persiapan
sampel yang luas.
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). Verification 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).

Transparansi dalam spesiasi daging yang digunakan dalam produk makanan adalah
kebutuhan yang semakin meningkat dan sangat penting untuk melindungi hak konsumen,
kepercayaan agama dan keberuntungan jerih payah (Ali, Hasyim, Mustafa, Che Man, Dhadi et
al, 2012;. Dosti, Ghasemi Dehkordi, & Rahimi, 2011; Fajardo, Gonzlez, Rojas, Garca, &
Martn, 2010). Verifikasi komponen yang dinyatakan dalam produk makanan juga diperlukan
untuk pencegahan praktik pemalsuan. Untuk tujuan ini, beberapa negara membuat peraturan
untuk menjamin bahwa produk makanan yang tersedia aman dan otentik. Oleh karena itu,
deteksi penipuan spesies dalam produk daging termasuk bakso penting bagi perlindungan
konsumen dan industri makanan (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 classified as restructured
meat. It can be prepared using beef, chicken, pork, or fish 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 profits.
Di Indonesia, salah satu makanan favorit yang dikonsumsi adalah meatball, atau dikenal
sebagai 'bakso' (Rohman, Sismindary, Erwanto, & Che Man, 2011). Saat ini, di Indonesia,
karena perbedaan harga yang tinggi antara daging babi dan sapi, praktek pemalsuan bakso
daging sapi dengan bakso daging babi terjadi. Bakso adalah olahan daging yang dihaluskan
yang dapat diklasifikasikan sebagai daging yang direstrukturisasi. Hal ini dapat disusun
dengan menggunakan daging sapi, ayam, babi, atau daging ikan, dan salah satu yang sangat
populer dan banyak ditemukan di pasar Indonesia adalah bakso sapi (Purnomo & Rahardyan,
2008). Kadang-kadang, penjual yang tidak bermoral menggantikan bakso daging sapi dengan
bakso daging babi untuk mendapatkan keuntungan ekonomis.
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 certification has been made
mandatory for all meat and meat based imported food products like meatball. Halal verification 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).
Penggantian daging sapi dengan daging babi adalah masalah serius tidak hanya untuk
alasan ekonomi tetapi juga untuk sudut pandang agama. Komunitas Muslim dan Yahudi tidak
diperbolehkan untuk mengkonsumsi produk makanan yang mengandung turunan babi seperti
daging babi. Dalam Islam dan Yahudi, babi tidak halal dan tidak kosher (Regenstein, Chaudry,
& Regenstein, 2003; Rohman & Che Man, 2012). Di Timur Tengah dan negara-negara Islam
lainnya, terutama di Asia Timur, sertifikasi halal telah diwajibkan untuk semua produk
makanan daging dan daging berbasis impor seperti bakso. Verifikasi halal membutuhkan
metode yang dapat diandalkan yang memastikan bahwa barang-barang non-halal seperti
daging babi tidak ada dalam produk makanan (Nakyinsige, Che Man, & Sazili, 2012).
Akibatnya, beberapa metode analisis telah dikembangkan, diusulkan dan digunakan untuk
analisis daging babi dalam produk makanan (Mursyidi, 2013).
Analysis of pork in meatball and other food products can be performed by DNA amplification
present in pork using polymerase chain reaction using different targets of amplification (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 spec-trometry 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).
Analisis daging babi di bakso dan produk makanan lainnya dapat dilakukan oleh amplifikasi
DNA yang terdapat dalam daging babi dengan menggunakan polymerase chain reaction
menggunakan target amplifikasi yang berbeda (Ali, Hasyim, Dhahi, et al, 2012;. Ali, Hasyim,
Mustafa, & Che Man, 2012; Ali, Hasyim, Mustafa, Che Man, Dhahi, et al, 2012), electronic nose
dan spectrometry kromatografi-mass gas dengan deteksi aroma dan senyawa volatil lemak
babi yang terdapat dalam bakso (Nur Juliana, Che Man, Mat Hashim. , & Mohamed, 2011), dan
spektroskopi Fourier transform inframerah (FTIR) dengan menganalisis lemak babi atau lard
secara keseluruhan dari materi yang diekstrak dari daging babi (Rohman, Sismindary, et al.,
2011).
FTIR spectroscopy coupled with chemometrics is promising analytical techniques to be used
in the halal verification 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 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 quantification and classification 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.
Spektroskopi FTIR dipasangkan dengan kemometrika menjanjikan teknik analisis yang
digunakan dalam penelitian verifikasi halal. Hal ini cepat, tidak merusak dan tidak melibatkan
persiapan sampel yang melelahkan. Dalam otentikasi halal, spektroskopi FTIR telah
dimanfaatkan untuk analisis lemak babi dalam campuran biner dengan lemak hewan lain
dengan bantuan kalibrasi multivariat yang dikombinasi dengan analisis diskriminan (Che Man
& Mirghani, 2001; Jaswir, Mirghani, Hassan, & Mohd Said, 2003), serta untuk analisis lemak
babi dalam formulasi kue dan cokelat menggunakan kalibrasi partial least square (Che Man,
Syahariza, Mirghani, Jinap, & Bakar, 2005; Syahariza, Che Man, Selamat, & Bakar, 2005).
Sebelumnya, kami telah menganalisis lemak babi dalam produk bakso (Rohman, Sismindary,
et al., 2011). Dalam penelitian itu, kami mengekstrak lemak babi dari lemak babi yang
terkandung dalam bakso di mana beberapa komponen lain mungkin ada dalam diekstrak
lemak babi. Dalam studi ini, kami mengembangkan spektroskopi FTIR yang dikombinasi
dengan partial least square dan analisis komponen utama untuk kuantifikasi dan klasifikasi
lemak babi dalam kuah bakso daging sapi. Karena komponen yang berbeda yang mungkin
ada dalam lemak babi yang diekstrak dari kuah bakso dan lemak babi yang diekstrak dari
lemak babi dalam bakso, oleh karenanya model kalibrasi harus dikembangkan.
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 filter

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.
Lemak babi (lard) dan lemak sapi yang diperoleh dengan proses rendering hewan yang
sesuai. Prosedur rendering sesuai dengan yang dilaporkan dalam Rohman dan Che Man
(2009). Jaringan adiposa babi dan ternak dipotong kecil-kecil menggunakan cutter komersial
agar dapat mengekstrak lemak babi dan lemak sapi secara efektif. Menggunakan Beaker
glass, jaringan potong dimasukkan ke dalam oven konvensional selama 3 jam pada 100 C.
Lemak yang mencair diregangkan dengan kertas saring. Residu air dihilangkan menggunakan
natrium sulfat anhidrat. Lemak yang diperoleh digunakan untuk analisis komposisi asam
lemak dan analisis spektra FTIR.
2.2.

Fatty acid composition

The composition of fatty acids composed of lard and beef fat was carried out using gas
chromatography with flame 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. Quantification analysis of FAMEs was performed using internal
normalization technique.
Komposisi asam lemak terdiri dari lemak babi dan lemak sapi dilakukan dengan
menggunakan kromatografi gas dengan detektor ionisasi nyala atau flame ionization detector
(GC-FID). Kondisi GC adalah seperti yang dilaporkan dalam Rohman et al. (2012). Sebagai
standar asam lemak metil ester atau fatty acid methyl esters (FAMEs), kami menggunakan 37
senyawa (C4 ke C24) dari Sigma Chemicals (St. Louis, MO, USA) untuk mengidentifikasi waktu
retensi FAME dalam lemak babi dan lemak sapi. Analisis kuantifikasi FAMEs dilakukan dengan
menggunakan teknik normalisasi internal.
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.
Satu set standar yang terdiri dari lemak babi dalam lemak sapi dibuat dengan
mencampurkan kedunya pada rentang konsentrasi 0-100% (v / v) dari lemak babi dalam
lemak sapi. Sampel yang dipilih, yang berbeda dari sampel kalibrasi, digunakan sebagai
sampel validasi. Lemak babi, lemak sapi bersama dengan campuran mereka diukur dengan
menggunakan spektrofotometer FTIR. Daerah-daerah spektral di mana variasi antara analit
yang diamati dipilih untuk mengembangkan kalibrasi multivariat dan analisis komponen
utama.
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.

Kaldu bakso diambil dari beberapa pasar di Yogyakarta. Sekitar 100 ml kaldu bakso diambil,
ditambah dengan 100 mL heksana, dan mengalami ekstraksi cair-cair dalam corong pemisah
atau separatory funnel. Campuran dikocok dengan kuat dan fase heksan mengandung lemak
diambil. Fasa air selanjutnya diekstrak dengan menggunakan 50 mL heksana. Fase heksana
dikumpulkan dan diuapkan di bawah vakum rotary evaporator pada suhu 60 C, dan residu
air dikeringkan dengan natrium sulfat anhidrat. Lemak yang diperoleh menjadi subjek
pengukuran FTIR.
2.5.

FTIR spectral acquisition

FTIR spectra of all samples was scanned in the mid infrared region of 4004000 cm-1 using
ABB MB3000 FTIR spectrophotometer (Clairet Scientific, Northampton, UK) equipped with
deuterated triglycine sulfate (DTGS) detector and KBR as beam splitter, with a resolution of 8
cm-1 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 reflectance (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.
Spektrum FTIR dari seluruh sampel dipindai di wilayah inframerah pertengahan 400-4000
cm-1 menggunakan ABB MB3000 FTIR spektrofotometer (Clairet Ilmiah, Northampton, Inggris)
yang dilengkapi dengan deuterated triglycine sulfat (DTGS) detektor dan KBR sebagai beam
splitter, dengan resolusi dari 8 cm-1 dan 32 pemindaian. Spectra diolah dengan
menggunakan Horizon MB FTIR software versi 3.0.13.1 (ABB, Kanada). Sampel ditempatkan
dalam kontak yang baik dengan horisontal jumlah pantulan dilemahkan (HATR) elemen (ZnSe
kristal) pada suhu kamar dikendalikan (20 C). Semua spektrum dijatah dengan latar
belakang spektrum udara. Setelah setiap scan, spektrum background referensi udara baru
diambil. Spektrum ini dicatat sebagai nilai absorbansi pada setiap titik data dalam rangkap
tiga.
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
classification 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 profiles can be used for differentiation of edible fats and oils due to its
capability to provide the fingerprint profiles 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 specified in
Codex Allimentarius (2003).
3.2.

FTIR spectra of lard and beef fat

The importance of IR spectroscopy in identification 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 cm-1, corresponding to stretching and bending vibrations of
functional groups. Peak assigned with (a) at 3007 cm -1 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 difficult to be differentiated, however, due to the
capability of FTIR spectra as fingerprint 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 cm -1 (m). The
wavenumber of3008 cm-1orig-inates from cis-olefinic 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.