International Journal of Food Properties

ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: https://www.tandfonline.com/loi/ljfp20

Use of Fourier Transform Infrared (FTIR)

Spectroscopy and Chemometrics for Analysis of
Lard Adulteration in “Rambak” Crackers

Yuny Erwanto, Afif Turindra Muttaqien, Sugiyono, Sismindari & Abdul

Rohman

To cite this article: Yuny Erwanto, Afif Turindra Muttaqien, Sugiyono, Sismindari & Abdul Rohman
(2016) Use of Fourier Transform Infrared (FTIR) Spectroscopy and Chemometrics for Analysis
of Lard Adulteration in “Rambak” Crackers, International Journal of Food Properties, 19:12,
2718-2725, DOI: 10.1080/10942912.2016.1143839

To link to this article: https://doi.org/10.1080/10942912.2016.1143839

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International Journal of Food Properties, 19:2718–2725, 2016
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print/1532-2386 online
DOI: 10.1080/10942912.2016.1143839

Use of Fourier Transform Infrared (FTIR) Spectroscopy and

Chemometrics for Analysis of Lard Adulteration in
“Rambak” Crackers

Yuny Erwanto1,3, Afif Turindra Muttaqien1, Sugiyono2, Sismindari3,4, and

Abdul Rohman3,4
1
Faculty of Animal Sciences, Gadjah Mada University, Yogyakarta, Indonesia
2
Faculty of Veterinary Medicine, Gadjah Mada University, Bulaksumur, Yogyakarta, Indonesia
3
Research Center of Halal Products, Gadjah Mada University, Yogyakarta, Indonesia
4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gadjah Mada University,
Yogyakarta, Indonesia

“Rambak” crackers are one of the traditional foods consumed among Indonesian people made from
various kinds of animal skin. The present study highlights the analysis of lard obtained from extraction
of “rambak” crackers using Fourier transform infrared (FTIR) spectroscopy in combination with
chemometrics of partial least square and principle component analysis. FTIR spectroscopy at wave-
number regions of 1200–1000 cm–1 was successfully used for quantification and classification of lard
in “rambak” crackers. The relationship between actual value of lard and Fourier transform infrared
predicted value has R2 value of 0.946 with low errors in calibration and validation models.
Furthermore, the chemometrics principle component analysis can be successfully used for determina-
tion of pig skin through analysis of lard in commercial “rambak” crackers. The developed method
(FTIR spectroscopy coupled with chemometrics) is rapid and reliable for quantification and classifica-
tion of lard in “rambak” crackers.

Keywords: “Rambak” crackers, Lard, Fourier transform infrared (FTIR) spectroscopy, Partial least
square, Principle component analysis.

INTRODUCTION

Krupuk “rambak” or “rambak” cracker, is one of the traditional foods in Indonesia that is made
from various kinds of animal skins such as buffalo, cattle, or pig. This food is traditionally hand
made with traditional processes. One of the interesting issues in the food industry is the
authenticity of food products. The authenticity of food products can involve the alteration of
the true labeling of food ingredients, in which the high value of raw materials are substituted by

Received 15 September 2015; accepted 15 January 2016.

Address correspondence to Yuny Erwanto, Division of Animal Products Technology, Faculty of Animal Sciences,
Gadjah Mada University, Jl. Fauna No. 3, Bulaksumur, Yogyakarta 55281, Indonesia. E-mail: erwantougm@gmail.com
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ljfp.

2718
 LARD DETECTION USING FTIR SPECTROSCOPY 2719

cheaper materials such as substitution of cow skin with pig skin.[1] The verification of labeled
components in any food products is necessary in order to prevent the adulteration practice. For
this purpose, some countries make regulation for assuring that food products available are safe
and authentic,[2] especially if food products contain pig derivatives like pig skin.
The presence of pig derivatives such as pig skin, lard, pork and porcine gelatin in any food products is
a serious matter for certain community,[3] because some religions like Islam, Judaism, and Hinduism
forbid their followers to consume any food products containing porcine and its derivatives.[4] Therefore,
it is necessary to evaluate the presence of these pig derivatives in any products using some instrumental
techniques. Such techniques used are differential scanning calorimetry,[5,6] gas chromatography-mass
spectrometry,[7] high-performance liquid chromatography,[8,9] electronic nose,[10,11] proton nuclear
magnetic resonance[12] and DNA-based methods using polymerase chain reaction.[13,14] Some of
these methods are too laborious and time consuming, consequently, an analytical technique offering
rapid and reliable must be used. One of the promising methods suitable for routine analysis is Fourier
transform infrared (FTIR) spectroscopy, especially in combination with some chemometrics techniques.
The use of FTIR spectroscopy in food analysis increased significantly. This is due to its
properties as fingerprint technique, which can be used for qualitative and quantitative
analyses.[15] Some researchers have reported the application of FTIR spectroscopy for
analysis of pig derivatives. Previous research used FTIR spectroscopy for rapid discrimina-
tion of pork in Halal and non-Halal Chinese ham sausages.[16] Previous research has also
shown the potential application of FTIR spectroscopy for analysis of lard in cake
formulation[17] and chocolate products.[18] Our group also developed FTIR spectroscopy in
combination with chemometrics for analysis of pork in beef meatballs,[19] lard in meatball
broth,[20] and rat’s meat in beef meatballs.[21] Differentiation of porcine and bovine gelatins
was successfully determined using FTIR spectroscopy.[22] Using literature review, there is
no publication reporting the employment of FTIR spectroscopy for identification and
quantification of crackers made from cow skin adulterated with pig skin. Therefore, in
this study, FTIR spectroscopy combined with chemometrics is employed for analysis of lard
in “rambak” crackers.

MATERIALS AND METHODS

In order for a wide application of species detection methods of commercial samples, the skins of
cow and pig were randomly obtained from some slaughter houses in Jogjakarta, Indonesia by
taking into account the different feeding of corresponding animals. The materials used for
making crackers formulation were purchased from local markets around Yogyakarta, Indonesia.
All solvents used for analysis were of pro analytical grade.

Preparation of “Rambak” Cracker

“Rambak” crackers were prepared by fresh skin from slaughter houses such as cow, buffalo, and
pig. The skin used was cleaned before frying. Skin was soaked overnight with a composition of
1 kg skin, 0.4 kg CaCO3, and 5 L water. The function of soaking is to make the hide swell, which,
in turn, is easier to remove the hair. After soaking is complete, the skin is washed by running
water until it is clean with no flavor. Subsequently, the skin is boiled at 100°C for 2 h. After that,
the skin is cut into smaller sizes and steamed with flavor for 1 h. The skin is dried using sunlight
for 2–3 days. The final product is ready to begin the frying process.
2720 ERWANTO ET AL.

Extraction of Lipid Fraction

“Rambak” crackers, either prepared from a laboratory or from commercial samples, were further
subjected to Soxhlet extraction. The extraction process involved the use of hexane as an extracting
solvent as described by Association of Official Analytical Chemists (AOAC).[23] The lipid
fraction yielded was further used for FTIR spectral measurement.

Preparation of Calibration and Validation Samples

The calibration sets were made by preparing pig skin and cow skin in “rambak” crackers with
different concentration of pig skin, namely 10, 20, 30, 40, 50, 60, 70, 80, and 90%. “Rambak”
crackers containing 100% pig skin and 100% cow skin were also prepared in order to observe
FTIR spectra differentiation. For validation or prediction models, another series of “rambak”
crackers prepared from the blending of pig skin and cow skin were made. The “rambak” crackers
were further subjected to Soxhlet extraction. The lipid fraction obtained was scanned using a
FTIR spectrophotometer. The spectral regions where the variations were observed were chosen for
developing the calibration model.

Analysis Using FTIR Spectroscopy

Measurement of FTIR spectra of lipid fraction extracted from calibration and validation samples
as well as commercial “rambak” crackers is performed using an ABB MB3000 FTIR spectro-
photometer (Clairet Scientific, Northampton, UK) in the mid-infrared region of 400–4000 cm−1.
This instrument is equipped with deuterated triglycine sulphate (DTGS) detector, 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 attenuated total reflec-
tance (ATR) accessory using 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.

Statistical Analysis
The quantitative analysis of lard (lipid fraction extracted from pig skin) was performed using
partial least square (PLS) calibration with the aid of Horizon MB software (Canada). The
validation samples were used to verify the calibration model. The values of root mean standard
error of calibration (RMSEC) and coefficient of determination (R2) were used as the validity
criteria for the calibration model. While, the root mean square error of prediction (RMSEP) and
R2 were used for validity criteria of validation model.[24] The classification of “rambak” crackers
made from pig skin and cow skin was carried with chemometrics of principle component analysis
(PCA) using Horizon MB software included in FTIR spectrophotometer.[25]

RESULTS AND DISCUSSIONS

FTIR Spectral Analysis

Lipid fraction obtained during Soxhlet extraction was analyzed using FTIR spectrophotometer at
mid infrared region (4000–650 cm–1). FTIR spectroscopy can be an ideal technique for analysis of
lipids, due to its property as fingerprint technique allowing an analyst to differentiate among
samples. IR spectra can be used as means for identification (qualitative analysis) and quantitative
 LARD DETECTION USING FTIR SPECTROSCOPY 2721

analysis based on Beer’s law.[15] The importance of IR spectroscopy for the qualitative analysis
comes from the much information contents obtained and the possibility to assign certain absorp-
tion bands related to the functional groups. In fats and oils, most of the peaks and shoulders of the
spectrum are attributable to specific functional groups present in fats and oils.[26]
Figure 1 revealed FTIR spectra of lipid fraction extracted from “rambak” cracker containing
100% cow skin (beef fat) and 100% pig skin (lard) at mid-infrared region (4000–650 cm–1). Both
spectra look very similar and show a typical absorption bands for common edible fats and oils.
The assignments of major peaks and shoulders were shown in Table 1. The main characteristic of

FIGURE 1 FTIR spectra of lipid fraction extracted from “rambak” cracker containing 100% cow skin (beef fat)
and 100% pig skin (lard) at mid infrared region (4000–650 cm–1).

TABLE 1
The functional groups responsible for infrared absorption of lard (extracted from “rambak” crackers contained
100% pig skin) and beef fat (extracted from “rambak” crackers contained 100% pig skin)

Assignment Wavenumbers (cm–1) Functional group responsible for IR absorption*

a 3,008 cis-olefinic C=H

b 2972 CH3 stretching asymmetric
c 2922 CH2 stretching asymmetric
d 1713 C=O carbonyl stretching
e 1460 CH2 bending
f 1417 CH rocking (bending) from cis-disubstituted alkenes
g 1378 CH3 bending
h 1227 C-O (eter) stretching
i 1162 C-O (eter) stretching
j 1,118 C-O (eter) stretching
k 1,096 C-O (eter) stretching
l 1033 C-O (eter) stretching
m 965 =CH from isolated trans-olefin
n 721 –CH2 rocking vibration

(*Vlachos et al., 2006).

2722 ERWANTO ET AL.

FTIR spectra is its fingerprint technique, as a consequence, FTIR spectra can be used as a means
for the differentiation of fats and oils.[27] Upon a closer scrutiny, the peaks at fingerprint regions
(1500–6650 cm–1) showed minor differences (peak heights), especially at wavenumbers of 1118
and 1096 cm–1 (assigned with j and k in Fig. 1) corresponding to the vibrations of C-H bending
and C-H deformation of fatty acids, respectively. Figure 2 showed the enlarged FTIR spectra at
fingerprint regions. The different peaks in terms of peak intensity was used as a means for
selecting the spectral regions for the quantification and classification of lard in “rambak” crackers
samples. Similar result also has been shown in our previous research that lard had an approximate
equal proportion of saturated acyl groups and oleic acyl group which was reflected in the lard
spectra, in which peaks of 1119 and 1100 cm–1 appeared as having the same height.[28]
Based on the fingerprint technique, meaning that there is no two compounds or samples having
the same spectra in terms of amount and intensity of peaks, FTIR spectroscopy can be used to
extract a difference among these fats. Upon a closer scrutiny, the minor differences (peak heights)
can be attainable at 1117 and 1097 cm−1 (l and m) corresponding to C–H bending vibration and
C–H deformation vibrations of fatty acids, respectively. Hence, these frequencies, which FTIR
spectra variations were observed, are used as a basis for choosing the spectral regions in the
quantification of pork fat in “rambak” crackers samples.[19]

Quantification of Lard in “Rambak” Crackers

Quantification of lard (lipid obtained from “rambak” crackers containing pig skin) in calibration
and validation samples is performed with the aid of multivariate calibration of PLS. Some
wavenumbers are optimized in order to find the optimum wavenumbers offering good correlation
between actual value of lard and FTIR predicted value. Finally, we used wavenumbers region of
1200–1000 cm–1 for quantification of lard due to its capability to offer the best prediction model
for the relationship between actual value of lard and FTIR predicted values. Besides, this
wavenumber also offer the highest coefficient of determination (R2) and the lowest values of
errors in calibration (RMSEC) and prediction (RMSEP).

FIGURE 2 The enlarged FTIR spectra at fingerprint regions (1500–650 cm–1) used for selecting wavenumbers for
quantitative analysis and classification of lard in “rambak” crackers.
 LARD DETECTION USING FTIR SPECTROSCOPY 2723

Figure 3 exhibited the calibration model for the relationship between actual value of lard
(x-axis) and FTIR predicted value (y-axis), as determined using multivariate calibration of
PLS using normal spectra at wavenumbers of 1200–1000 cm–1. The coefficient of determina-
tion (R2) obtained is high, i.e., 0.946 meaning that the calibration models can describe the
accuracy of 94.6%. In addition, the calibration error expressed with RMSEC is low (2.77%).
The calibration model was further evaluated using validation or validation samples. The
values of R2 (0.997) and RMSEP (2.77%) were obtained. From this result, it is obvious
that FTIR spectroscopy combined with multivariate calibration of PLS provide the accurate
and precise results with high R2 values and low errors (RMSEC and RMSEP values) for
analysis of lard in “rambak” crackers.

Classification of “Rambak” Crackers with Pig and Cow Skin

The chemometrics of PCA was used as means for the classification of “rambak” crackers with
pig skin and cow skin. The wavenumber regions for PCA were also optimized based on its
capability to separate between pig skin and pig cow present in “rambak” crackers. Finally, the
wavenumbers used for quantitative analysis (1200–1000 cm–1), was chosen for PCA. Figure 4
revealed the score plot of PCA of pig skin, cow skin contained in “rambak”crackers, as well
as commercial “rambak” crackers, describing the projection of samples defined by the first
principle component (PC 1) and the second principle component (PC 2). Using this projection,
“rambak” crackers containing pig skin, cow skin, and commercial “rambak” crackers are well
separated. This means that PCA can accomplish the classification among them. Based on this
profile, it can be stated that commercial samples (region B) do not contain pig skin in the
products.

FIGURE 3 The relationship between actual value (x-axis) and FTIR predicted value (y-axis) of lard, obtained
from “rambak” crackers containg pig skin at wavenumbers 1200–1000 cm–1 with the aid of multivariate calibration
of partial least square.
2724 ERWANTO ET AL.

FIGURE 4 The Score plot of first principal components (PC1) and second principal components (PC2) of
“rambak” crackers made from 100% cow skin (A) 100% pig skin (B), and selected commercial samples (Sa, Sb).

CONCLUSIONS

The non-destructive technique for the detection and quantification of pig skin adulteration in
commercial “rambak” crackers based on lard analysis could be developed using FTIR spectro-
scopy combined with PLS (for quantification) and PCA (for classification). PLS calibration model
revealed good calibration and validation models as indicated by high value of R2 and low value of
RMSEC and RMSEP. Furthermore, PCA using absorbance at wavenumbers 1200–1000 cm–1 is
capable of classification among samples as illustrated by clear separation among the evaluated
samples.

FUNDING

This research was supported by project grant from the directorate of higher education, Ministry of
Higher Education and Culture, with Contract No. 159/LPPM/2015.

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