International Journal of Food Properties

ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: http://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,
DOI: 10.1080/10942912.2016.1143839

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

Accepted author version posted online: 17

Feb 2016.

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 1 RUNNING HEAD: LARD DETECTION USING FTIR SPECTROSCOPY

2 USE OF FOURIER TRANSFORM INFRARED (FTIR)

3 SPECTROSCOPY AND CHEMOMETRICS FOR
4 ANALYSIS OF LARD ADULTERATION IN

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RAMBAK CRACKERS

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5

6 Yuny Erwanto1,3, Afif Turindra Muttaqien1, Sugiyono2, Sismindari3,4 and Abdul Rohman3,4

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7 Faculty of Animal Sciences, Gadjah Mada University, Yogyakarta, 55281, Indonesia

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2
8 Faculty of Veterinary Medicine, Gadjah Mada University, Bulaksumur, Yogyakarta, 55281
9 Indonesia
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10 Research Center of Halal Products, Gadjah Mada University, Yogyakarta, 55221, Indonesia.

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Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gadjah Mada University,
Yogyakarta, 55221, Indonesia.
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13 *Corresponding author.

14 email: erwantougm@gmail.com (Yuny Erwanto)

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15 Abstract

16 Rambak crackers is one of the traditional food consumed among Indonesian people made from
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17 various kinds of animal skin. The present study highlights the analysis of lard obtained from
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18 extraction of rambak crackers using FTIR spectroscopy in combination with chemometrics of

19 partial least square (PLS) and principle component analysis (PCA). FTIR spectroscopy at
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20 wavenumber regions of 1,200 1000 cm-1 was successfully used for quantification and

21 classification of lard in rambak crackers. The relationship between actual value of lard and

22 FTIR predicted value has R2 value of 0.946 with low errors in calibration and validation models.

23 Furthermore, the chemometrics PCA can be successfully used for determination of pig skin

20
 1 through analysis of lard in commercial rambak crackers. The developed method (FTIR

2 spectroscopy coupled with Chemometrics) is rapid and reliable for quantification and

3 classification of lard in rambak crackers.

4 Keywords: Rambak crackers, Lard, FTIR spectroscopy, Partial least square, Principle

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5 component analysis.

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INTRODUCTION
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7 Krupuk rambak or rambak cracker, is one of the traditional food in Indonesia made from

9
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various kinds of animal skin such as buffalo, cattle or pig skin. This food is traditionally hand

made with traditional process. One of the interesting issues in food industry is the authenticity of
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10 food products. The authenticity of food product can involve the alteration of the true labeling of

11 food ingredients, in which the high value of raw materials are substituted by cheaper materials
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12 such as substitution of cow skin with pig skin.[1] The Verification of labeled components in any

13 food products is necessary in order to prevent the adulteration practice. For this purpose, some
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[2]
14 countries make regulation for assuring that food products available are safe and authentic,
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15 especially if food products contain pig derivatives like pig skin.

16 The presence of pig derivatives such as pig skin, lard, pork and porcine gelatin in any food
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17 products is a serious matter for certain community,[3] because some religions like Islam, Judaism

18 and Hinduism forbid their followers to consume any food products containing porcine and its

19 derivatives.[4] Therefore it is necessary to evaluate the presence of these pig derivatives in any

20 products using some instrumental techniques. Such techniques used are differential scanning

21
 1 calorimetry,[5, 6]
gas chromatography-mass spectrometry,[7] high performance liquid

2 chromatography,[8, 9]
electronic nose, [10, 11]
proton nuclear magnetic resonance[12] and DNA-

3 based methods using polymerase chain reaction.[13, 14] Some of these methods are too laborious

4 and time consuming, consequently, an analytical technique offering rapid and reliable must be

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5 used. One of the promising methods suitable for routine analysis is Fourier transform infrared

6 (FTIR) spectroscopy, especially in combination with some Chemometrics techniques.

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7 The use of FTIR spectroscopy in food analysis increased significantly. This is due to its

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8 properties as fingerprint technique, which can be used for qualitative and quantitative

9 analyses.[15] Some researchers have reported the application of FTIR spectroscopy for analysis of

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pig derivatives. Previous research used FTIR spectroscopy for rapid discrimination of pork in

Halal and non-Halal Chinese ham sausages.[16] Previous research has also shown the potential
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11
[17]
12 application of FTIR spectroscopy for analysis of lard in cake formulation and chocolate
[18]
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13 products. Our group also developed FTIR spectroscopy in combination with chemometrics
[19] [20]
14 for analysis of pork in beef meatball, lard in meatball broth and rats meat in beef
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[21]
15 meatball. Differentiation of porcine and bovine gelatins was successfully determined using

16 FTIR spectroscopy. [22] Using literature review, there is no publication reporting the employment
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17 of FTIR spectroscopy for identification and quantification of crackers made from cow skin

18 adulterated with pig skin. Therefore, in this study, FTIR spectroscopy combined with
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19 Chemometrics is employed for analysis of lard in rambak crackers.

20

22
 1 MATERIALS AND METHODS

2 In order for widely application of species detection methods of commercial samples, the skin of

3 cow and pig were randomly obtained from some slaughter houses in Jogjakarta, Indonesia by

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4 taking into account the different feeding of corresponding animals. The materials used for

5 making crackers formulation were purchased from local market around Yogyakarta, Indonesia.

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6 All solvents used for analysis were of pro analytical grade.

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7 Preparation of Rambak Cracker

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Rambak crackers were prepared by fresh skin from slaughter houses such as cow, buffalo and

9 pig. Skin used to be clean before frying. Skin was soaked overnight with composition of 1 kg
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10 skin, 0.4 kg CaCO 3 , and 5 liter water. The function of soaking is to make swelling hide and

11 easily to unhairing. After soaking is complete, the skin is washed by running water until clean
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12 and no flavor. Subsequently, skin is boiled at 100oC for 2 hours. After that, skin is cut into small
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13 size and steamed with flavor until 1 hour. The skin is dried using sunlight dry for 2-3 days. The

14 final product is ready to be frying process.

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15 Extraction of Lipid Fraction

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16 Rambak crackers, either from prepared laboratory or from commercial samples, were furtther

17 subjected to Soxhlet extraction. The extraction process involved the use of hexane as an

18 extracting solvent as described by Association of Official Analytical Chemists.[23] The lipid

19 fraction yielded was further used for FTIR spectral measurement.

23
 1 Preparation of Calibration and Validation samples

2 The calibration sets were made by preparing pig skin and cow skin in rambak crackers with

3 different concentration of pig skin, namely 10, 20, 30, 40, 50, 60, 70, 80 and 90%. Rambak

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4 crackers containing of 100% pig skin and 100% cow skin was also prepared in order to observe

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5 FTIR spectra differentiation. For validation or prediction models, another series of rambak

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6 crackers prepared from the blending of pig skin and cow skin were made. The rambak crackers
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7 were further subjected to Soxhlet extraction. The lipid fraction obtained was scanned using FTIR

8 spectrophotometer. The spectral regions where the variations were observed were chosen for

9 developing calibration model. an

10 Analysis Using FTIR Spectroscopy
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11 Measurement of FTIR spectra of lipid fraction extracted from calibration and validation samples
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12 as well as commercial rambak crackers is performed using ABB MB3000 FTIR

13 spectrophotometer (Clairet Scientific, Northampton, UK) in the mid infrared region of 4004000
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14 cm1. This instrument is equipped with deuterated triglycine sulphate (DTGS) detector, with a
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15 resolution of 8 cm1 and 32 scanning. Spectra were processed using Horizon MB FTIR software

16 version 3.0.13.1 (ABB, Canada). The samples were placed in good contact with attenuated total
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17 reflectance (ATR) accessory using ZnSe crystal at controlled ambient temperature (20 oC). All

18 spectra were rationed against a background of air spectrum. After every scan, a new reference air

19 background spectrum was taken. These spectra were recorded as absorbance values at each data

20 point in triplicate.

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 1 Statistical Analysis

2 Quantitative analysis of lard (lipid fraction extracted from pig skin) was performed using partial

3 least square calibration with the aid of Horizon MB software (Canada). The validation samples

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4 were used to verify the calibration model. The values of root mean standard error of calibration

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5 (RMSEC) and coefficient of determination (R2) were used as the validity criteria for the

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6 calibration model. While, root mean square error of prediction (RMSEP) and R2 were used for
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validity criteria of validation model.[24] The classification of rambak crackers made from pig

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7

8 skin and cow skin was carried with chemometrics of principle component analysis (PCA) using

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Horizon MB software included in FTIR spectrophotometer.[25]

RESULTS AND DISCUSSIONS

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10
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11 FTIR Spectral Analysis

12 Lipid fraction obtained during Soxhlet extraction was analyzed using FTIR spectrophotometer at
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13 mid infrared region (4,000 650 cm-1). FTIR spectroscopy can be an ideal technique for analysis
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14 of lipids, due to its property as fingerprint technique allowing an analyst to differentiate among

15 samples. IR spectra can be used as means for identification (qualitative analysis) and quantitative
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16 analysis based on Beers law.[15] The importance of IR spectroscopy for the qualitative analysis

17 comes from the much information contents obtained and the possibility to assign certain

18 absorption bands related to the functional groups. In fats and oils, most of the peaks and

19 shoulders of the spectrum are attributable to specic functional groups present in fats and oils.[26]

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 1 Figure 1 revealed FTIR spectra of lipid fraction extracted from rambak cracker containing

2 100% cow skin (beef fat) and 100% pig skin (lard) at mid infrared region (4,000 650 cm-1).

3 Both spectra look very similar and show a typical absorption bands for common edible fats and

4 oils. The assignments of major peaks and shoulders were shown in Table 1. The main

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5 characteristic of FTIR spectra is its fingerprint technique, as a consequence, FTIR spectra can be

6 used as a means for the differentiation of fats and oils.[27] Upon a closer scrutiny, the peaks at

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fingerprint regions (1500 6650 cm-1) showed minor differences (peak heights), especially at
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8 wavenumbers of 1118 and 1096 cm-1 (assigned with j and k in Figure 1) corresponding to the

9 vibrations of C-H bending and C-H deformation of fatty acids, respectively. Figure 2 showed the

10

11
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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
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12 in rambak crackers samples. Similar result also has been shown in our previous research that

13 lard had approximately equal proportion of saturated acyl groups and oleic acyl group which was
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14 reflected in the lard spectra, in which peaks of 1119 and 1100 cm-1 appeared as having the same

15 height. [28]
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16 Based on the fingerprint technique, meaning that there is no two compounds or samples having
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17 the same spectra in terms of amount and intensity of peaks, FTIR spectroscopy can be used to

18 extract a difference among these fats. Upon a closer scrutiny, the minor differences (peak
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19 heights) can be attainable at 1117 and 1097 cm1 (l and m) corresponding to CH bending

20 vibration and CH deformation vibrations of fatty acids, respectively. Hence, these frequencies,

21 which FTIR spectra variations were observed, are used as a basis for choosing the spectral

22 regions in the quantification of pork fat in rambak crackers samples. [19]

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 1 Quantification of Lard in Rambak Crackers

2 Quantification of lard (lipid obtained from rambak crackers containing pig skin) in calibration

3 and validation samples is performed with the aid of multivariate calibration of Partial least

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4 square (PLS). Some wavenumbers are optimized in order to find the optimum wavenumbers

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5 offering good correlation between actual value of lard and FTIR predicted value. Finally, we

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6 used wavenumbers region of 1,200 1,000 cm-1 for quantification of lard due to its capability to
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7 offer the best prediction model for the relationship between actual value of lard and FTIR

8 predicted values. Besides, this wavenumber also offer the highest coefficient of determination

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(R2) and the lowest values of errors in calibration (RMSEC) and prediction (RMSEP).

10 Figure 3 exhibited the calibration model for the relationship between actual value of lard (x-axis)
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11 and FTIR predicted value (y-axis), as determined using multivariate calibration of PLS using

12 normal spectra at wavenumbers of 1,200 1,000 cm-1. The coefficient of determination (R2)
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13 obtained is high, i.e. 0.946 meaning that the calibration models can describe the accuracy of

14 94.6%. In addition, the calibration error expressed with root mean square error of calibration
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15 (RMSEC) is low (2.77%). The calibration model was further evaluated using validation or
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16 validation samples. The values of R2 (0.997) and root mean square error of prediction (2.77%)

17 were obtained. From this result, it is obvious that FTIR spectroscopy combined with multivariate
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18 calibration of PLS provide the accurate and precise results with high R2 values and low errors

19 (RMSEC and RMSEP values) for analysis of lard in rambak crackers.

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 1 Classification of Rambak Crackers with Pig and Cow Skin

2 The chemometrics of principal component analysis (PCA) was used as means for the

3 classification of rambak crackers with pig skin and cow skin. The wavenumber regions for

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4 PCA were also optimized based on its capability to separate between pig skin and pig cow

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5 present in rambak crackers. Finally, the wavenumbers used for quantitative analysis (1200

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6 1000 cm-1), was chosen for PCA. Figure 4 revealed the score plot of PCA of pig skin, cow skin
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7 contained in rambakcrackers, as well as commercial rambak crackers, describing the

8 projection of samples dened by the rst principle component (PC 1) and the second principle

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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
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11 classication among them. Based on this profile, it can be stated that commercial samples

12 (region B) do not contain pig skin in the products.

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13 CONCLUSIONS
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14 Nondestructive technique for the detection and quantification of pig skin adulteration in
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15 commercial rambak crackers based on lard analysis could be developed using fourier

16 transform infrared (FTIR) spectroscopy combined with partial least square (for quantification)
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17 and principle component analysis (PCA) (for classification). PLS calibration model revealed

18 good calibration and validation models as indicated by high value of R2 and low value of

19 RMSEC and RMSEP. Furthermore, PCA using absorbance at wavenumbers 1200-1000 cm-1 is

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 1 capable of classification among samples as illustrated by clear separation among the evaluated

2 samples.

3 ACKNOWLEDGEMENT

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4 This research was supported by project grant from the directorate of higher education, Ministry

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5 of Higher Education and Culture, with Contract No. 159/LPPM/2015.
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 1 Rohman. Differentiation of lard from other edible oils by means of Fourier transform infrared

2 spectroscopy and chemometrics. Journal of the American Oil Chemists' Society 2011, 88,187

3 192.

4 .

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 1 Figure 1. FTIR spectra of lipid fraction extracted from rambak cracker containing 100% cow
2 skin (beef fat) and 100% pig skin (lard) at mid infrared region (4,000 650 cm-1).

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 1 Figure 2. The enlarged FTIR spectra at fingerprint regions (1,500 650 cm-1) used for selecting
2 wavenumbers for quantitative analysis and classification of lard in rambak crackers.

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 1 Figure 3. The relationship between actual value (x-axis) and FTIR predicted value (y-axis) of
2 lard, obtained from rambak crackers containg pig skin at wavenumbers 1,200 1000 cm-1
3 with the aid of multivariate calibration of partial least square.

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

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 1 Table 1. The functional groups responsible for infrared absorption of lard (extracted from
2 rambak crackers contained 100% pig skin) and beef fat (extracted from rambak crackers
3 contained 100% pig skin)

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Assignment Wavenumbers Functional group responsible for IR

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(cm-1) absorption*

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a 3,008 cis-olenic C=H

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b 2972
an CH 3 stretching asymmetric
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c 2922 CH 2 stretching asymmetric
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d 1713 C=O carbonyl stretching

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e 1460 CH 2 bending
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f 1417 CH rocking (bending) from cis-

disubstituted alkenes
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g 1378 CH 3 bending

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 h 1227 C-O (eter) stretching

i 1162 C-O (eter) stretching

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j 1,118 C-O (eter) stretching

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k 1,096 C-O (eter) stretching

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l 1033 C-O (eter) stretching

m 965
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=CH from isolated trans-olen
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n 721 CH 2 rocking vibration
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1 (*Vlachos et al., 2006).

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