Food Measure

DOI 10.1007/s11694-016-9402-4

ORIGINAL PApER

Application of mid-infrared spectroscopy and PLS-Kernel

calibration for quick detection of pork in higher value meat mixes
MahmoudAbu-Ghoush1 IsmailFasfous2 YahyaAl-Degs2 MuradAl-Holy1
Ayman A.Issa2 Anas Y.Al-Reyahi3 Abdel AzizAlshathri4

Received: 5 January 2016 / Accepted: 23 August 2016

Springer Science+Business Media New York 2016

Abstract Analytical methods for meat-species identification and detection of adulteration are always needed for quality control and the safety of consumers. A novel application
of PLS-Kernel with MIR for quick detection of pork in beef
mixes down to 1.4wt% is outlined. PLS-Kernel algorithm
showed an excellent performance for handling many variables spectral data, which makes it suitable for food analysis
by IR. Raw spectral data indicated a nonlinear relationship
between pork level and IR band intensities of proteins and
fats in the mixes. The ratios A
( amid-I ) / A1745 cm1
1654 cm 1

(A

+ A1450 cm1 / A1175 cm1

were correlated with

pork level to establish a useful analytical signal for detection purposes. Chemometric analysis was carried out on
9001900cm1 as an informative spectral range for protein.
PLS-Kernel model is recommended over PCR and PLS for
handling many variables in IR data due to the lower computation cycles and less-memory storage. The proposed
method would replace advanced techniques for quick pork
detection in minced meat mixes.
1395 cm 1

(C=O band of ester), A

( amid-II ) / A1745 cm1 , and
154 0 cm 1

Yahya Al-Degs
yahya@hu.edu.jo
1

Faculty of Allied Health Sciences, Department of Clinical

Nutrition and Dietetics, Hashemite University, Zarqa, Jordan

Faculty of Sciences, Department of Chemistry, Hashemite

University, Zarqa, Jordan

Preparatory Year Deanship, Prince Sattam bin Abdulaziz

University, Al-Kharj, SaudiArabia

College of Agricultural and Food Sciences, Food Science

Department, King Faisal University, Al-Hofuf, SaudiArabia

13

M. Abu-Ghoush et al.

Graphical abstract

Fresh beef
(local price 8$/kg)

Fresh pork
(local price 4$/kg)

Beef is often mixed with low-price meat like pork for more commercial
benefits. Pork adulteration is possible in highly consumed meatballs or minced
meat as reported in the current literature

Minced beef containing 20%wt pork Sensitive analytical

techniques are often needed to detect pork adulteration

Meat-species detection often requires

advanced DNA-based or
chromatographic-based methodologies

Expensive and lengthy procedures with

modest detection limit in certain matrices

Keywords Minced meat Meat-pork adulteration

PLS-Kernel Clustering Food surveillance

Introduction
Constant vigilance of food is an important issue for food
dealers and consumers. Assessment of food authenticity
and detection of adulteration are essential tasks for food
and nutrition scientists. Moreover, awareness of consumers
towards processed and fast food is increasing as consumers often need to know the basic ingredients of the product
[1]. Pig derivatives including lard and pork are prohibited to

13

Another practical alternative is coupling

tremendous IR data with PLS calibration for
detection of restructured meat mixes

Application of MIR spectroscopy and PLS-Kernel

calibration for quick pork detection down to 1.4
wt% is outlined in this work

be consumed by Muslims and Jews. Derivatives of pig are

less costly than other animals like lamb and beef and this is
largely attributed to the lower feeding and raising costs of
pigs [1]. For this reason, adding pork to beef or lamb meats is
a plausible act of adulteration [1, 2]. Adding meat of kangaroo (a non domestic animal) to beef meat was also reported
[3]. Lard is also employed as adulterant in much expensive
edible oils [2]. Moreover, pork meat has lower protein content than camel, sheep, and buffalo meats and this is a good
commercial reason for mixing pork with other meats [4]. In
fact, there is a large demand on halal food. Halal means
not prohibited food from religious standpoint [1]. The international trade in halal meat is estimated to be 150billion
dollars in 2010 [5]. European Commission (EC) legislation

Application of mid-infrared spectroscopy and PLS-Kernel calibration for quick detection of pork in
Table 1 Design of calibration and validation sets used in the study
Calibration set
Beef (g)
10.00a
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0b

Validation set

Pork (g)

Pork level
(wt%)

0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00

0
10
20
30
40
50
60
70
80
90

10.00

100

Beef (g) Pork (g) Pork level

(wt%)
9.50
8.50
7.50
6.50
5.50

0.50
1.50
2.50
3.50
4.50

5
15
25
35
45

Masses were recorded to the nearest 0.01g using electronic balance

Pure beef used as control sample in the study

Pure pork used as control sample in the study

(178/2002) on food safety confirms clearly that each stakeholder in a food supply chain must be able to detect all ingredients making up the foodstuffs [6].
From religious and marketing standpoints on halal food
industry, it is essential to develop fast, sensitive, and accurate analytical methods to detect any meat adulteration. The
reported analytical methods employed for pork adulteration
detection are Fourier transform infrared (FTIR) spectroscopy [7, 8], separation-based techniques [9, 10], electronic
nose [11], and the less frequently used differential scanning
calorimetry [12]. Examination of muscle extracts using
electrophoretic, immunological, and DNA-based procedures have manifested good success to detect pork in other
valuable meats [13].

IR spectroscopic methods (including near infrared (NIR)

400012,500cm1 and mid infrared (MIR) 4004000cm1)
are preferable over other methods due to their rapidness,
low cost, affordability in most laboratories, and noninvasive
procedure [1, 13, 14]. Downey and co-workers have tested
NIR and MIR spectroscopic methods for quick detection of
different meat mixes with minimum experimental efforts
[15]. However, the sensitivity and the selectivity of spectroscopic methods have significantly improved when coupled
with multivariate calibration [1]. Accordingly, nonlinear
iterative partial least squares was used to extract the proper
analytical information from MIR and NIR data for accurate
detection of pork and lard in different food matrices [1].
Kurniawati and coworkers have combined MIR data (1000
1200cm1) with partial least squares (PLS) and principal
components regression (PCR) for accurate determination of
lard in meatballs [16]. In a similar study, Nurrulhidayah and
co-workers have applied PLS for detection of beef fat in
high value foodstuff [17]. Beside calibration, principal competent analysis (PCA) and other clustering methods were
also used for clustering of different meat mixes artificially
adulterated with pork [18].
Modern FTIR instruments can generate large number of
signals for the sample; accordingly, the large X matrices (i.e.,
explanatory variables matrices) become larger in size which
needs more advanced variants of partial least squares PLS to
end up with accurate results with short computation time and
less-storage-capacity [19]. In fact, nonlinear iterative partial
least squares NIPALS is the most adopted algorithm for pork
and lard detection in different food matrices [1, 6, 8]. There
are many variants of PLS that have no practical applications
in food analysis or even food authentication studies including
Kernel, SIMPLS, and Bidiagonalization variants. Generally,
the earlier algorithms are different in their mechanisms for
running chemical analysis. For example, NIPALS is suitable

Fig. 1 MIR spectra of beef,

pork, 10% pork mix, 50%
pork mix, and 90% pork mix
plotted over the spectral range
9001900cm1 (IR spectra
of the rest of mixes were not
included for more clarity)

13

for modeling many variables-X but it requires long computational time and more memory-storage [1921]. However,
SIMPLS may be faster than NIPALS but it is not suitable for
many variables-X matrices. On the other hand, Kernel-PLS
is considered as an adjustable algorithm which can fit systems of many variables or even many objects (samples) by
creating condensed/smaller matrices [22, 23].
The main aim of this work was to assess the resolving
power of PLS-Kernel method for handling complex and nonlinear Mid-IR data generated for beef adulterated with different levels of pork (590wt%). The clustering efficiency of
Kernel algorithm for adulterated samples is also manifested.
Calibration performance of PLS-Kernel against commonly
used NIPALS is outlined and discussed.

Materials and methods

Samples conditioning for IR measurements
Beef and pork meats were randomly purchased from different meat stores. The meats had the same cut which was taken
from the leg of the animal. The meats were offered as fresh
products to the consumers as claimed on the external label.
The meats were dried by a tissue to remove external blood
residues and other liquids. All fats were carefully removed
before preparing mixes. The samples were minced and
homogenized. The mixes were prepared by adding variable
amounts of pork to beef to a final level of 590wt%. The
mixtures were mixed in electric grinder and homogenized.
The samples were dried as a thin layer (about 0.5cm thickness) in Petri dishes before IR measurements. In all cases,
each mix is divided into three identical portions in preparation for IR measurements and the average of measurements
was reported.
Calibration and validation sets created for multivariate
calibration
Calibration and validation sets were carefully designed to
end up with accurate and reliable results. The compositions
of both sets are shown in Table1.
As shown in Table1, different levels of pork were considered (1090wt%) in calibration set and (545wt%) in
validation set. The variation in pork level is necessary to
detect the spectral features over mixes. Based on our preliminary IR tests, the spectra of beef and 1% pork mix were
almost identical. Accordingly, detection of 1% adulteration
is not possible by IR technique. For validation set, five mixes
were provided with pork levels not included in calibration
set and this is necessary to check up the performance of the
proposed analytical method.

13

M. Abu-Ghoush et al.

Spectral measurements
PerkinElmer Dynascan Interferometer AVI (USA) was
used to measure IR spectra of samples over the range
4004000cm1. Samples were placed in a small crystal
cup (50mm in diameter and 10mm in depth) and sealed
before scanning. The temperature of sample holder was
controlled at 25.0C. Spectral data were provided in absorbance or %transmittance mode. The spectra of each sample
were taken as an average of 16 successive scans and this is
accomplished in 45s. The spectra were subtracted against
background air spectrum. As mentioned earlier, three identical portions of each mix were prepared for IR measurements and the average of these replicates was reported. The
resolution of IR data was set at 2.0cm1, which makes 1551
data point/spectrum. At the end of each scanning, the cup
was carefully cleaned with a detergent, washed with distilled water, dried with lint-free tissue, cleaned with ethanol,
and finally dried with lint-free tissue. Due to their importance in the test, five identical portions were prepared from
controlled samples (beef and pork) and scanned as outlined
earlier.
From IR data of samples, the absorbance ratios of the
bands A1654 cm1 / A1745 cm1 , A1540 cm1 / A1745 cm1 , and

(A

1395 cm 1

+ A1450 cm1 / A1175 cm1 were obtained using the

base-line method. Spectral range 9001900cm1 was used

to run multivariate calibration methods.
Multivariate calibration, samples clustering and figures
of merit
Initially, IR data were exported from the instrument as
Excel files. All numerical analysis was carried out using
Matlab (The Language of Technical Computing, Mathworks Company, USA). Before running PLS-Kernel as outlined in Sect.Materials and methods, dependent variables
(IR data) were placed in Xcal (11501) matrix and independent variables (pork levels) were placed in ycal (111) vector. Both Xcal and ycal were used to train the model and to
find calibration vector b as outlined in Sect.Materials and
methods. For comparison purposes, the same calibration
(11 samples) and validation (5 samples) sets were employed
to train and testify NIPALS algorithm as outlined in literature [21]. The overall performance of the models for pork
detection was assessed by estimating relative error of prediction [24]:
1/ 2

n (y
yi , nomi ) 2

i =1 i , pred

REP % = 100
n
2

y
(
)
i =1 i,nomi

Application of mid-infrared spectroscopy and PLS-Kernel calibration for quick detection of pork in

where ypred, ynomi and n are predicted pork level (%), nominal
pork level (%) and number of samples in the set.
The theoretical background of PLS-Kernel, clustering of
samples, and net-analyte signal NAS calculations were provided in Appendix.

Results and discussion

IR spectra of pork-beef mixes and the importance of
multivariate calibration
IR spectra of controlled meats and some mixes are shown in
Fig.1. The spectra of other mixes were removed for more
clarity.
Visual inspection of the spectra revealed that beef has
the following IR bands: 1745 (short), 1654 (strong), 1540
(strong), 1450 (medium), 1395 (medium), 1240 (short),1175
(broad/short) and 1083 (short) cm1. IR data indicated that
beef has high portion content with trace amounts of fats or
lipids. The low intensity of 1745cm1 (stretching vibration
of C=O bond in ester) and 1175cm1 (stretching vibration
of CO bond in ester) is an evidence of low fat content as
studied in beef samples [13, 25, 26]. The content of nucleic
acids is higher in beef compared to pork as inferred from
the higher intensities of 1240 and 1083cm1 band, which
accounted for the symmetric and antisymmetric vibrations
of PO2 in nucleic acids [25, 26].
The high intensity of 1654cm1 (C=Ostretching vibration in peptide bondamid I) and 1540cm1 (NH stretching vibration in peptide bondamid II) is a strong evidence
of the high protein content in beef. The bands appeared at
1450 and 1395cm1 are due to symmetric and antisymmetric vibrations of CH bond in protein [13, 25]. The following characteristic IR bands were observed for controlled
pork sample: 1745 (medium), 1654 (strong), 1540 (strong),
1450 (short), 1395 (short), 1240 (short), 1175 (medium) and
1083 (short) cm1. The earlier observations confirmed that
pork sample has good protein content with reasonable level
of fat as confirmed from the higher intensities (compared
to beef) of 1745 and 1175cm1 IR bands. Compared to
controlled beef sample, pork has lower protein content as
indicated from the lower intensities of 1654cm1 (amid-I)
and 1540cm1 (amide-II) bands. Moreover, band intensities at 1745 and 1175cm1 are relatively higher than those
observed in beef indicating the higher fat content in controlled pork. The lower intensities of the bands 1395 and
1450cm1 (symmetric and antisymmetric vibrations of CH
bond in protein) appeared in pork spectrum would indicate
the lower protein content. In general, spectral features of
mixes were closer to beef at lower pork doses (<20%) and
at much higher doses; the obtained spectrum is closer to
pork.

Table 2 Influence of pork level on the ratios of different IR protein/

fat bands
Pork level (wt%)

A1654/A1745 A1540/A1745 (A1395+A1450)/A1175

0
10
20
30
40
50
60
70
80
90
100

15.3
11.4
9.0
8.0
7.0
6.0
5.2
4.8
4.0
3.5
3.1

8.6
6.5
5.4
4.5
3.8
2.9
2.5
2.3
1.7
1.6
1.6

6.4
5.2
3.8
2.8
1.8
1.1
1.0
0.9
0.8
0.7
0.7

It is important to mention that both controlled meat samples do not contain large levels of fat due to the absence
of the strong band which is often observed around 1175
1200cm1for pure fat [1, 8]. These observations strongly
indicated that protein level is higher in beef and this is in
agreement with the reported protein levels of 15.7 and 11.9%
for beef and pork, respectively [27]. Upon adding pork to
beef, the intensities of the original bands of beef have been
reduced as shown in Fig.1. At 10% pork adulteration (spectrum 2), protein content has decreased (lower intensities in
1654 and 1540cm1 bands) while fat level is not affected (no
change in 1745 and 1175cm1intensities). While increasing
the pork dosage in the mix, protein level is further reduced
and fat level became higher. For example, at 90% pork adulteration (spectrum 5 in Fig.1) band intensities of fat (1745
and 1175cm1) are comparable to those observed in controlled pork sample. Adding large amounts of pork to beef
ends up with a serious protein reduction. Theoretically, the
differences in the spectra are due to the differences in the
chemical constituents including fatty or nucleic acids concentration and distribution in both meats. It is interesting to
mention that IR spectra of beef and 1% pork mix have identical shapes and this indicated that such low level of adulteration is not detectable by IR, however, 1% adulteration is
commercially less likely.
A convenient way to measure the level of protein to fat is
to estimate the ratios of characteristics IR bands of protein to
those of fat [13]. The correlation of the estimated ratios with
pork levels is helpful to find a quantitative relationship for
detection of pork in beef mixes. The bands appeared at 1654
and 1540cm1 were assigned for protein while 1745 and
1175cm1 were assigned for fat. The ratios were estimated
using the base-line method and the final results are provided
in Table2.
The results in Table2 indicated that increasing pork dosage has ended up with a sample of lower protein content
but higher fat content compared to the original sample. For

13

example, increasing pork level from 10 to 90% has resulted

in significant reduction in protein as the ratioA1654/A1745
reduced from 11.4 to 3.5. Similar observations were obtained
using the ratio A1540/A1745, however, A1654/A1745 was more
sensitive to pork in the mix. The ratio (A1395+A1450)/A1175
also measures the relative protein to fat level as it takes into
account the stretching vibration of CH bond in protein
to CO vibration of ester bond. The results are interesting
and present a complex and nonlinear relationship between
this ratio and added pork. A large reduction in the ratio was
Fig. 2 The score plot u1 (first
score vector) and u2 (second
score vector) as obtained by
Kernel-PLS analysis

Fig. 3 Calibration vector b

furnished by Kernel-PLS and
used for pork prediction in new
samples

13

M. Abu-Ghoush et al.

observed by increasing the pork content up to 40% level and

at higher levels the ratio stabilized and slightly changed. As
noted in Fig.1, adding more pork to the mix decreases the
intensity at 1175cm1 band and increases the intensities at
1395 and 1450cm1 bands. Accordingly, detection of pork
in beef mixes is possible using IR data if a suitable relationship between pork level and any of the earlier ratios is
established. Univariate calibration, in fact, would be used to
predict pork in new samples but over certain ranges 3060%
for A1654/A1745, 2050% for A1540/A1745 and 1040% for

Application of mid-infrared spectroscopy and PLS-Kernel calibration for quick detection of pork in

(A1395+A1450)/A1175. H owever, none of the ratios is applicable to quantify pork over the studied range which extends
from 5 to 90%. For accurate pork detection, more advanced
multivariate calibration tools are needed to include all IR
data and extract the useful analytical information needed for
proper calibration. Due to the large size of calibration data
X (11501), PLS-Kernel was applied to create smaller data
matrix in preparation for numerical analysis.
Clustering of samples and earlier detection of pork
adulteration
The samples were graphically clustered by plotting the first
score vector of the samples u1 against the second one u2
as obtained from PLS-Kernel model. Initially, the model
was applied on X matrix containing all samples. Analysis
showed that only three PLS variables were needed to explain
the variation in IR data. The clustering results are shown in
Fig.2.
As shown in Fig.2 reasonable performance of the applied
model for clustering beef-pork mixes with quick detection of
pork meats in the food product. As indicated from the solid
points in the plot, the model has high performance for separation of pure beef and pure pork from each other and this
mainly attributed to the significant variations in corresponding IR signal. Furthermore, samples containing pork over the
range 1590% were also appeared in separate cluster in the
plot. The clustering performance of the method seems to be
sensitive to pork level where 5 and 10% samples appeared
as one cluster. In general, the applied method was efficient
for clustering beef, pork and mixes containing pork in large
excess of 15%. As shown in Fig.1, 1% pork adulterated
sample was miss-classified as it appeared just next to beef
sample. Kuswandi and co-worker applied linear discriminate
analysis to end up with accurate classification of meatball
Table 3 Prediction of pork in validation set by Kernel-PLS
Nominal pork level %

Kernel-PLSa

NIPALSb

5.0
15.0
25.0
35.0
45.0
Statistical indicators

5.2
14.9
25.2
34.8
45.2
REP%: 3.7
R: 0.9994

4.9
15.9
22.1
32.1
46.8
REP%:
7.1
R: 0.9822

a
Calibration conditions: Kernel matrix size 1111, spectral range
9001900cm1, 501 spectral points/sample, 3-PLS latent variables
as obtained from cross-validation technique
b

Calibration conditions: Xcal matrix size 11501, y111 spectral range

9001900cm1, 501 spectral points/sample, 5-PLS latent variables as
obtained from cross-validation technique

samples initially adulterated with pork [6]. PCA is often used

for clustering purposes [1], however, the application of PLSKernel for clustering of beef and pork-beef mixes has not
been reported in the literature.
PLS-Kernel calibration and pork prediction in
independent/validation samples
It is important to mention that the 5% pork mix was created to check up the calibration performance of PLS-Kernel
for small levels and to assess LOD and LOQ of the proposed
multivariate calibration method. It is necessary to recall that
pork adulteration is commercially feasible at levels higher
than 10% [6, 8, 11]. Initial analysis using raw IR data and
first or second derivatives of the signals indicated comparable outputs of the models and there was no need for
derivatization. Moreover, the best calibration results were
accomplished using spectral range 9001900cm1 and,
as mentioned earlier, this part of the spectrum contains the
most informative IR signals of protein. The rest parts of the
spectrum 400900 and 19004000cm1 were not included
in the calibration. In a similar study, the spectral range
10001200cm1 with PLS calibration was reported to be
efficient for accurate detection of pork in meatball [8]. The
final results of pork detection in independent or validation
samples along with NIPALS, REP%, and correlation coefficient r are all summarized in Table3.
Generally, both methods were able to predict pork down
to 5% with acceptable prediction error. PLS-Kernel outperformed common NIPALS with lower REP% value of 3.7
and 7.1, respectively. Although both methods have comparable analytical performance, PLS-Kernel is more flexible
toward large matrices as the one involved in the current case
as numerical analysis started with a small condensed 1111
matrix. On the other side, more computation time and efforts
were needed when using other models (PLS or PCR) due to
the large size of Xcal matrix (11501). As already known,
multivariate calibration is accomplished over two main steps
known as calibration and validation steps. In calibration
step, the model is trained by finding the optimum number of
factors. Each algorithm has its own steps of calculations. In
fact, more steps are needed in PLS than in PCR as concentration vector is included in the former during building the
model [19, 2224]. More calculation steps will require more
time and high storage capacity to save the generated data.
Compared to NIPALS (a common PLS variant) and PCR,
PLS-Kernel is operated with fewer calculation steps to find
calibration vector. As outlined in the manuscript, the basic
idea in PLS-Kernel is compressing the large matrix into
smaller one and this will end up with less computational time
and effort. Cross-validation test indicated a better and faster
performance of PLS-Kernel compared to NIPALS. As summarized in Table3, only three PLS variables were needed

13

to build the model and estimate calibration vector b, while,

five variables were required to run regression by NIPALS for
pork prediction in new samples. Calibration vector furnished
by PLS-Kernel is depicted in Fig.3.
As shown in Fig.3b has comparable spectral features to
pork (see Fig.1) and this is a good indication for the excellent performance of the model for extracting the proper IR
signal of pork from the complex meatmatrices. The closeness
between the generated plot and true IR of pure pork sample
is expected as multivariate calibration tools have the ability
to extract the spectral shapes of calibrated solutes beside the
quantitative relationships needed for determination of calibrated solute (pork in the current case) in new matrices.
The applicability of the proposed analytical procedure
could potentially applied for other commercial minced beef
mixes which would contain other meats including horse,
lamb and other low-value meats.
In fact, limit of detection (LOD) and limit of quantification (LOQ) are the most significant figures of merit to be
reported for a new analytical procedure. Based on net-analyte
signal NAS calculations as outlined earlier, LOD and LOQ
for the proposed IR method were 1.4 and 4.6%, respectively.
In fact, the proposed chemometric method offered a simple,
quick, and accurate methodology for detection and quantification of pork in beef mixes down to 1.4%. The reported
LOD is comparable (even better in some cases) to those seen
in the literature [1]. The best detection limit for pork in beef
was 1wt% and reported using liquid chromatography [9].

Conclusions
With minimum experimental efforts, pork is detected down
to 1.4% using MIR spectral data and calibration by PLSKernel. The relationship between pork level in pork-beef
mixes and informative IR bands was complex and nonlinear
which requires advanced multivariate calibration methods.
PLS-Kernel was efficient and fast for handling many variables X-matrix (501 variables in the current case) and reducing computation cycles compared to common NIPALS. With
REP% value of 3.7%, the model showed an excellent analytical performance for quick pork adulteration in beef mixes
over a wide range 590%. Clustering of adulterated samples
is also achieved using sample score vectors that furnished by
PLS-Kernel algorithm.

Appendix: Theoretical background

PLS-Kernel calibration
Partial least squares (PLS) is an efficient tool for developing a quantitative relationship between several predictor or

13

M. Abu-Ghoush et al.

dependent variables X (IR signals in this work) and a property of interest y (the independent variables or pork contents
in this work). Mathematically, the relationship between X
and y is given as [1921]: y=Xb, where y, X, and b are
pork nominal level (wt%) in calibration samples or mixes
arranged in a vector, the matrix of IR data of calibration samples measured at different wavenumbers, and the calibration
sensitivity which is necessary for estimating pork content in
new or uncalibrated samples. PLS is an efficient numerical
tool to find b which is often accomplished using different
variants of PLS [20, 21]. In general, the dimensions of the
earlier quantities are X (I samplesJ variables), y (I samples1), and b (J variables1). The values of I and J in this
work are 11 and 501, respectively. The most adopted algorithm in food analysis is NIPALS which available in many
commercial software packages (like MVC1 and TOMCAT).
The mechanism of NIPALS is outlined elsewhere [20]. A
brief summary on PLS-Kernel is provided for the readers
in the following section. There are two versions of Kernel
algorithm, the first one can handle matrices of many samples
compared to variables (I>>J) and the other one (which is
suitable for the current analytical system) is proposed for
many variables X-matrices where J>>I (i.e., for IR data)
[19, 22, 23]. In Kernel algorithms, condensed matrices
are created from X and y which is an essential step. In the
adopted algorithm, two condensed matrices are created XXt
and yyt. Kernel matrix is then estimated as: XXtyyt of dimension 1111. The main steps of the algorithm are [22, 23]:
1. The eigenvector of Kernel matrix is taken as the first X
score vector t1. The y score vector is then estimated as:
u1=yytt1
2. The next step is to update the association matrices by
eliminating the explained variable as following:
G1 = I t1t1t (I identity matrix )
X1X1t = G1XX t G1
y1y1t = G1yy t G1
The above three steps save us to retrain to the original
large matrices and calculation of association matrices
are necessary at the start of the algorithm only. As can
be seen, the matrices involved in Kernel algorithm
are sampler (size 1111) than the original matrix
(11501) and this ensures less computation time and
memory storage.
3. The next t and u vectors are estimated as outlined above
using the updated matrices. The calibration vector
needed to detect pork in new samples is estimated from
the weight and the loading matrices (W, P and Q) as following [19]:

Application of mid-infrared spectroscopy and PLS-Kernel calibration for quick detection of pork in

W = Xt U

LOD = 3   /  s k * 

( ) (T X)

The minimum quantifiable amount of pork in the mix is estimated as [29]:

( ) (T y )

LOQ = 10   /  s k * 

P = Tt T

Q = Tt T

 tep 3 is repeated until the optimum number of PLSS

variables is estimated using cross-validation technique
[20].
It should be mentioned that all vectors in W should be
normalized to find b:
1

b = W ( PW ) Q
Note that b vector has the dimension of 5011 which
should explain for the spectral features of pork that
extracted by PLS-Kernel. Level of pork is predicted
from unknown IR spectrum aun as following [22, 23]:
cun = aun b
Clustering of samples by PLS-Kernel algorithm
For clustering purposes, score vectors of y (i.e., u1 and u2)
were graphically plotted to detect the variations among samples. Score vectors were obtained from PLS-Kernel using all
mixes (16 samples).
Net-analyte signal calculations and figures of merit
The analytical performance of multivariate calibration is
assessed by carrying out net-analyte signal NAS calculations. NAS is a suitable method to characterize the analytical
figures of merit related to the multivariate calibration [24,
28]. For classical multivariate calibration, the basic equation
that is needed to estimate figures of merit is [24, 28, 29]:
s k * = [I S k S+ k ]s k
where S is the matrix of sensitivities collected for all other
solutes, sk is the sensitivity vector of the analyte, and sk* is
the estimated net part of the kth component that is orthogonal to the other constituents [29]. NAS is necessary to find
meaningful parameters to assess the analytical method like
limit of detection (LOD) and limit of quantification (LOQ).
In the following equations, stands for Euclidian norm of
a vector. LOD which gives the minimum detectable amount
of pork is estimated as [24, 29]:

where represents the instrumental noise which is estimated by recording five IR spectra of the blank (i.e., background air spectrum). Then the norms of blank readings
(NASblank) are estimated and is taken as the standard
deviation of the estimated norms [29].

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