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DOI 10.1007/s11694-016-9402-4
ORIGINAL PApER
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
Yahya Al-Degs
yahya@hu.edu.jo
1
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
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
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
0.50
1.50
2.50
3.50
4.50
5
15
25
35
45
(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].
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.
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
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.
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
13
M. Abu-Ghoush et al.
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
13
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.
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)
( ) (T y )
LOQ = 10 / s k *
P = Tt T
Q = Tt T
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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