Journal of Food Engineering 169 (2016) 309e320

Contents lists available at ScienceDirect

Journal of Food Engineering

journal homepage: www.elsevier.com/locate/jfoodeng

Three dimensional chemometric analyses of hyperspectral images for

beef tenderness forecasting
Govindarajan Konda Naganathan a, Kim Cluff a, Ashok Samal b, Chris R. Calkins c,
David D. Jones a, George E. Meyer a, Jeyamkondan Subbiah a, d, *
a
Biological Systems Engineering, University of Nebraska, Lincoln, NE 68583-0726, USA
b
Computer Science and Engineering, University of Nebraska, Lincoln, NE 68588-0115, USA
c
Animal Science, University of Nebraska, Lincoln, NE 68583-0908, USA
d
Food Science and Technology, University of Nebraska, Lincoln, NE 68583-0726, USA

a r t i c l e i n f o a b s t r a c t

Article history: A prototype on-line hyperspectral imaging system (l ¼ 400e1000 nm) was developed and used to ac-
Received 7 June 2015 quire images of exposed ribeye muscle on hanging beef carcasses (n ¼ 274) at 2-day postmortem in a
Received in revised form commercial beef packing plant. After image acquisition, a strip steak was cut from each carcass and
5 August 2015
vacuum packaged. After aging for 14 days, the steaks were cooked and Warner-Bratzler shear force
Accepted 3 September 2015
values were collected as a measure of tenderness. Four different principal component analysis-based
Available online 6 September 2015
dimensionality reduction methods were implemented to reduce information redundancy in beef
hyperspectral images. Textural features extracted from the 2-day hyperspectral images were modeled
Keywords:
Instrument grading
using Fisher's linear discriminant (FLD), support vector machines (SVM), and decision tree (DT) models to
Principal component analysis predict 14-day aged, cooked beef tenderness. Based on a true validation procedure using 101 samples,
Partial least squares analysis the FLD model yielded a tender certification accuracy of 86.7%. In addition, wavelengths corresponding to
Fisher's linear discriminant model myoglobin and its derivatives (541, 577, and 635 nm), beef aging (541, 577, 635, 756, and 980 nm), protein
Support vector machines (910 nm), fat (928 nm), and water (739, 756, and 988 nm) were identified.
Decision tree © 2015 Elsevier Ltd. All rights reserved.

1. Introduction
1.1. Beef tenderness
Beef tenderness is an important palatability attribute strongly
related to consumer satisfaction. In a number of studies, it has been
shown that consumers can discern tenderness variations and a
significant portion of them is willing to pay a premium for guar-
anteed tender products (Feuz et al., 2004; Lusk et al., 1999; 2001;
Shackelford et al., 2001). The United Stated Department of Agri-
culture (USDA) has established beef grading standards to classify
beef carcasses into quality grades such as Prime, Choice, and
Standard (USDA, 1997). The grading system is based primarily on
the degree of marbling, which is shown to have only weak corre-
lation with tenderness (Jeremiah, 1996; Wheeler et al., 1994). In
addition, based on the National Beef Quality Audit, 90% of the
* Corresponding author. Kenneth E. Morrison Distinguished Professor of Food
Engineering, 212 L.W. Chase Hall, University of Nebraska, Lincoln, NE 68583-0726,
USA.
E-mail address: jeyam.subbiah@unl.edu (J. Subbiah).
carcasses fall into two quality grades namely USDA Choice and
Select (McKenna et al., 2002). Within this narrow range, USDA
quality grading has been found ineffective in assessing tenderness
variations. George et al. (1999) reported the odds of obtaining a
slightly tough or tougher rating for supermarket beef was 20e25%
for Choice and Select-grade strip steaks, respectively.
In the U.S. beef marketing system, beef products leave the
packing plants about two to three days after animal slaughter and
presented to consumers in about 24 days at retail and 30 days at
food service facilities (Savell et al., 2006). The time between animal
slaughter and consumer presentation is called the “storage” or
“aging” period, during which several biochemical processes occur
within beef muscle that improves tenderness. A significant portion
of this improvement in tenderness occurs within 14 days after
slaughter. Also, tenderness is a property of cooked meat. Therefore,
the beef industry needs a non-destructive technique that can scan
fresh beef at 2 to 3-day postmortem and forecast the subsequent
14-day cooked-beef tenderness. A technology to nondestructively
predict beef tenderness in a real-time packing plant environment is
not yet available (Rosenvold et al., 2009; Yancey et al., 2010).

http://dx.doi.org/10.1016/j.jfoodeng.2015.09.001
0260-8774/© 2015 Elsevier Ltd. All rights reserved.
310 G. Konda Naganathan et al. / Journal of Food Engineering 169 (2016) 309e320
1.2. Hyperspectral imaging (HSI)
A number of technologies such as colorimeter (Wyle et al.,
1999), video image analysis (Howard et al., 2010; Subbiah, 2004;
Vote et al., 2003; Wyle et al., 2003), spectroscopy (Mitsumoto
et al., 1991; Rosenvold et al., 2009; Rust et al., 2008; Shackelford
et al., 2005; Yancey et al., 2010), hyperspectral imaging (ElMasry
et al., 2012; Konda Naganathan et al., 2008a; Konda Naganathan
et al., 2008b; Konda Naganathan, 2011), and hyperspectral scat-
tering (Cluff et al., 2008; Wu et al., 2012; Xia et al., 2007), have been
implemented for beef tenderness prediction. Tenderness in beef is
related to two major components: muscle structure and biochem-
ical activity (Webb et al., 1964). The video image analysis systems
can only capture beef muscle structure information because they
have a high spatial resolution. Similarly, the spectroscopic systems
can capture only beef biochemical information because they have a
high spectral resolution. In contrast, hyperspectral imaging (HSI)
has the ability to capture both the structural and biochemical in-
formation because it is a combination of video image analysis and
spectroscopy and has high spatial and spectral resolution. Hence,
the likelihood of accurately predicting beef tenderness with
hyperspectral imaging could be much greater.
1.3. Hyperspectral image analysis challenges
Hyperspectral image analysis for beef tenderness prediction in-
cludes the following steps: image calibration, dimensionality
reduction, textural feature extraction, and development of tender-
ness prediction models (Subbiah et al., 2014). The dimensionality
reduction is a critical step because of the huge amount of data stored
in a hyperspectral image and presence of data redundancy or multi-
collinearity. Similarly, identifying image features that can reliably
discriminate tenderness, developing robust discriminant models,
and imbalanced proportion of tender to tough samples and vice versa
in the dataset are a few of the challenges in the tenderness prediction
model development process. These challenges are explained in the
subsequent sections. This study addresses these challenges by eval-
uating different dimensionality reduction methods and discriminant
models in the context of beef tenderness forecasting.
1.3.1. Dimensionality reduction
A typical hyperspectral image of a beef sample is shown in
Fig. 1a. It is a three dimensional data cube with two spatial axes (X
and Y) and a spectral or wavelength axis (l). The hyperspectral
image is a collection of l number of gray level images or wave-
length bands, each of size X  Y. It can also be considered as a
collection of X  Y number of spectra, each of size 1  l. A typical
beef reflectance spectrum of size 1  l is shown in Fig. 1b. In a
typical hyperspectral image, the spectral axis contains hundreds of
wavelength bands sampled at narrow intervals. The presence of
hundreds of wavelength bands increases the size of a hyperspectral
image, while the narrow sampling increases the correlation be-
tween neighboring bands leading to data redundancy or multi-
collinearity between bands.
Principal component analysis (PCA) is the most commonly used
chemometric method to reduce the dimensionality of one dimen-
sional spectral data such as the one shown in Fig. 1b. In the one
dimensional PCA, a set of N spectra, each of size 1  l, is first
collected from N different beef samples and PCA is conducted on
the original dataset of size N  l. The PCA procedure transforms the
correlated original data to uncorrelated data called PCA scores,
which are linear combinations or weightings of the original data
(Cluff et al., 2013; Johnson, 1998). With the transformed data or PCA
scores, the original data can be expressed in fewer dimensions. In
other words, the original data of size N  l is transformed to PCA
scores of size N  l, where l << l. In addition to transforming the
original data, the PCA methods output two parameters called Eigen
values and Eigen vectors or loading vectors that are very useful in
interpreting the results of the chemometric analysis. The Eigen
values provide the variation explained by each PCA band, whereas
the Eigen or loading vectors provide the weighting function used to
obtain the PCA scores.
The biggest challenge in translating the one dimensional PCA to
three dimensional PCA for hyperspectral images is the data size. In
the three dimensional PCA, the data size is N  X  Y  l. The one
dimensional dataset is the equivalent of the three dimensional
hyperspectral image data without the spatial information X and Y.
Considering a value of 100 for both X and Y, the three dimensional
dataset (hyperspectral image) is 10,000 times bigger than the one
dimensional dataset (spectral data). Even a single hyperspectral
image of size X  Y  l representing a single sample can be bigger
than an entire one dimensional dataset of size N  l representing N
samples. Because of this reason, a single hyperspectral image can be
perceived as a dataset and PCA can be implemented on individual
hyperspectral images separately. The Eigen values and vectors of
such PCA procedure are different for each sample. Therefore,
interpretation of the PCA results becomes a challenge because there
is no baseline. In this study, the challenges associated with imple-
menting three dimensional PCA on hyperspectral images were
addressed in the context of beef tenderness prediction.
1.3.2. Discriminant models
Even after reducing the dimensionality of the hyperspectral
images with chemometric methods, the textural feature extraction
step creates a large number of image features that are input to
discriminant models to provide a binary output: tender or tough.
Large number of features in comparison to the number of samples,
correlation among the features, and imbalanced data (dispropor-
tionate mix of tender and tough samples) are a few challenges in
developing a robust tenderness discriminant model. Also, the
relationship between the image features to the outcome may be
linear or non-linear. In this study, these challenges were addressed
by selecting a few significant image features and using them in
different types of discriminant models.
1.4. Objectives
The objectives of this study were to (1) implement four different
chemometric dimensionality reduction methods, three mutations
of PCA and partial least squares analysis (PLSA), for three dimen-
sional hyperspectral image data and evaluate them for beef
tenderness prediction and (2) compare three discriminant models
namely Fisher's linear discriminant (FLD) model, support vector
machines (SVM) model, and decision tree (DT) model, for their
ability to discriminate beef samples into two tenderness categories:
tender and tough.
2. Materials and methods
2.1. Prototype on-line spectrograph hyperspectral image acquisition
system
A prototype on-line spectrograph HSI system (Fig. 2) was
developed for collecting reflectance images of ribeye muscle
(longissimus dorsi) on hanging beef carcasses in beef packing plants.
The system consisted of a 12-bit line scan camera (Model: MV1-
D1312-160-CL-12, Photonfocus, Lachen, Switzerland) with a pixel
resolution of 1312  1082, a spectrograph (V10E, Specim Spectral
Imaging LTD., Oulu, Finland) sensitive in the visible-near infrared
region (VNIR) ranging from 400 to 1000 nm, 17 mm focal length lens
 G. Konda Naganathan et al. / Journal of Food Engineering 169 (2016) 309e320 311

Fig. 1. Three dimensional hyperspectral image cube (X  Y  l) and one dimensional reflectance spectrum (1  l) of beef ribeye muscle.

Fig. 2. Prototype on-line spectrograph hyperspectral image acquisition system and its use in acquiring hyperspectral images of beef ribeye muscle on a hanging beef carcass. Parts:
(1) camera, (2) spectrograph, (3) lens, (4) mirror scanner assembly, (5) lighting dome, (6) locating plates, and (7) handle.

(XENOPLAN 1.4/17-0903, Schneider Optics, Kreuznach, Germany), a
mirror scanner assembly, and a computer. The line scan camera
scans a single spatial line of an object and the spectrograph dis-
perses the line into two dimensional focal plane array image with a
spatial and spectral axis. To obtain a three dimensional hyper-
spectral image of an object, the object is usually moved at a pre-
defined speed in bench-top spectrograph HSI systems (Konda
Naganathan et al., 2008a; Konda Naganathan et al., 2008b). How-
ever, such design may not work to acquire a hyperspectral image of
a beef ribeye muscle on a hanging beef carcass because of the ne-
cessity to move the entire carcass. Therefore, the mirror scanner
assembly was designed with a mirror positioned at 45 angle and a
stepper motor. The mirror was moved to scan the entire ribeye area
as opposed to moving the entire beef carcass. The placement of the
mirror at a 45 angle allowed the reflected signal from the beef
ribeye muscle to pass at a 90 angle onto the spectrograph. This
design made the system compact enough to fit within the ribbed
space between the ribeye muscle and hindquarter of the carcass,
which is directly above the exposed ribeye. Also, the mirror was
attached to the stepper motor to scan the ribeye muscle. A lighting
dome was fabricated and attached with the mirror scanner as-
sembly. Six 50-W tungsten halogen lamps were placed at the bot-
tom of the dome, facing upwards. The dome was painted with
white reflectance coating (Munsell, Edmund Optics, Barrington, NJ)
to provide uniform diffuse lighting over the entire ribeye area. In
addition, locating plates were provided at the bottom of the dome
to firmly and positively hold the camera module onto the exposed
portion of the carcass during image acquisition.
2.2. Samples and hyperspectral image acquisition
Prior to image collection, the system was calibrated for wave-
lengths using Eq. (1) provided by the spectrograph manufacturer,
where, l is wavelength (nm) and BN is band or channel number.
312 G. Konda Naganathan et al. / Journal of Food Engineering 169 (2016) 309e320
Using the spectral calibration equation (Eq. (1)), it was determined
that only 784 channels (139e922) out of the available 1082 spectral
channels were useful because the remaining channels fell outside
of the spectrograph's sensitive region of 400e1000 nm. Therefore,
the system was set up to acquire only the 784 useful channels. In
addition, the 784 channels were binned by a factor of two to yield
392 spectral bands with a band interval of 1.5 nm.
l ¼ 0:00005  ðBNÞ2 þ 0:7087  ðBNÞ þ 302:22
A total of 274 beef hyperspectral images representing 274 beef
carcasses were acquired at 2-day postmortem using the
spectrograph-based HSI system. Beef carcasses representing “A”
maturity and USDA quality grades of Prime, Choice, Select, and
Standard were imaged at a commercial slaughterhouse in the U.S.
Carcasses were randomly selected from the flow of carcasses
approximately 48 h after slaughter. Live and carcass weights were
not recorded, but carcasses represented standard sampling from
commercial cattle. Approximate mean carcass weight could be
from 800 to 900 lbs and approximate mean live weight could be
from 1300 to 1400 lbs. Cattle were selected from a Midwest beef
plant that slaughtered only beef cattle; no dairy cattle are slaugh-
tered at this facility. It was not possible to identify breed of each
carcass sampled. Animals were humanely stunned (using a pneu-
matic impact stunner) and processed under USDA inspection.
Carcasses were sampled throughout the production shift (from
approximately 5000 carcasses), representing well over 100
producers.
It took about 8 s to acquire a hyperspectral image. In addition to
the beef images, dark and white reference images were acquired at
approximately 45e60 min intervals. The white images were collected
using a Teflon plate, whereas the dark images were collected by
closing the camera lens with a cap. The reference images were used to
calibrate the beef images using Eq. (2). A sample selection method
based on Mahalanobis distance was carried out to divide the samples
into training and validation sets (Konda Naganathan et al., 2008a;
Shenk and Westerhaus, 1991). The training set had 173 samples,
whereas the validation set had 101 samples.
2.3. Reference tenderness scores
After image acquisition, strip loin steaks were removed from the
13th rib region of the carcasses for shear force measurements. The
steaks were aged for 14 days. Following aging, the steaks were
cooked in an impingement oven to an internal temperature of
70  C. After cooling, six 12-mm diameter cores were sampled
randomly from each steak. Force required to shear the core was
recorded using a Warner-Bratzler attachment on an Instron Uni-
versal Testing Machine. Peak shear-force values from the six
replicate cores were averaged to obtain the shear-force tenderness
reference value (AMSA, 1995). The samples having Warner-Bratzler
shear force (WBSF) values greater than 44 N were considered as
tough.
2.4. Hyperspectral image processing
Only the hyperspectral image acquisition was done on-line.
Raw Beef Image  Dark Reference Image
Calibrated Beef Image ¼
White Reference Image  Dark Reference Image
Image calibration and processing algorithms were imple-
mented off-line using ENVI (ITTVIS, Boulder, CO), MATLAB (The
MathWorks Inc., Natick, MA), and SAS (SAS Institute Inc., Cary,
NC).
2.4.1. Distortion correction
As mentioned earlier, a mirror was used to scan the ribeye
(1) muscle. This setup caused a geometric distortion along the vertical
dimension of the image. To characterize and correct the distortion,
the following procedure was followed. A 10  10 mm grid that
covered the entire field of view, was printed on a letter sized paper
using a laser printer. Then the grid was pasted on an acrylic sheet to
hold it flat. The grid plate was imaged following the same proce-
dure used for acquiring hyperspectral images of beef. Shown in
Fig. 3a is the distorted image of the grid. The horizontal lines
appeared straight and they were parallel to each other across the
entire image. The vertical line at the middle of the image looked
straight and undistorted. The vertical lines away from the center of
the image were distorted. The amount of distortion increased as the
distance increased with respect to the center of the image. A
distortion with the above described characteristics is called barrel
distortion. However, the distortion was only along the vertical axis
of the image.
A distortion correction routine was developed to correct the
distorted image. First, a set of reference lines was determined by
drawing vertical lines passing through the intersection points of
the distorted lines and the middle row of the image. The inter-
section points were precisely determined by thresholding the
distorted image (Fig. 3b). For better visualization purpose, only
one distorted vertical line was isolated from the entire image
and its zoomed in view is presented in Fig. 3c, where the solid
line represents a distorted vertical line, dotted horizontal line
represents the middle row of the image, and the dotted vertical
line represents the ideal distortion corrected line (or reference
line). Using the pixel indices of the distorted and reference lines,
a distortion correction or mapping function was developed to
correct the distorted image. The correction procedure provided
the horizontal shift distance for each pixel. The distortion cor-
rected image is shown in Fig. 3d. The error in the distortion
correction routine was calculated by comparing the coordinates
of the corrected lines to that of the reference lines and the error
was less than or equal to two pixels at any given instance.
2.4.2. Image calibration and region-of-interest (ROI) selection
Reflectance images were obtained by subtracting the dark image
from the raw beef image and dividing by the dark subtracted Teflon
image (Eq. (2)). By doing this calibration procedure, hyperspectral
image variations due to illumination and sensor response due to
environmental conditions such as temperature, were minimized.
After the image calibration, a region-of-interest (ROI) of size 256
pixels  128 pixels  392 bands was manually selected within the
ribeye muscle. Also, the first 32 bands corresponding to a wave-
length range from 400 to 447 nm were considerably noisy that they
were discarded. Therefore, the ROI image size was reduced to 256
pixels  128 pixels  360 bands. Further image processing steps
were performed on these ROI hyperspectral images.
(2)
 G. Konda Naganathan et al. / Journal of Food Engineering 169 (2016) 309e320 313

Fig. 3. Images of a grid pattern before and after distortion correction.

2.4.3. Dimensionality reduction methods or chemometric analyses of CPCA method was used to determine pork quality attributes such
Four different dimensionality reduction methods e sample as drip loss, pH, and color (Qiao et al., 2007).
principal component analysis (SPCA), chemometric principal In the MPCA method, the 173 images in the training set were
component analysis (CPCA), mosaic principal component analysis mosaicked together to create a large image (Fig. 6). In other words,
(MPCA), and partial least squares analysis (PLSA) e were employed the 173 images, each of size 256  128  360, were transformed
to reduce the spectral dimensionality of the hyperspectral images into 5,668,864 (173  256  128) spectra, each of size 1  360. In
of beef. It is important to note that the naming convention used to
denote the dimensionality reduction methods are not universal.
The distinction among the dimensionality reduction methods was
primarily based on how the hyperspectral image data were trans-
formed and presented to standard PCA and PLSA algorithms. Before
conducting the dimensionality reduction procedure, the spectra
were mean centered in all the methods.
In the SPCA method, each ROI hyperspectral image was
considered as a separate data set and the Eigen values and Eigen
vectors obtained from the SPCA procedure were specific to that
image (Fig. 4). The SPCA loading vectors varied from one image to
the other. Similarly, the PCA scores or pixel values of the SPCA
bands of a particular image were computed from the loading vec-
tors specific to that image. Each ROI hyperspectral image of size
256  128  360 was transformed to 32,768 (256  128) spectra,
each of size 1  360, and presented to the PCA algorithm. This type
of analysis explains within-sample variation and ignores between-
sample variations. Konda Naganathan et al. (2008b) used the SPCA
method on 14-day hyperspectral images to predict 14-day aged,
cooked beef tenderness. In this study, the SPCA method was applied
on 2-day beef hyperspectral images to forecast 14-day aged, cooked
beef tenderness.
The CPCA method utilized all the images in the training set
(n ¼ 173) and provided common Eigen values and vectors (Fig. 5).
The 173 hyperspectral images, each of size 256  128  360, were
transformed to 173 spectra, each of size 1  360, by averaging on
both spatial dimensions. This analysis ignores within-sample Fig. 4. Flowchart showing the steps of sample principal component analysis (SPCA) on
variation and focuses on between-sample variation. Similar type three dimensional hyperspectral images.
314 G. Konda Naganathan et al. / Journal of Food Engineering 169 (2016) 309e320
Fig. 5. Flowchart showing the steps of chemometric principal component analysis
(CPCA) on three dimensional hyperspectral images.
contrast to the SPCA method, the MPCA method yielded common
Eigen values and loading vectors. This method explains both
within- and between-sample variations, as all the spatial and
spectral information within- and between-samples was used in
determining the loading vectors. Since the number of spectra was
Fig. 6. Flowchart showing the steps of mosaic principal component analysis (MPCA)
on three dimensional hyperspectral images.
very large, more computation time (few hours depending on the
hardware specifications of the computer used for analysis) and
sophisticated hardware were required as compared to the other
two methods. However, this step could be implemented off-line
and needs to be completed only once for a certain population of
carcasses. Thus, this method does not need to be computed real-
time.
All the PCA methods required only the image data and they did
not require the tenderness references (dependent variable) in the
dimensionality reduction process. In other words, the PCAs
methods were optimized to create the PCA bands such that they
explain the variation contained in the image data (independent
variables) alone, while the variation in the tenderness scores
(dependent variables) was not considered in the analysis. In
contrast, the PLSA method can consider the image data and the
tenderness scores in the analysis. Therefore, the PLSA method,
similar to the one presented by Konda Naganathan et al. (2008a),
was also implemented and compared against the PCA methods. The
PLSA method was very similar to the CPCA method in terms of data
transformation except that it required the tenderness scores or
WBSF values in computing the Eigen values and vectors.
For each dimensionality reduction method, the corresponding
Eigen or loading vectors were exported to ENVI software. A batch
processing algorithm was written to create PCA and PLSA bands by
multiplying the loading vectors with the spectral profile of each
pixel in the hyperspectral image. Further analyses such as feature
extraction were carried out on the PCA and PLSA bands.
2.4.4. Feature extraction
From each PCA or PLSA band, six different hyperspectral image
feature sets were evaluated for beef tenderness assessment: (1)
descriptive statistical features, (2) wavelet features (Subbiah,
2004), (3) gray level co-occurrence matrix features (Konda
Naganathan et al., 2008a; Konda Naganathan et al., 2008b; Li
et al., 1999), (4) Gabor features (Manjunath and Ma, 1996), (5)
Laws features (Laws, 1980), and (6) local binary pattern features
(Ojala et al., 1996). A total of 68 features (6 descriptive statistical
features þ 4 wavelet features þ 4 gray level co-occurrence matrix
features þ 4 Gabor features þ 14 Laws features þ 36 local binary
pattern features) were extracted from each PCA or PLSA band. A
detailed description of the algorithms and equations used to extract
these features is explained elsewhere (Konda Naganathan, 2011;
Konda Naganathan et al., 2015). When developing tenderness
discriminant models, these features were pooled together and
significant features were selected.
2.4.5. Discriminant models
The tenderness discriminant models e FLD (Krzanowski, 1988),
SVM (Cristianini and Shawe-Taylor, 2000), and DT model (Breiman
et al., 1984), were developed in MATLAB. All the models were
trained with a leave-one-out cross-validation procedure with equal
priors and linear kernels. The models were designed to provide
binary outputs such as tender or tough. For the SVM model, a least
squares-based method was used to optimize the feature selection.
For the DT model, a classification tree was used. The hyperspectral
image features were input to these models, which then classify
each sample into two tenderness categories: tender or tough. Prior
to developing tenderness prediction models, a stepwise feature
selection algorithm (STEPDISC procedure in SAS) was followed to
identify image features that have most tenderness discrimination
power. A P-value of 0.15 was used to test the significance level of a
feature during both forward addition and backward elimination.
2.4.6. Evaluation
The tenderness prediction models classify samples into two
 G. Konda Naganathan et al. / Journal of Food Engineering 169 (2016) 309e320 315

tenderness categories: tender or tough. A commonly used metric to

evaluate performance of such discriminant models is overall ac-
curacy, ratio between the number of correctly classified samples
and total number of samples. Though the overall accuracy measure
is useful in some classification problems, it is not adequate to
explain the performance or usefulness of a beef tenderness pre-
diction model because it does not differentiate the two types of
misclassifications: tender into tough and tough into tender. The
latter one has more serious outcomes than the first one. So, in this
study, tender certification accuracy (TECA) and a custom defined
metric called accuracy index (AI) (Eq. (3)) were used to compare the
tenderness prediction models.

1
Accuracy Index ðAIÞ ¼ ½TEIA þ 2  TOIA þ 2  TECA þ TOCA
6
 100
(3)

Fig. 7. Scree plot showing the variation explained by the first ten principal component
where, TEIA is tender identification accuracy which is the ratio of analysis (PCA) and partial least squares analysis (PLSA) bands. SPCA e Sample principal
number of samples correctly predicted as tender to the total component analysis; CPCA e Chemometric principal component analysis; MPCA e
Mosaic principal component analysis; PLSA e Partial least squares analysis.
number of “true” tender samples; TOIA is tough identification ac-
curacy which is the ratio of number of samples correctly predicted
as tough to the total number of “true” tough samples; TECA is
tender certification accuracy which is the ratio of number of sam-
577, 635, 739, 756, 910, 928, 972, and 988 nm. The wavelengths
ples correctly predicted as tender to the total number of samples
541, 577, and 635 nm were related to myoglobin and its de-
predicted as tender; and TOCA is tough certification accuracy which
rivatives such as deoxy-, oxy-, and met-myoglobin (Liu et al.,
is the ratio of number of samples correctly predicted as tough to the
2003; Millar et al., 1996). Myoglobin is the important pigment
total number of samples predicted as tough. In some research areas,
protein responsible for beef color. Also, these wavelengths were
the evaluation metric TEIA is denoted as (100 e beta error or false
shown to explain the variations related to the aging of beef (Liu
negative error), whereas TECA is denoted as (100 e alpha error or
et al., 2003). The reflectance intensity at 541, 577, and 756 nm
false positive error) by considering the true tender samples as
decreases as aging period increases, whereas the intensities at
“positive” samples.
635 and 980 nm increase as age increases. Also, the wavelengths
910 and 928 nm have been related to protein and fat, respectively.
3. Results and discussions
Similarly, the wavelengths 739, 756, and 988 nm have been
related to water.
3.1. Samples

Out of the 173 samples in the training set, there were 133 (76.9%)
tender samples and the rest of the 40 (23.1%) samples were tough.
Similarly, the validation set had 79 (78.2%) tender samples and 22
(21.8%) tough samples. The WBSF values in the training set ranged
from 20 to 65 N with a mean value of 37 N. The validation set had a
WBSF range of 25e64 N with a mean value of 38 N. This implies
that the Mahalanobis distance-based sample selection algorithm
was able to divide the samples into two sets that had similar WBSF
distributions.
3.2. Eigen values and loading vectors
The first ten Eigen values for the four dimensionality reduction
methods used in this study are presented in Fig. 7. There were no
considerable differences among these four methods. The first three
to five principal components could be optimal in all four methods.
The first three SPCA, CPCA, MPCA, and PLSA bands explained
97.63%, 99.34%, 98.66%, and 99.01%, respectively, of the variations in
the hyperspectral images. When the first five bands were consid-
ered, the variations explained by these methods were 97.95%,
99.90%, 99.22%, and 99.85%. So, the first five loading vectors were
used to construct five PCA or PLSA bands.
The first five loading vectors for the four different dimen-
sionality reduction methods are shown in Fig. 8. The peaks, val-
leys, and slope changes of these loading vectors have been related
to molecular vibrations of the constituents of beef. Such transi-
tions were observed at the following wavelengths: 509, 541, 560,
3.3. PCA and PLSA bands
The PCA and PLSA bands of the four dimensionality reduction
methods are shown in Fig. 9. These bands were generated by a
linear combination of each image pixel's spectrum with the loading
vectors. The shape and amplitude of the loading vectors dictate the
pixel values of the PCA and PLSA bands. For instance, the loading
vector 1 of the SPCA and PLSA methods were inversely related and
so were the SPCA and PLSA bands (Fig. 9). Similarly, the loading
vector 5 of the SPCA method was close to zero for all wavelengths
and the fifth SPCA band looked dark, which indicated that the pixel
values were close to zero as well (Fig. 9).
3.4. Optimal number of PCA or PLSA bands
In this study, a total of 36 tenderness prediction models (4
dimensionality reduction methods such as SPCA, CPCA, MPCA, and
PLSA  3 different number of PCA or PLSA bands such as 3, 4, and 5
bands  3 discriminant models such as FLD, SVM, and DT) were
developed and compared. First, for a given combination of the
dimensionality reduction method and discriminant model, the
number of PCA bands that yielded the highest AI value in true
validation was selected. For example, for the SPCA-FLD combina-
tion, the five band model achieved an AI value of 63.8% in true
validation (Table 1), which was higher than that of the three or four
band models. The optimal number of bands for the SPCA, MPCA,
and PLSA models were the same in all three discriminant models.
316 G. Konda Naganathan et al. / Journal of Food Engineering 169 (2016) 309e320

Fig. 8. First five Eigen or loading vectors of four different dimensionality reduction methods. SPCA e Sample principal component analysis; CPCA e Chemometric principal
component analysis; MPCA e Mosaic principal component analysis; PLSA e Partial least squares analysis.

However, for the CPCA method, the optimal number of bands var-
ied with respect to the discriminant model. The optimal number of
bands for the CPCA-FLD, CPCA-SVM, and CPCA-DT combinations
were five, three, and four, respectively.
3.5. Assessment of the tenderness prediction models
The tender certification accuracy and accuracy index values of
the discriminant models in cross-validation and true validation are
provided in Table 1. It also summarizes the optimal number of PCA
or PLSA bands and number of features used to build the prediction
 G. Konda Naganathan et al. / Journal of Food Engineering 169 (2016) 309e320 317

Fig. 9. Principal component analysis (PCA) and partial least squares regression analysis (PLSA) bands generated by four different dimensionality reduction methods. SPCA e Sample
principal component analysis; CPCA e Chemometric principal component analysis; MPCA e Mosaic principal component analysis; PLSA e Partial least squares analysis.

Table 1
Tenderness prediction results based on the dimensionality reduction methods and discriminant models.

Dimensionality No. of PCA or No. of Tender certification Tender certification Accuracy index Accuracy index
reduction method PLSA bands features accuracy (cross-validationa) accuracy (true validationb) (cross-validationa) (true validationb)

Fisher's linear discriminant (FLD) model

SPCA 5 5 88.8 84.8 71.9 63.8
CPCA 5 9 90.9 86.7 77.5 66.8
MPCA 3 5 88.9 85.0 72.2 64.1
PLSA 4 4 91.0 82.0 76.1 59.1
Support vector machines (SVM) model
SPCA 5 5 89.4 84.1 72.6 62.9
CPCA 3 7 89.2 84.3 73.0 63.1
MPCA 3 5 89.2 83.7 72.1 62.2
PLSA 4 4 91.5 81.0 76.2 57.9
Decision tree (DT) model
SPCA 5 5 85.0 79.0 66.4 54.2
CPCA 4 8 79.4 83.1 56.9 61.0
MPCA 3 5 80.6 82.3 58.5 59.5
PLSA 4 4 85.2 83.9 66.5 62.4

SPCA e Sample principal component analysis; CPCA e Chemometric principal component analysis; MPCA e Mosaic principal component analysis; PLSA e Partial least squares
analysis.
a
Cross validation was performed with 173 samples.
b
True validation was performed with 101 samples.
318 G. Konda Naganathan et al. / Journal of Food Engineering 169 (2016) 309e320
models. The tenderness certification accuracy and accuracy index
values in true validation were used as a basis to compare the
tenderness prediction models. When comparing the models
developed in this study with the one published in the literature, AI
values were calculated if they were not reported in the original
article.
Overall, the CPCA-FLD model had the highest tender certifica-
tion accuracy and AI value of 86.7% and 66.8%, respectively, in true
validation. Corresponding values in cross-validation were 90.9%
and 77.5%. The CPCA-FLD model used nine features derived from 5
PCA bands. The MPCA-FLD model followed the CPCA-FLD model in
terms of overall performance with a tender certification accuracy of
88.9% and 85% in cross-validation and true validation, respectively.
The AI values of the MPCA-FLD model were 72.2% and 64.1% in
cross-validation and true validation, respectively. It used only five
features from 3 PCA bands.
For the SPCA, CPCA, and MPCA methods, the FLD models per-
formed better than the SVM and DT models. For the PLSA method,
the DT model outperformed the other models. The tender certifi-
cation accuracy of the SPCA-FLD, CPCA-FLD, MPCA-FLD, and PLSA-
DT models were 84.8%, 86.7%, 85%, and 83.9%, respectively, in true
validation. The corresponding AI values of these models were
63.8%, 66.8%, 64.1%, and 62.4%, respectively. Aside from differences
in the number of features and bands used in building the models,
the PLSA method needed the WBSF values in determining the
loading vectors, whereas the SPCA, CPCA, and MPCA did not.
3.6. Comparison of the dimensionality reduction methods and
discriminant models
The combined effect of the dimensionality reduction method and
discriminant model was discussed above. To determine the effect of
the dimensionality reduction method independent of the discrim-
inant model and vice versa, average tender certification accuracy
and AI values were computed for each dimensionality reduction
method and discriminant model by averaging corresponding met-
rics. For example, to compute the average tender certification ac-
curacy of the SPCA method, the tender certification accuracies of the
SPCA-FLD, SPCA-SVM, and SPCA-DT were averaged. Similarly, the
average tender certification accuracy of the FLD model was
computed by averaging the tender certification accuracies of the
SPCA-FLD, CPCA-FLD, MPCA-FLD, and PLSA-FLD models. The same
procedure was followed to compute the average AI values.
The CPCA method had the highest average tender certification
accuracy and AI value of 84.7% and 63.6%, respectively, in true
validation. The CPCA method was followed by the MPCA, SPCA, and
PLSA methods in terms of average tender certification accuracy and
AI value. Similarly, the FLD model outperformed the other two
models with an average tender certification accuracy of 84.6% and
AI of 63.4% in true validation. The FLD model was followed by the
SVM and DT models. The results of this analysis agreed with the
earlier finding of superior performance of the CPCA-FLD model
compared to the other combinations.
Of the three PCA methods, the SPCA method performed worse
than the other two methods. As mentioned earlier, the loading
vectors of the SPCA were different for each hyperspectral image or
beef samples because each hyperspectral image was considered as
a separate data set and PCA was conducted. There were two distinct
groups of loading vectors and one group was the exact opposite of
the other. To show an example, the first loading vector of two beef
samples and their SPCA bands are provided in Fig. 10. The groupings
did not relate to either WBSF values or marbling scores of the
sample.
In terms of computational time, the MPCA method required
significantly more time than the other methods to determine the
Fig. 10. Sample principal component analysis (SPCA): First Eigen or loading vector and
SPCA bands of two different beef samples.
loading vectors because of the large image size due to image
mosaicking (Fig. 6). The CPCA and PLSA methods required the least
amount of time to create the loading vectors because these two
methods operated on a one dimensional data by averaging the
spatial pixels (Fig. 5). It is important to note that the loading vectors
of the CPCA, MPCA, and PLSA methods were common for all the
images. Real time applications using either of these three methods
do not need to re-compute the loading vectors for a new set of
samples that were not used in creating the loading vectors. During
the calibration process, the loading vectors can be saved to a file.
Real time applications can read the loading vectors from the file and
use them to create PCA or PLSA bands, which will significantly
reduce the total time needed to process an image. In contrast, the
SPCA method may not be suitable for real time applications because
the loading vectors need to be computed for each image in a new
data set.
3.7. Comparison with the bench-top hyperspectral imaging system
The tenderness prediction models developed in this study were
compared against the ones in the literature on the basis of the AI
values. When the AI values were not reported in the original arti-
cles, the AI values were computed based on the information pro-
vided. In this study, the SPCA-FLD model developed using five PCA
bands had an AI value of 71.9% and 63.8% in cross-validation and
true validation (Table 1), respectively. In a similar study, SPCA-
based hyperspectral features were used by Konda Naganathan
et al. (2008b) to classify beef samples into three tenderness cate-
gories: tender, intermediate, and tough. When the three-category
classification model was converted into two-category classifica-
tion model by combining tender and intermediate categories into
one group, the model had an accuracy of 100% based on leave-one-
out cross-validation. The study conducted by Konda Naganathan
et al. (2008a) used the 14-day image features to predict 14-day
tenderness, whereas the current study used 2-day image features
to forecast 14-day tenderness. This also implies that tenderness
forecasting is more complex than predicting current status of
tenderness. Finally, they acquired images in controlled conditions
with minimal experimental errors using a bench-top hyperspectral
imaging system to acquire images of beef steaks that were excised
from beef carcasses. The hyperspectral imaging system used in this
study acquired hyperspectral images beef ribeye muscle on
hanging beef carcasses in a packing plant environment and
 G. Konda Naganathan et al. / Journal of Food Engineering 169 (2016) 309e320
therefore the images incorporated more experimental errors that
were encountered in real packing plant environment.
The PLSA-DT model developed in this study had an AI value of
62.4% in true validation. In a similar study, Konda Naganathan et al.
(2008a) used PLSA features in a canonical discriminant model to
predict beef tenderness and obtained an AI value of 77.5%. They
used a NIR hyperspectral imaging system (l ¼ 900e1700 nm), ac-
quired beef images off-line, and reported results based on cross-
validation, whereas the current study used a VNIR hyperspectral
imaging system (l ¼ 400e1000 nm), acquired beef images on-line,
and reported results based on true validation. Also, the sample size
and tenderness distributions were different.
3.8. Comparison with the other tenderness prediction instruments
The MPCA-FLD model developed in this study had an AI value of
64.1% in true validation. In a study funded by National Cattlemen's
Beef Association, an acousto-optic tunable filter (AOTF)-based
hyperspectral imaging system was used and MPCA features were
modeled using an FLD model to classify beef samples into two
tenderness categories: tender or tough. The AOTF system had an AI
value of 70% (Lorenzen et al., 2009). The AOTF system had higher
spatial resolution (0.16 mm) than that of the spectrograph system
used in this study (0.52 mm). Slightly poorer performance of the
spectrograph-based system to the AOTF system might be due to the
lower spatial resolution. These results are in agreement with the
recent findings that high resolution images could yield better beef
tenderness discrimination when compared to that of low resolution
images (Goldberg, 2011; Howard et al., 2010; Jackman et al., 2009;
Subbiah, 2004). In the same NCBA study, in addition to the AOTF
system, two other instruments e one based on video image analysis
(Beefcam) and the other based on near infrared spectroscopy, were
also tested head-to-head. The Beefcam and spectroscopic system
had an AI value of 53.9% and 58.3%, respectively. These results
indicated that hyperspectral imaging was better than the BeefCam
and spectroscopic systems.
4. Conclusions
Three dimensional chemometric analyses in conjunction with
discriminant models were developed to analyze beef hyperspectral
images for forecasting beef tenderness. Hyperspectral images
(n ¼ 274) of exposed ribeye muscle on hanging beef carcasses were
acquired at 2-day postmortem using a prototype on-line spectro-
graph hyperspectral imaging (HSI) system. Warner-Bratzler shear
force values were collected at 14-day postmortem as a measure of
tenderness. Four different dimensionality reduction methods:
sample principal component analysis (SPCA), chemometric prin-
cipal component analysis (CPCA), mosaic principal component
analysis (MPCA), and partial least squares analysis (PLSA), and three
different discriminant models: Fisher's linear discriminant (FLD)
model, support vector machines (SVM) model, and decision tree
(DT) model, were implemented. The best tenderness prediction
results were obtained by the CPCA and FLD combination with a
tender certification accuracy of 86.7% and accuracy index value of
66.8%, in true validation using 101 samples. Within the dimen-
sionality reduction methods, the performance of the CPCA method
was the best followed by the MPCA, SPCA, and PLSA methods.
Similarly, within the discriminant models, the FLD models per-
formed better than the SVM and DT models. In addition, key
wavelengths corresponding to myoglobin and its derivatives (541,
577, and 635 nm), beef aging (541, 577, 635, 756, and 980 nm),
protein (910 nm), fat (928 nm), and water (739, 756, and 988 nm)
were identified.
319
Acknowledgments
The authors would like to appreciate National Cattlemen's Beef
Association and Nebraska Agricultural Experiment Station for
providing funding for conducting this study. The authors are also
thankful to the beef industry for providing access to the plant and
the lab for the slice shear force measurements of the beef samples.
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