Computers and Electronics in Agriculture 124 (2016) 234–242

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Computers and Electronics in Agriculture

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

Original papers

Nondestructive determination of cocoa bean quality using FT-NIR

spectroscopy
S. Sunoj a,⇑, C. Igathinathane b, R. Visvanathan c
a
Department of Food and Agricultural Process Engineering, Agricultural Engineering College and Research Institute, Tamil Nadu Agricultural University, Coimbatore, TN 641003, India
b
Department of Agricultural and Biosystems Engineering, North Dakota State University, 1221 Albrecht Boulevard, Fargo, ND 58102, USA
c
Anbil Dharmalingam Agricultural College and Research Institute, Tamil Nadu Agricultural University, Tiruchirappalli, TN 620009, India

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

Article history: A study was conducted to develop nondestructive calibration models to predict cocoa bean’s quality
Received 7 March 2016 parameters, such as fermentation index, pH and total polyphenol content using Fourier transform (FT)
Received in revised form 12 April 2016 near infrared (NIR) spectroscopy. Cocoa pods were stored for 0, 7, 14, and 21 days as a pretreatment
Accepted 16 April 2016
method and the effect on quality parameters were investigated. The beans from the stored pods were
Available online 26 April 2016
fermented by heap method. Fermentation index of all treatments ranged between 0.535 and 1.242, pH
values between 4.26 and 6.13, and total polyphenol content varied between 6.48 and 15.58 mg g
1.
Keywords:
The samples were scanned in the NIR region of wavenumber 12,500–3600 cm
1 for the spectral analysis.
Cocoa bean
Fermentation
Results obtained from conventional quality analysis were correlated with spectra of respective treatment
FT-NIR spectroscopy for calibration models development. Partial least square regression technique was used to develop the
PLS regression calibration models. Scanned spectra were preprocessed with vector normalization, multiplicative scatter
Nondestructive correction, and first derivative techniques. The calibration models fitted the prediction of fermentation
index and total polyphenols well (R2  0:80), compared to that of pH (R2 < 0:80). The proposed method
was quick (<1 min) when compared to the conventional method (28 h). The FT-NIR spectroscopy
method of analysis produced rapid and accurate quality attributes of fermenting cocoa, and can be
employed to similar food products.
Published by Elsevier B.V.

1. Introduction
Among plantation crops, cocoa (Theobroma cacao L.) is a popular
crop that serves as the raw material for chocolate manufacturing
industries. It is also used as a flavor enhancer in confectioneries,
baking and dairy products industries. The ideal climate for growing
cocoa is hot, rainy, and tropical conditions. Cocoa trees are a
delicate and sensitive, and producers must protect the crop from
the wind and sun by planting them as an intercrop between other
trees, for example, coconut and nutmeg trees. Ivory Coast leads in
the production of cocoa beans, while other leading cocoa producers
are Ghana, Indonesia, Nigeria, and Cameroon (Worldatlas, 2016).
Out of total global cocoa production, Africa is the major contributor
with 68% of world’s production. The total cocoa production has
increased from 3.3 Mt in 2000 to 4.5 Mt in 2014 (FAOSTAT, 2016).
⇑ Corresponding author at: Department of Agricultural and Biosystems Engineer-
ing, North Dakota State University, 1221 Albrecht Boulevard, Fargo, ND 58102, USA.
E-mail addresses: sunoj.shajahan@ndsu.edu, sunojshajahan@gmail.com
(S. Sunoj).
The supply chain of cocoa beans is very long, complex, and it
involves several stakeholders. It starts from producers, who grow,
harvest, extract the beans from pod, ferment, dry, and supply the
beans. Beans are then collected by the local agents, traders, and
buying stations until they reach the chocolate manufacturing unit
(Saltini et al., 2013). The quality of cocoa beans wholly depends on
the producers, who performs the fermentation, during which flavors
are formed. Traditionally, fermentation of cocoa beans is carried out
using heap method, and beans rest in heaps for six days. Prior to
fermentation, the cocoa pods are subjected to preconditioning by
storing the pods for a predetermined duration. Several researchers
have reported that fermenting the beans obtained from stored pods
resulted in improved chocolate flavor by reducing astringency and
bitterness (Afoakwa, 2011; Nazaruddin et al., 2006).
Due to the high demand for cocoa beans, there are chances of
adulteration while supplying the beans to the traders. One possi-
bility of adulteration is to mix the under-fermented beans with
fermented ones. In such a scenario, the quality estimation becomes
important and care should be exercised in measurements. A rapid
tool to estimate quality parameters will be a great asset to buyers
and processors.

http://dx.doi.org/10.1016/j.compag.2016.04.012
0168-1699/Published by Elsevier B.V.
 S. Sunoj et al. / Computers and Electronics in Agriculture 124 (2016) 234–242
The quality parameters of the beans mainly include fermenta-
tion index, pH, and total polyphenols (Afoakwa, 2011). The fermen-
tation index is the degree of fermentation of the beans and it is a
measure of ‘‘browness.” The color of cocoa beans is due to the pres-
ence of anthocyanin pigments (Afoakwa et al., 2013). During the
initial stages of fermentation, the cocoa beans are purple, and later
during fermentation the anthocyanin pigments are decomposed
and form other condensation products, which result in brown
color. The pH during and at the end of fermentation has a major
role in flavor development. Beans of higher pH (5.5–5.8) are con-
sidered unfermented, with low fermentation index, and those of
lower pH (4.75–5.19) are considered as well-fermented. Chocolates
made from cocoa beans of high pH (5.5–5.8) resulted with astrin-
gency due to the presence of strong flavor (Jinap et al., 1995). Cocoa
beans are a rich source of polyphenols, which serve as antioxidant.
The presence of polyphenols imparts astringency and bitterness to
the cocoa beans. Polyphenols in food products have gained its
importance due to the advantageous role in the prevention of
cancer and cardiovascular diseases.
At present conventional (wet chemistry) methods are used to
determine fermentation index, pH, and total polyphenol content
in cocoa beans. These methods, however, are time-consuming,
expensive, and destructive. Therefore, there is an immediate need
for rapid and nondestructive tool to evaluate these quality
parameters. Application of Fourier transform near infrared (FT-
NIR) spectroscopy in the food industry for rapid quality measure-
ments was well researched. The FT-NIR spectroscopy can be a
useful tool for rapid estimation of the quality, since it requires no
sample preparation and evaluation performed within a minute
(Tripathi et al., 2010; Subramanian and Rodriguez-Saona, 2008).
Several researchers have utilized the NIR technology in prediction
of pH of food products (Balage et al., 2015; Giovenzana et al., 2014;
Li et al., 2013), and estimation of polyphenols (Giovanelli et al.,
2014; Bala and Singh, 2013; Sinija and Mishra, 2009).
Only a few studies have looked into the cocoa quality aspects,
such as quantification of fat, nitrogen, and moisture in cocoa
powder (Veselá et al., 2007), prediction of fermentation level and
quantification (Hue et al., 2014; Krähmer et al., 2015), quantifica-
tion of pH and fermentation index (Teye et al., 2015), and identifi-
cation of cocoa beans adulteration (Teye et al., 2014). However, all
these studies were performed by grinding the sample (semi-
destructive). It will be efficient if the quality of the beans were
evaluated using the whole beans avoiding the grinding operation,
which will become a beneficial ready-to-apply tool for traders to
obtain the beans quality quickly. The literature on assessing the
quality parameters of plantation crops using FT-NIR spectroscopy
are scarce. Therefore, a research was formulated with the objective
to evaluate the major quality parameters, such as fermentation
index, pH, and total polyphenol content in whole cocoa beans in
a nondestructive manner using FT-NIR spectroscopy.
2. Materials and methods
2.1. Cocoa beans sample
Ripe cocoa pods of mixed F1 progeny variety were obtained
from a local producer (Mill Gounder Thottam, Semmedu village,
Coimbatore, Tamil Nadu, India, coordinates: 11.01°N, 76.95°E).
Damaged and diseased pods were manually picked and rejected.
Pods were transported to the laboratory on the same day of harvest
for experiments. The pods were pretreated by storing about 200
pods at ambient temperature (25–30°) and relative humidity
(70–85%) for a period of 0, 7, 14, and 21 days. Stored cocoa pods
(50) from each treatment period were then split open and the
cocoa beans were fermented by heaping over a platform and
235
covered with plantain leaves. During fermentation, approximately
150 g of samples were collected each day to inspect the changes in
quality parameters. The samples were dried under the sun to a
final moisture content of below 8% (w.b.) to obtain the cocoa beans
resembling the final product. Dried samples were packed in ziplock
bags, labeled, and stored in a dark room at ambient temperature
before experiments. In total, cocoa samples from 24 treatments
were collected for the experiments (Fig. 1). This included the
samples from 1st day of fermentation (under-fermented) to 6th
day of fermentation (well-fermented) cocoa beans at interval of
one day for the different pod storage days. This six days period
covered the entire range of fermentation followed by the farmers.
These whole cocoa beans were used for spectral measurements,
while for carrying out chemical analysis, the beans were ground
to a fine powder. To avoid becoming a mash, the beans were frozen
with liquid nitrogen to make them brittle prior to grinding.
2.2. Chemical analysis
The chemical analysis for quality of cocoa beans followed in the
study involved destructive procedures. Cocoa nibs were collected
by dehusking the dried samples. Samples were prepared by grind-
ing the cocoa nibs and other necessary filtration or mixing opera-
tions were performed before the actual measurements. Various
standard methods of chemical quality analysis employed in the
study are described as follows.
2.2.1. Fermentation index
Fermentation index was estimated using the method described
by Gourieva and Tserevitinov (1979). Cocoa nibs were ground and
about 0.1 g was extracted with 50 ml mixture of methanol:HCl
(97:3). The mixture was placed in a refrigerator at 82 °C, and
allowed to stand for 20 h and then filtered. Absorbance of the
filterate was read at wavelengths 460 and 530 nm using UV–vis
Spectrophotometer (Model No.: 108, Systronics, India). Fermenta-
tion index greater than 1 is considered as well-fermented, while
less than 1 as under-fermented.
Absorbance at 460 nm
Fermentation index ¼ ð1Þ
Absorbance at 530 nm
2.2.2. Measurement of pH
The pH of cocoa beans was determined according to the method
described in AOAC (1995). About 5 g of ground beans was blended
for 2 min with 45 ml of hot distilled water (60 ± 5 °C). The homoge-
nate was filtered and cooled to below 25 °C immediately; the pH
was measured using a digital pH meter (Model: 1012E, Environ-
mental Scientific Instruments, India). Prior to measuring the pH,
the instrument was calibrated with buffer at pH 4 and 7.
2.2.3. Total polyphenols
Cocoa bean’s total polyphenols were estimated using Folin–
Ciocalteu reagent (Singleton and Rossi, 1965). Total polyphenol
content was expressed in terms of mg g
1 of the ground bean.
2.3. FT-NIR spectroscopy
The spectral data were recorded in diffuse reflectance mode
from an FT-NIR spectroscopy system (MATRIX-I, Bruker optics,
Germany) using integrating sphere. Tungsten halogen lamp was
used as the light source. A Michelson interferometer, equipped
with lead sulphide (PbS) detector, detected the interference of
the light beam passing through the employed sample. Interfero-
gram collected by the detector was converted into a spectrum by
Fourier transformation of the interferogram. The system was
operated by Spectral Acquisition and Processing Software (OPUS
236 S. Sunoj et al. / Computers and Electronics in Agriculture 124 (2016) 234–242
Fermentaon days
POD STORAGE DAYS
Fig. 1. Changes in cocoa beans with the effect of pod storage days and fermentation days.
7.2, Bruker optics, Germany). Spectra obtained were at a resolution
of 8 cm
1 over a wavenumber range of 12,500–3600 cm
1 with
64 scans per spectrum. This range in wavelength will be
800–2778 nm, as wavelength is the inverse of a wavenumber.
Multivariate analysis was carried out using QUANT software (ver-
sion 7.2, Bruker optics, Germany), which performs partial least
square (PLS) regression technique for developing calibration
models.
2.4. Data preprocessing
The OPUS 7.2 software provides 11 important spectral prepro-
cessing methods, including: unprocessed, constant offset elimina-
tion, straight line subtraction (SLS), vector normalization (VN),
min–max normalization, multiplicative scatter correction (MSC),
first derivative, second derivative, first derivative + SLS, first
derivative + VN, and first derivative + MSC. Though each prepro-
cessing has its own effect on the spectra and in calibration model
development, the choice of best spectral preprocessing is purely
based on trial and error. The advantage of preprocessing of spectra
is to reduce noise and extract useful information from the spectra
(Jha and Garg, 2010). The suitability of the developed model was
based on relevant model performance parameters as defined
subsequently.
2.5. Calibration model development
The calibration model was developed using PLS algorithm in the
QUANT software. The PLS model was validated by ‘‘leave-one-out”
cross-validation method, wherein the same samples were used
both for developing the model and validating. In this procedure,
one sample was left out and the model was developed with the
remaining data. The value for the left out sample was predicted
based on the model developed and prediction residual was com-
puted. This process was repeated until every data was left out once,
then all the prediction residuals were combined to compute the
validation results; coefficient of determination (R2 ), root mean
square error for cross-validation (RMSECV), and residual predictive
deviation (RPD).
The spectra were recorded with the whole cocoa beans prior to
chemical analysis. The number of spectra also plays a major role to
make a robust calibration model. Cocoa bean samples collected on each
day of fermentation was dried and used for spectral measurements as
discussed earlier (Section 2.1). The spectra for each treatment were
recorded thrice by shuffling the sample cup that altered the orientation
of bean in the sample cup. This random orientation in a way will help
standardize the variation in presenting the bean particles to spectral
measurements. A total of 72 spectra (24 samples  3 replications) for
the cocoa samples were used for building the calibration model. The
conventional chemical analyzes were also replicated thrice and their
results were fed to the respective spectra, which were recorded prior
to conventional analysis. Several statistical measures, defined
hereunder, were used to perform the cross-validation of the developed
calibration models and measure their performance.
2.5.1. Sum of squared errors
Difference between experimental and predicted value is
residual value. The squared sum of residuals is termed as sum of
squared errors (SSE) and is given as:
X
n
SSE ¼
i
y i Þ2
ðy ð2Þ
i¼1

i is the
where n is the number of samples in the calibration set, y
predicted value for sample i, and yi is the experimental value
obtained by conventional analysis for sample i.
2.5.2. Root mean square error for cross-validation
The square root of the mean of SSE is termed as the root mean
square error for cross-validation (RMSECV). RMSECV is a good
indicator of model accuracy and it determines the possibilities of
errors in model predictions. It is calculated from Eq. (2) as:
rffiffiffiffiffiffiffiffi
SSE
RMSECV ¼ ð3Þ
n
As it can be observed from the Eq. (3), the RMSECV value
decreases as n increases. Though it does not have any range of
acceptable values, the model with lower value is considered
optimum (Jiang et al., 2015).
 S. Sunoj et al. / Computers and Electronics in Agriculture 124 (2016) 234–242 237

2.5.3. Coefficient of determination 3. Results and discussion

The coefficient of determination ðR2 Þ, expressed as a dimension-
less fraction, of a model is an indicator of how well the data follows
the statistical model. It gives the percentage of reliability of the
model which is obtained by regression. Higher R2 values indicate
better reliability. It can be calculated using Eq. (2) as:
!
SSE
2
R ¼ 1
Pn
i¼1 ðym
yi Þ2
where ym is the mean value of the selected quality attribute of all
samples obtained by conventional analysis. As a thumb rule, the
models with R2 values between 0.65 and 0.80 are used for approx-
imate quantification; values between 0.81 and 0.90 are considered
as good prediction models, while the values above 0.90 are consid-
ered as excellent calibration model for prediction (Saeys et al.,
2005).
2.5.4. Residual predictive deviation
To evaluate the accuracy of developed calibration model, resid-
ual predictive deviation (RPD) is another performance measure
which compares the variation in the samples with respect to the
prediction variation. RPD is calculated as the ratio of standard devi-
ation (SD) of reference value and RMSECV.
Standard deviation of reference values
RPD ¼
Standard values of cross-validation
The calibration models with RPD value less than 2 is considered
insufficient for application, between 2 and 2.5 is considered for
approximate quantification, values between 2.5 and 3 as a good
model, while calibration models with RPD values more than 3
can be considered an excellent and most reliable for analytical
tasks (Saeys et al., 2005).
2.5.5. Bias of NIR predictions
Bias value was estimated using the equation reported by
Ferreira et al. (2013) and Teye et al. (2015). It estimates the differ-
ence between experimental value and NIR predictions with usual
notation:
1X n
Bias ¼
i Þ
ðy
y
n i¼1 i
Bias can either be positive or negative. Positive values indicate
that the model was overestimating, while negative values indicate
otherwise. Larger bias values indicate that the NIR predictions vary
significantly from the experimental values (Cantor et al., 2011).
The number of latent variables (LV) is essential to estimate
whether the model is over-fitting or under-fitting. Too many LVs
would result in over-fitting, while fewer LVs lead to under-
fitting. The optimum number of LVs must not exceed one-sixth
the number of samples used for calibration (Kemsley, 1998;
Westad and Marini, 2015). Hence, in the present study 10 LVs were
used for model development.
3.1. Cocoa quality by chemical analysis
3.1.1. Fermentation index variation
The results of fermentation index, pH and total polyphenols of
cocoa beans obtained by conventional chemical analysis are
presented in Table 1. The minimum value of fermentation index
ð4Þ (0.54) was recorded for the pods stored for 0 day on 1st day of
fermentation, while maximum value (1.24) was recorded for beans
stored for 14 days at 6th day of fermentation. The fermentation
index below 1 was considered unfermented and index above 1
was considered fully fermented (Gourieva and Tserevitinov,
1979). For complete fermentation, the fermentation time of 6 days
was required for the pods stored for 0 and 7 days, while the pods
stored for 14 and 21 days took only 4 days to get fully fermented.
The results indicate that increasing fermentation time and pod
storage duration had a significant effect on fermentation index
Fig. 2. Earlier investigations (Kongor et al., 2013; Nazaruddin
et al., 2006) also observed a similar effect on fermentation index
due to pod storage.
3.1.2. Variation of pH
Measured pH of cocoa beans by chemical analysis and the
variation with fermentation days for different days of storage are
also presented in Table 1 and Fig. 2. With days of fermentation
ð5Þ there is a general trend of reduction up to 3 or 4 days from the ini-
tial (5.76–6.11) and then an increase towards the final 6 days
(5.19–6.13); but an overall mild reduction was observed for differ-
ent stored samples. Furthermore, a sharp decrease in the pH,
where the pods were stored for 0 and 7 days, and a gradually
decreasing trend for the pods stored for 14 and 21 days were
observed. This was due to the decrease in pulp volume per seed
because of the water evaporation and inversion of sucrose, which
diminishes total sugar content leading to reduced acid production
during fermentation. The decreasing and increasing trend of pH
during the fermentation days was due to the fact that the adhering
pulp was converted into acid and alcohol, which ceased fermenta-
tion (Afoakwa et al., 2013). After 4th day onwards, the adhering
pulp was released in the form of sweat, which released the acid
and alcohol produced during fermentation and thus pH increased.
In the case of pods stored for 14 and 21 days, the cocoa beans when
ð6Þ
extracted, had lesser pulp adhering to the seeds. However, the
acidic beans had a direct impact on the flavor of chocolate, which
produced astringency taste and seemed to cause problems while
manufacturing chocolate. After pod storage treatments, the beans
obtained after fermentation had slightly higher pH (i.e. less
acidity). These results thus indicated that after 14 and 21 days of
pod storage, the cocoa beans were less acidic than other two pod
storage treatments.
Table 1
Cocoa beans quality parameters measured by conventional and FT-NIR spectroscopy
methods.

Quality parameter Conventional method FT-NIR spectroscopy

2.5.6. Statistical analysis Rangea Mean SDb Rangea Mean SDb
A two-way analysis of variance (ANOVA) was followed to study
Fermentation 0.54–1.24 0.87 0.19 0.53–1.19 0.89 0.17
the effect of fermentation time and pod storage duration on fer- index
mentation index, pH and total polyphenols of cocoa beans. Least pH 4.26–6.13 5.53 0.56 4.06–6.17 5.32 0.48
significant difference (LSD) was applied for the treatment which Total polyphenols 6.48–15.58 11.08 2.41 7.02–16.23 11.15 2.29
had a significant effect (p < :01). ANOVA and LSD were performed (mg g
1)

using AGRESS (Version 7.01, Pascal international software a

Values obtained from pods stored for 0, 7, 14, and 21 days.
b
solutions). SD – standard deviation (decimals).
238 S. Sunoj et al. / Computers and Electronics in Agriculture 124 (2016) 234–242

0 days 7 days 14 days 21 days 0 days 7 days 14 days 21 days

1.4 7
(a) (b)
6.5
1.2
Fermentation index

6
1

pH
5.5
0.8
5

0.6
4.5

0.4 4
1 2 3 4 5 6 1 2 3 4 5 6
Fermentation days Fermentation days

0 days
day 7 days 14 days 21 days

17
(c)
15
Total polyphenols, mg g-1

13

11

5
1 2 3 4 5 6
Fermentation days

Fig. 2. Changes in (a) fermentation index, (b) pH, and (c) total polyphenols during the fermentation.

3.1.3. Total polyphenol variation 3.2. FT-NIR spectral characteristics

Total polyphenol contents for all days of storage studied had
different starting values but decreased in a similar manner with The spectra obtained in the entire spectral range corresponding
days of fermentation (Fig. 2) and the value ranges with mean are to wavenumbers of 12,500–3600 cm
1 for the nondestructive
presented in Table 1. In general, as the days of storage increased quality analysis of cocoa beans are shown in Fig. 3. It is evident that
the total polyphenol decreased at any level of fermentation. all the samples exhibited a similar trend in absorbance, but each
Considering the fermentation effect, a reduction of 25% and 32% sample varied depending on its orientation, surface characteristics,
from the initial total polyphenol contents of 14.84 and 9.53 mg g
1 and most importantly its biochemical constituents. PLS regression
for 0 and 21 days or storage, respectively. A similar decreasing algorithm relates the spectral data with the component of interest
trend was observed with the other days of storage (7 and 14) with in the whole NIR region and determines which region is best fit for
fermentation duration. that particular component.
The decrease in polyphenol could be due to the diffusion of The prominent peaks were caused due to the stretching of
polyphenols into fermentation sweat. Also, the reduction in pulp hydrogen groups from CAH, OAH, and NAH in the sample
volume during pod storage might facilitate the oxidation and (Krähmer et al., 2015; Teye et al., 2015). These observed peaks at
polymerization of polyphenols. The decreasing trend of polyphe- different wavenumber range were caused due to the following
nols (Fig. 2) was similar to the result obtained by Nazaruddin vibrations (Fig. 3):
et al. (2006). Thus, pod storage caused a significant reduction in
the polyphenol compounds, which will reduce the astringency 8100–8600 cm
1: 2nd overtones of CAH groups
and bitterness in cocoa and cocoa products made from the 6700–6900 cm
1: 1st overtones of CAH groups
respective cocoa beans and is considered desirable. The statistical 5100–5300 cm
1: 2nd overtones of C@O groups
analysis (ANOVA) showed, there was a highly significant effect 4000–4800 cm
1: Combinations of amides
(p < :01) on all quality attributes due to pod storage and fermenta-
tion time as well as their interaction (Table 2).

Peaks at 5600–5800 cm
1 and 4000–4200 cm
1 were absent,

which was caused due to 1st overtones, and combination vibra-
tions of CAH stretching (CH2 groups), respectively. The water
Table 2
ANOVA of pod storage and fermentation time on quality parameters.
absorption bands should be eliminated in order to reduce the
interference with the chemical structures of our component of
Source Fermentation index pH Total polyphenols interest. Therefore, in the present study, the water absorption
Pod storage (P) 1114.00a 775.34a 12221.83a regions (10,500–9250, 7100–6800, and 5300–4950 cm
1) were
Fermentation time (F) 3890.98a 340.96a 3066.94a excluded. The remaining regions in five bands such as 9000–
Interaction (P  F) 59.76a 64.22a 141.74a
7450, 6900–6030, 6030–5500, 4950–4500, and 4500–3850 cm
1
a
F-ratio values significant at 1% level. were only selected for the calibration model development using
 S. Sunoj et al. / Computers and Electronics in Agriculture 124 (2016) 234–242 239

Fig. 3. FT-NIR spectra obtained for cocoa beans in the whole region.

the ‘Interactive region selection’ option provided within the OPUS
software. As observed, the prominent peaks were observed at
around 4000, 5200, 6800, and 8200 cm
1 within the NIR region
(Fig. 3). Since the number of samples were less (72 samples),
cross-validation method was followed for calibration model devel-
opment (Westad and Marini, 2015). The cross validation builds the
model using ‘‘leave-one-out” technique and validates the model
with the left out sample. The method used 71 remaining samples
for model building and validated with the left out sample and
repeated until all the samples were validated once. It is possible
to have a more robust model with increased number of samples,
which in turn improve predictions of the models. The spectral pat-
tern obtained were similar to the previous studies (Krähmer et al.,
2015; Teye et al., 2015).
3.3. Calibration models for cocoa beans quality
The FT-NIR spectra were preprocessed by vector normalization,
multiplicative scatter correction (MSC), and first derivative (FD) for
the calibration models development, and R2 and RMSECV were
used as performance parameters (Table 3). A simplified regression
model based on the best preprocessing method for each quality
parameter was also developed, plotted (Fig. 4), and discussed.
3.3.1. Fermentation index calibration models
Model prediction results agreed well with values obtained by
chemical analysis (Table 1). The first derivative preprocessing
was found suitable for fermentation index models than other
two preprocessing methods. The OPUS software considers the vari-
ation of absorbance in spectra, and is directly related to the con-
centration of the component (Tripathi and Mishra, 2009). Model
performance statistical parameters, such as R2 (Eq. (4)), RMSECV
(Eq. (3)), RPD (Eq. (5)) values of the fermentation index model were
0.88, 0.06, and 2.74, respectively. No outliers were detected in
model development. The linear regression plot between experi-
mental values of fermentation index and FT-NIR predicted values
(Fig. 4) show very good linear fit (R2 ¼ 0:88). Also, the bias value
of
0:0004 indicated the calibration model was underestimating.
Spectral regions which had the maximum influence on the
regression model fit can be found out by PLS loading spectra
(Bala and Singh, 2013). The spectral regions between 6033 and
5493, and between 4953 and 3849 cm
1 were found responsible
for predicting fermentation index of cocoa beans (Fig. 5a). The
model prediction results with R2 > 0:80 and RPD in the 2.5–3.0
range were considered reliable and the model could be used as a
good prediction model for fermentation index in cocoa beans.
Although, Teye et al. (2015) and Hue et al. (2014) obtained higher
prediction estimates with R2 > 0:95 and RMSE < 0:9, these exper-
iments were conducted with ground cocoa beans providing an
ideal sample. But in the present study whole cocoa beans were
used, hence, a lower prediction was expected. Other promising
results (Jiang et al., 2015; Giovenzana et al., 2014) have also shown
that FT-NIR spectroscopy could be a reliable tool in predicting the
fermentation levels of food products.

Table 3
FT-NIR spectroscopy calibration model performance statistics results of cocoa beans quality using various preprocessing methods.

Preprocessing method Fermentation index pH Total polyhenols

R2 RMSECV RPD Bias R2 RMSECV RPD Bias R2 RMSECV RPD Bias

Vector normalization 0.83 0.09 1.82 – 0.58 0.35 1.52 – 0.84 0.93 2.53
0.0408
Multiplicative scatter correction 0.86 0.07 2.35 – 0.65 0.31 1.72 – 0.82 0.98 2.40 –
First derivative 0.88 0.06 2.74
0.0004 0.76 0.26 2.05 0.015 0.79 1.06 2.22 –

RMSECV – root mean square error for cross-validation.

RPD – residual predictive deviation.
Bias was calculated only for the best model.
240 S. Sunoj et al. / Computers and Electronics in Agriculture 124 (2016) 234–242

Fermentation index pH
6.50
1.300

NIR predicted value

6.00
NIR predicted value

1.100
5.50

0.900
5.00

R² = 0.88 R² = 0.76
0.700 RMSECV = 0.06 4.50 RMSECV = 0.26
RPD = 2.74 RPD = 2.05
y = 0.899x + 0.089 y = 0.775x + 1.185
0.500 4.00
0.500 0.700 0.900 1.100 1.300 4.00 4.50 5.00 5.50 6.00 6.50
Experimental values Experimental values

Total polyphenols, mg g-1

16
NIR predicted value

14

12

10
R² = 0.84
8 RMSECV = 0.93
RPD = 2.53
y = 0.883x + 1.335
6
6 8 10 12 14 16
Experimental values

Fig. 4. Linear regression plot of calibration between experimental and predicted values by FT-NIR spectroscopy.

Fig. 5. Spectral loadings obtained by PLS regression (a) fermentation index, (b) pH, and (c) total polyphenols.
 S. Sunoj et al. / Computers and Electronics in Agriculture 124 (2016) 234–242
3.3.2. Calibration models for pH
The model preprocessed with the first derivative method for pH
variation resulted in a better performance with higher R2 value of
0.76 and lower RMSECV value of 0.26 compare to the other meth-
ods (Table 3). The bias value of 0.015 obtained for pH indicated the
calibration model was overestimating. There were no outliers
detected in the model development. But the RPD value was only
2.05, which signifies that the model could be used only for approx-
imate quantification (Saeys et al., 2005). The lack of better perfor-
mance can be seen from the scatter data of the linear plot (Fig. 4),
and this explained the nature of variation of pH values as observed
earlier (Fig. 2b), when compared to the other parameters that
exhibited uniform trends. PLS spectral loadings (Fig. 5b), showed
the spectral influence were in the regions of 9003–8223, 6904–
5493, and 4953–4721 cm
1.
It should be noted that the NIR spectroscopy simply records the
variation in absorbance due to the molecular interaction at differ-
ent wavenumbers and specifically relates to the chemical compo-
sition of the sample. Therefore, NIR spectroscopy cannot measure
the pH directly as a pH meter does. But still, the model can be
improved, by increasing the number of samples and narrowing
the range of values. The prediction of pH with higher coefficient
2 0:06 6 RMSECV 6 1:06; and 1:52 P RPD P 2:74) were found
of determination (R > 0:80) was obtained by Krähmer et al.
(2015) and Teye et al. (2015) for ground cocoa samples, Balage
et al. (2015) for pork, Giovenzana et al. (2014) for beer, and Li
et al. (2013) for pears. These studies considered more than 200
samples (compared to 72 in the present study) and obtained better
R2 values.
3.3.3. Total polyphenols calibration models
The total polyphenol content predicted by FT-NIR spectroscopy
was in the range of 7.02–16.23 mg g
1, which compared closely
with the chemical analysis range of 6.48–15.58 mg g
1 (Table 1).
The linear regression plot (Fig. 4) also produced better reliability
of developed model with R2  0:84 and standard error of cross-
validation value of 0.93. Evaluated RPD value of 2.53 showed that
this can be a good model for prediction of polyphenol content. The
analysis showed that the bias value of
0.0408 for total polyphe-
nols indicated the model was underestimating. Also, there were
no outliers detected in model development. For the total polyphe-
nols, the best model was obtained when the spectra were prepro-
cessed with vector normalization (Table 3). The PLS loading spectra
(Fig. 5c) clearly indicated that the prediction region between
6904.3 and 5492.6 cm
1 was the best for quantifying polyphenols
in cocoa beans. Prediction of phenols in mustard also showed max-
imum influence in a similar range of 6102–5446 cm
1 (Bala and
Singh, 2013). Recent studies also reported that FT-NIR as a rapid
tool for estimating total polyphenol content (Aliakbarian et al.,
2015; Giovanelli et al., 2014).
The calibration models developed showed that FT-NIR method
could be reliable. The estimations of all three quality attributes
(fermentation index, pH, and total polyphenols) by chemical meth-
ods took approximately 28 h (24 h for fermentation index, 5 min
for pH, and 3.5 h for total polyphenols). However, for FT-NIR
method, once the calibration methods are developed for individual
quality attributes, a single scan over the cocoa beans would pro-
duce the analyzed results in a minute or less. Moreover, the cocoa
beans used for FT-NIR calibration model development were simple
dried beans used without grinding, making it a purely nondestruc-
tive method.
3.4. Suggestions for future work
The other quality indicators in cocoa, such as fat content, mold
infestations, and beans germination can also be studied using
241
FT-NIR spectroscopy. During the chocolate manufacturing process,
the polyphenol content is responsible for imparting the unique fla-
vor to the chocolate. The effect of different processes on the
polyphenol content during the chocolate manufacturing process
can be another potential area of future studies. The present calibra-
tion modeling can be extended to a wide range of parameters and
with different varieties of cocoa beans. Such future research
outputs will be of great advantage for the traders and chocolate
manufacturing industries, as calibration models for wide range of
parameters and cocoa varieties reduce the quality assessment time
and produce reliable results as demonstrated in this research.
4. Conclusions
The present study successfully demonstrated the potential of
FT-NIR spectroscopy in predicting the quality parameters, namely
fermentation index, pH, and total polyphenols of cocoa beans non-
destructively. FT-NIR spectroscopy calibration models were devel-
oped based on higher coefficient of determination (R2 ), lower root
mean square error of calibration (RMSECV) and residual predictive
deviation (RPD) values; and the models (0:76 6 R2 6 0:88;
acceptable. The models prediction and values obtained through
conventional chemical methods in an overall sense agreed well
for the quality parameters studied. It was found that FT-NIR spec-
troscopy method was more suitable for certain quality attributes
(fermentation index and total polyphenols) than others (pH), and
increasing the number of samples improve the models and predic-
tions. These models can be applied for general screening and rapid
quality estimation (<1 min compared to 28 h of conventional
method), which results in saving significant time and effort in mea-
surements. The research can be potentially extended to other
cocoa beans quality parameters and cocoa varieties.
Acknowledgements
Thanks to R. Pandiselvam, doctoral student, and Dr. R. Arul
Mari, Assistant Professor, Department of Food and Agricultural Pro-
cess Engineering, Agricultural Engineering College and Research
Institute, Tamil Nadu Agricultural University, India for the help
throughout this study.
References
Afoakwa, E.O., 2011. Cocoa cultivation, bean composition and chocolate flavor
precursor formation and character. In: Chocolate Science and Technology. John
Wiley & Sons, pp. 12–34.
Afoakwa, E.O., Kongor, J.E., Takrama, J.F., Budu, A.S., Mensah-Brown, H., 2013. Effects
of pulp preconditioning on total polyphenols, o-diphenols and anthocyanin
concentrations during fermentation and drying of cocoa (Theobroma cacao)
beans. J. Food Sci. Eng. 3, 235–245.
Aliakbarian, B., Casale, M., Paini, M., Casazza, A.A., Lanteri, S., Perego, P., 2015.
Production of a novel fermented milk fortified with natural antioxidants and its
analysis by NIR spectroscopy. LWT-Food Sci. Technol. 62, 376–383.
AOAC, 1995. Association of Official Analytical Chemists, Official Methods of Analysis
of AOAC International.
Bala, M., Singh, M., 2013. Non destructive estimation of total phenol and crude fiber
content in intact seeds of rapeseed–mustard using FT-NIR. Indust. Crops Prod.
42, 357–362.
Balage, J.M., E Silva, S.D.L., Gomide, C.A., Bonin, M.D.N., Figueira, A.C., 2015.
Predicting pork quality using Vis/NIR spectroscopy. Meat Sci. 108, 37–43.
Cantor, S.L., Hoag, S.W., Ellison, C.D., Khan, M.A., Lyon, R.C., 2011. NIR spectroscopy
applications in the development of a compacted multiparticulate system for
modified release. AAPS PharmSciTech 12, 262–278.
FAOSTAT, 2016. <http://faostat3.fao.org/download/Q/QC/E> (Accessed on: April 20,
2016).
Ferreira, D.S., Pallone, J.A.L., Poppi, R.J., 2013. Fourier transform near-infrared
spectroscopy (FT-NIRS) application to estimate brazilian soybean [Glycine max
(L.) Merril] composition. Food Res. Int. 51, 53–58.
Giovanelli, G., Sinelli, N., Beghi, R., Guidetti, R., Casiraghi, E., 2014. NIR spectroscopy
for the optimization of postharvest apple management. Postharvest Biol.
Technol. 87, 13–20.
242 S. Sunoj et al. / Computers and Electronics in Agriculture 124 (2016) 234–242
Giovenzana, V., Beghi, R., Guidetti, R., 2014. Rapid evaluation of craft beer quality
during fermentation process by Vis/NIR spectroscopy. J. Food Eng. 142, 80–86.
Gourieva, K., Tserevitinov, O., 1979. Method of evaluating the degree of
fermentation of cocoa beans. USSR patent 646.
Hue, C., Gunata, Z., Bergounhou, A., Assemat, S., Boulanger, R., Sauvage, F., Davrieux,
F., 2014. Near infrared spectroscopy as a new tool to determine cocoa
fermentation levels through ammonia nitrogen quantification. Food Chem.
148, 240–245.
Jha, S., Garg, R., 2010. Non-destructive prediction of quality of intact apple using
near infrared spectroscopy. J. Food Sci. Technol. 47, 207–213.
Jiang, H., Zhang, H., Chen, Q., Mei, C., Liu, G., 2015. Identification of solid state
fermentation degree with FT-NIR spectroscopy: comparison of wavelength
variable selection methods of CARS and SCARS. Spectrochim. Acta A: Molec.
Biomolec. Spectrosc. 149, 1–7.
Jinap, S., Dimick, P., Hollender, R., 1995. Flavour evaluation of chocolate formulated
from cocoa beans from different countries. Food Control 6, 105–110.
Kemsley, E.K., 1998. Case studies. In: Kemsley, E.K. (Ed.), Discriminant Analysis and
Class Modelling of Spectroscopic Data. Wiley, Chichester, UK, pp. 110–164.
Kongor, J.E., Takrama, J.F., Budu, A.S., Mensah-Brown, H., Afoakwa, E.O., 2013. Effects
of fermentation and drying on the fermentation index and cut test of pulp pre-
conditioned Ghanaian cocoa (Theobroma cacao) beans. J. Food Sci. Eng. 3, 625–
634.
Krähmer, A., Engel, A., Kadow, D., Ali, N., Umaharan, P., Kroh, L.W., Schulz, H., 2015.
Fast and neat–determination of biochemical quality parameters in cocoa using
near infrared spectroscopy. Food Chem. 181, 152–159.
Li, J., Huang, W., Zhao, C., Zhang, B., 2013. A comparative study for the quantitative
determination of soluble solids content, pH and firmness of pears by Vis/NIR
spectroscopy. J. Food Eng. 116, 324–332.
Nazaruddin, R., Seng, L., Hassan, O., Said, M., 2006. Effect of pulp preconditioning on
the content of polyphenols in cocoa beans (Theobroma cacao) during
fermentation. Indust. Crops Prod. 24, 87–94.
Saeys, W., Mouazen, A.M., Ramon, H., 2005. Potential for onsite and online analysis
of pig manure using visible and near infrared reflectance spectroscopy. Biosyst.
Eng. 91, 393–402.
Saltini, R., Akkerman, R., Frosch, S., 2013. Optimizing chocolate production through
traceability: a review of the influence of farming practices on cocoa bean
quality. Food Control 29, 167–187.
Singleton, V., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic-
phosphotungstic acid reagents. Am. J. Enol. Viticulture 16, 144–158.
Sinija, V., Mishra, H., 2009. FT-NIR spectroscopy for caffeine estimation in instant
green tea powder and granules. LWT-Food Sci. Technol. 42, 998–1002.
Subramanian, A., Rodriguez-Saona, L., 2008. Fourier transform infrared (FTIR)
spectroscopy. In: Sun, D. (Ed.), Infrared Spectroscopy for Food Quality Analysis
and Control. Academic Press, New York, pp. 145–178.
Teye, E., Huang, X., Lei, W., Dai, H., 2014. Feasibility study on the use of fourier
transform near-infrared spectroscopy together with chemometrics to
discriminate and quantify adulteration in cocoa beans. Food Res. Int. 55, 288–
293.
Teye, E., Huang, X., Sam-Amoah, L.K., Takrama, J., Boison, D., Botchway, F., Kumi, F.,
2015. Estimating cocoa bean parameters by FT-NIRS and chemometrics
analysis. Food Chem. 176, 403–410.
Tripathi, S., Mishra, H., 2009. A rapid FT-NIR method for estimation of aflatoxin B1
in red chili powder. Food Control 20, 840–846.
Tripathi, S., Patel, K., Bafna, A., 2010. Nondestructive determination of curcuminoids
from turmeric powder using FT-NIR. J. Food Sci. Technol. 47, 678–681.
Veselá, A., Barros, A.S., Synytsya, A., Delgadillo, I., Čopíková, J., Coimbra, M.A., 2007.
Infrared spectroscopy and outer product analysis for quantification of fat,
nitrogen, and moisture of cocoa powder. Anal. Chim. Acta 601, 77–86.
Westad, F., Marini, F., 2015. Validation of chemometric models – a tutorial. Anal.
Chim. Acta 893, 14–24.
Worldatlas, 2016. Top 10 cocoa producing countries. <http://www.worldatlas.com/
articles/top-10-cocoa-producing-countries.html> (Accessed on: April 20, 2016).