Food Control 115 (2020) 107302

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Comparison of different sample preparation techniques for NIR screening T

and their influence on the geographical origin determination of almonds
(Prunus dulcis MILL.)
Maike Arndta,1, Marc Rurikb,f,1, Alissa Dreesa, Katharina Bigdowskia, Oliver Kohlbacherb,c,d,e,f,
Markus Fischera,∗
a
Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146, Hamburg, Germany
b
Applied Bioinformatics, Center for Bioinformatics, Sand 14, 72076, Tübingen, Germany
c
Quantitative Biology Center, University of Tübingen, Auf der Morgenstelle 10, 72076, Tübingen, Germany
d
Biomolecular Interactions, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076, Tübingen, Germany
e
Translational Bioinformatics, University Medical Center Tübingen, Sand 14, 72076, Tübingen, Germany
f
Institute for Bioinformatics and Medical Informatics, University of Tübingen, Sand 14, 72076, Tübingen, Germany

ARTICLE INFO ABSTRACT

Keywords: The aim of the present study was to conduct a systematic and in-depth comparison of four different sample
Almonds preparation techniques for near-infrared (NIR) spectroscopy analysis of almonds and evaluation of their suit-
NIR ability for the geographical origin determination. Although it is generally known that the sample preparation has
Sample preparation an impact on the NIR screening, there is no scientific consensus on a commonly accepted procedure. In this work,
Geographical origin
64 almond samples from six countries were analyzed as whole and bisected nuts as well as in a ground and
Support vector machine
Chemometrics
freeze-dried state (after grinding). In order to assess the suitability for future applications, both the labor effort
for sample preparation and the classification accuracy were evaluated. Using support vector machine (SVM)
classification we obtained a classification accuracy of 80.2% (±1.9%) on the validation set for the determination
of origin of freeze-dried almonds. The other three sample preparations result in at least 8.3 percentage points
lower classification accuracies. Nonetheless, the analysis of whole and bisected almonds is more suitable for an
initial rapid screening due to a lower overall required work effort. The results confirm the influence of the
sample preparation techniques in NIR screening and pave the way for future widespread analytical applications.

1. Introduction
Fourier transform near-infrared (FT-NIR) spectroscopy is a highly
versatile and powerful method in verifying the authenticity of food raw
materials. It has been used to address a wide range of food-related
questions such as the determination of adulteration in olive oil or
honey, of the geographical origin of fish, to distinguish varieties of wine
and quality issues such as mycotoxin contamination (Cozzolino, 2016;
Rodriguez-Saona, Giusti, & Shotts, 2016). In general, FT-NIR spectro-
scopy is a quick analytical method since no extraction is required prior
to analysis. Due to its simple handling and rapid analysis, FT-NIR
spectroscopy is a cost-efficient, non-polluting and thus green analytical
method (Armenta, Garrigues, & de la Guardia, 2008; Gałuszka,
Migaszewski, & Namieśnik, 2013). Therefore, various analytical
methods based on NIR spectroscopy, e.g. the determination of water
content in meat (Porep, Kammerer, & Carle, 2015), are already estab-
lished in industrial routine laboratories.
Even though FT-NIR spectroscopy is generally a fast method, the
sample preparation can differ significantly. Depending on the chosen
sample preparation technique – i.e. for nuts simply using whole or bi-
sected nuts or more elaborately prepared materials like ground or
freeze-dried nuts – time requirement can vary from almost immediate
spectra recording to around 72 h (Richter, Rurik, Gurk, Kohlbacher, &
Fischer, 2019; Zhang, Jiang, Liu, Mei, & Huang, 2017). Currently,
however, there is no universally established form of sample preparation
of food materials for the determination of origin. While in recent studies
Biancolillo et al. and Moscetti et al. analyzed whole hazelnuts, Vitale
et al. bisected pistachios prior to analysis (Biancolillo et al., 2018;
Moscetti, Radicetti, Monarca, Cecchini, & Massantini, 2015; Vitale
et al., 2013). In other laboratories even ground walnuts (Gu et al.,

∗
Corresponding author.
E-mail address: markus.fischer@chemie.uni-hamburg.de (M. Fischer).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.foodcont.2020.107302
Received 2 February 2020; Received in revised form 5 April 2020; Accepted 6 April 2020
Available online 11 April 2020
0956-7135/ © 2020 Elsevier Ltd. All rights reserved.
M. Arndt, et al.
2018) or freeze-dried (after grinding) asparagus were utilized for FT-
NIR measurements (Richter et al., 2019). NIR screening of freeze-dried
(after grinding, hereinafter referred to as just freeze-dried) nuts has – to
the best of our knowledge – never been applied, despite it being a
promising sample preparation technique due to the removal of poten-
tially superimposing water bands in the resulting spectra.
This study shall provide a systematic and in-depth comparison and
evaluation of the above-mentioned forms of sample preparation (whole,
bisected, ground, and freeze-dried) in terms of their suitability for the
determination of origin of almonds (Prunus dulcis MILLER). The prediction
of the geographical origin of almonds is of great economic interest as
the country of harvest determines the market price. While almonds
from the USA cost about 5 US$/kg, almonds from the Mediterranean
region can cost up to 9 US$/kg (related to worldwide export 2018, UN
Comtrade Database, 2018). Although a financial profit could be gen-
erated by misdeclaration of the origin of almonds (Manning, 2016),
there is still no reliable and routinely applicable method of origin de-
termination to counteract this type of food fraud. The potential of NIR
analysis for this application has, however, been hinted at in a recent
publication (Firmani, Bucci, Marini, & Biancolillo, 2019) presenting an
FT-NIR-based method for differentiating “Avola Almonds” from other
Italian almonds.
The aim of this study is therefore to investigate the influence of
various sample preparations on the FT-NIR analysis of almonds. Whole,
bisected, ground, and freeze-dried almonds are compared regarding
their processing effort, analysis time, required sample volume, re-
producibility, and, in particular, their ability to classify the geo-
graphical origin. So, almonds from various economically relevant pro-
duction countries were acquired and analyzed in all four sample
preparations via a non-targeted FT-NIR spectroscopy approach. The
data were evaluated by multivariate analysis, especially principal
component analysis (PCA) and support vector machine (SVM) classifi-
cation.
2. Materials and methods
2.1. Almond sample acquisition
In order to assess the suitability for geographical origin determi-
nation, a total of 64 authentic almond samples from six different
countries were analyzed. The samples were acquired directly from the
producers and exporters. With the exception of the Australian samples,
which stem from 2019 crops, all almonds were harvested in 2018. Due
to the seasonal differences in the southern hemisphere, however, a six-
month discrepancy of the harvest time is inevitable. In an attempt to
cover a broad spectrum of almonds, 17 different varieties (e.g. Non
Pareil, Padre, Butte, Tuono) and additionally even bitter almonds were
chosen.
2.2. Sample preparation
100–1000 g of the acquired almond raw material, shelled and un-
shelled, were shock frosted in liquid nitrogen for 5 min each and sub-
sequently stored at −20 °C. If necessary, the almonds were cracked
manually under dry ice cooling (−78.5 °C) to safely remove remaining
endocarp. The samples were stored at −20 °C. Each of the 64 acquired
authentic almond samples were eventually analyzed after undergoing
the four sample preparation techniques which are listed in the fol-
lowing sections.
2.2.1. Preparations for analyzing whole almonds
Five almonds with intact tegument (brown skin) were selected from
the available sample material. If the morphology of the seeds visually
differed greatly within the sample, almonds were chosen which also
differed in shape and/or color in order to cover the sample's variance as
best as possible. The almonds were thawed at room temperature
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Food Control 115 (2020) 107302
(22 °C ± 2 °C) before the FT-NIR analysis. After the analysis, the al-
monds were bisected to check for visible damage due to the ability to
detect internal damages via NIR-spectroscopy (Nakariyakul, 2014).
2.2.2. Preparations for analyzing bisected almonds
The selection of the five almonds was conducted as described in the
previous section. After thawing (22 °C ± 2 °C), the almonds were
manually bisected widthways resulting in a plane contact surface ap-
plicable for FT-NIR analysis.
2.2.3. Preparations for analysis of ground almonds
At least 100 g of whole frozen almonds were ground to homo-
geneous powder by using a knife mill (Grindomix GM 300, Retsch,
Haan, Germany). In order to prevent frictional heat, the grinding was
carried out using dry ice (at least twice the sample mass). The ground
material was stored at −20 °C for a minimum of 24 h to ensure
quantitative dry ice sublimation. Prior to FT-NIR analysis, 1.25 g
(±0.1 g) of the ground material was thawed at 22 °C (±2 °C) in closed
glass vials (52.0 mm × 22 mm x 1.2 mm, Nipro Diagnostics Germany
GmbH, Ratingen, Germany).
2.2.4. Preparations for analysis of freeze-dried almonds
The ground almonds (obtained after grinding process described in
Section 2.2.3.) were freeze-dried using a freeze dryer (Beta 1–8
LSCplus, Martin Christ Freeze Dryers GmbH, Osterode, Germany) to
decrease the water content to about 1% by weight. About 60 g ground
almonds (incl. dry ice) were freeze-dried for 24 h. Subsequently, the
ground material was manually stirred and freeze-dried for an additional
24 h. Thereafter, 1.25 g (±0.1 g) of freeze-dried almond powder was
thawed at 22 °C (±2 °C) in closed glass vials (52.0 mm × 22 mm x
1.2 mm, Nipro Diagnostics Germany GmbH, Ratingen, Germany).
2.3. NIR spectroscopy
The thawed samples were analyzed using a FT-NIR spectrometer
with an integration sphere (TANGO, Bruker Optics, Bremen, Germany).
The spectra were acquired in reflectance mode with 50 scans per
spectrum with a resolution of 2 cm−1. A wavenumber range of 11,550
until 3950 cm−1 was selected. Data acquisition was carried out via
OPUS software (Bruker Optics, Bremen, Germany).
Spectra of the ground and freeze-dried samples were acquired inside
glass vials (see Section 2.2.3), while the whole and bisected samples
were put directly on the instrument's surface.
Depending on the form of sample preparation, different numbers of
replicates are required for a sufficient coverage of the almonds’ basic
populations. While the ground and freeze-dried samples required only
five recorded spectra, analysis of whole and bisected almonds was
conducted with six spectra per almond (amounting to a total of 30
spectra per sample). A more detailed description of the four different
procedures can be taken from Table 1. All samples were measured at
room temperature (22 °C ± 2 °C).
2.4. Spectra pre-processing
To avoid overfitting and enable comparability, multiplicative
scatter correction (MSC), smoothing, first order derivative, reduction of
variables, and median formation were applied as data pre-processing
steps.
At first, MSC was used in order to equalize additive and multi-
plicative scattering effects using the mean spectrum of each sample
preparation technique as reference. The mentioned scattering effects
lead to differences in the spectra caused by inhomogeneous physical
effects and not – as is desired – by geographical origin. To put it more
precisely, the morphology of whole and bisected almonds can result in
different scattering as the shape of the seeds or the surface structure of
the tegument varies. Moreover, different morphologies result in a
M. Arndt, et al.
Table 1
Spectra acquisition of the four sample preparation techniques for representing
the almonds’ basic population.
whole bisected ground freeze-dried
required 30 30 5 5
spectra
sample 5 almonds 5 almonds 1.25 g ± 0.1 g 1.25 g ± 0.1 g in
volume in glass vials glass vials
procedure three spectra per three spectra thorough shake thorough shake
side per almond, per almond (vertically and (vertically and
120° turn half, 120° turn horizontally) horizontally)
between every between every between the between the
measurement measurement replicates, replicates,
uniform filling uniform filling
level level
different amount of reflected light causing baseline shifting. Scattering
effects caused by the inhomogeneous physical effects are also observed
analyzing ground or freeze-dried samples as the particle size is not
perfectly uniform.
In addition, the first-order gap-segment derivatives were calculated
to eliminate offset and baseline drifts (gapDer function implemented in
the R package prospectr 0.1.3, using a derivative order m of 1, a
smoothing window s of 11 and a window size w of 11; Stevens &
Ramirez-Lopez, 2014). Gap-segment derivatives use a window-based
approach that first averages the points in a smoothing window centered
around the current measurement point, then calculate the derivative by
taking the difference between two points separated by a given gap size
(Norris & Williams, 1984; Rinnan, Van Den Berg, & Engelsen, 2009).
The high resolution of 2 cm−1 used during spectra acquisition re-
sulted in 3725 variables that often contain similar information for ad-
jacent wavenumbers. For this reason, the average of five contiguous
wavenumbers was taken (binning function implemented in the R
package prospectr 0.1.3), reducing 3693 (after the first derivative) to
739 variables.
The final step of data pre-processing was to take the median of each
wavenumber of all measured spectra for the same sample (30 spectra
for whole and bisected samples and five spectra for ground and freeze-
dried samples). The median was used instead of the arithmetic average
in order to minimize the influence of potential outliers.
2.5. Multivariate data analysis and classification models
Multivariate data analysis was conducted separately for each al-
mond sample preparation. Each preparation leads to one data set con-
taining the results of all 64 acquired almond samples.
First, a principal component analysis (PCA) was applied to visualize
the present data. The PCAs were performed on the pre-processed and
centered data (mean = 0). Subsequently, the supervised learning al-
gorithm support vector machine (SVM) was applied for classifying the
acquired data regarding the almond samples' geographical origin. SVM
is a state-of-the-art classification method that can be used to train both
linear and non-linear classifiers (Cortes & Vapnik, 1995). In this study
SVM was performed using LIBSVM (Chang & Lin, 2011), a publicly
available library that implements methods for the training and classi-
fication of SVMs. LIBSVM was accessed using the e1071 interface (R
Core Team, 2019, R version 3.6.0; Meyer et al., 2019, ‘e1071’ version
1.7–2). As SVM is a binary classifier, for multiclass classification the
one-versus-one approach implemented in LIBSVM was used, i.e. a
binary classifier is created for each pair of classes. A new data point is
then classified by taking the most frequent class label.
In order to avoid overfitting and simultaneously optimize the model
parameters, a nested cross-validation was conducted (Krstajic,
Buturovic, Leahy, & Thomas, 2014; Meyer et al., 2019; Varma & Simon,
2006). This approach uses an inner cross-validation to optimize model
3
Food Control 115 (2020) 107302
parameters and an outer cross-validation to assess the model perfor-
mance. First, the data set was separated into five equal parts by stra-
tified random sampling regarding the geographical origin. Four parts
were used to train the model in an inner cross-validation and one is set
aside as a test set. This process was repeated five times so that each part
operated as a test set once (5-fold outer cross-validation). For each fold
of the outer cross-validation, an inner 10-fold cross-validation was
performed within the respective training set to optimize the model
parameters that are then validated using the set-aside test set of the
outer cross-validation. The entire nested cross-validation was repeated
20 times with different random stratified splits into training and test set
to offset potential bias from their composition.
3. Results and discussion
3.1. Spectra interpretation
Fig. 1 compares the median spectra of the four processing forms
after MSC (9000–3950 cm−1, median value of all samples of each
sample preparation method, see Fig. S1 for whole and unprocessed
spectra). A precise peak assignment cannot be achieved, since peak
overlaps are unavoidable when complex matrices like almonds are
analyzed. Nevertheless, the spectra of the bisected, ground as well as
the freeze-dried almonds show similar trends in absorbance which can
mainly be attributed to the predominant lipids. The HC]CH band oc-
curs at approximately 8550 cm−1 (C–H, second overtone) and is most
likely caused by the carbon–carbon double bonds of the almonds’ un-
saturated fatty acids. The aliphatic hydrocarbon part of the lipids ab-
sorbs in various areas: at about 5800 cm−1, the C–H stretch first
overtone of methylene is located while the symmetric CH2 bond vi-
brations (C–H, stretch first overtone) appear at around 5680 cm−1.
Another lipid associated absorbance is located at around 4330 cm−1
which is caused by the C–H bending (second overtone). Besides lipids,
some other characteristic bands were observed. At around 4855 cm−1,
protein vibrations are located due the amide combination band of
CONH2. The broad absorbance band in a range of 7100–6100 cm−1 is
composed of the N–H stretching (first overtone) of proteins and the
water associated absorbance (O–H symmetric and asymmetric
stretching combination, first overtone). Furthermore, a combination
band of water appears at a wavenumber of around 5155 cm−1 due to
the O–H stretching and H–O–H bending combination (Buijs & Choppin,
1963; Shenk, Workman, & Westerhaus, 2001; Weyer & Workman,
2012).
Examining the NIR spectra of Fig. 1, it becomes evident that the
median spectrum of the whole almonds' analysis differs from the other
spectra: apart from the overall lower absorbance values, the spectra
also exhibits a partially disparate absorbance pattern. While the slightly
weaker lipid bands are still observed, the absorbance in the range of
4960–4510 cm−1 is significantly increased. When visually comparing
the whole almond samples with samples of the other preparations, the
most apparent difference is the tegument. As in NIR spectroscopy the
samples surface majorly impacts the measurement, the tegument's
contribution to the spectra is – for obvious reasons – at its maximum in
the spectra recorded from whole almonds. Hence, it seems likely that
the band from 4960 to 4510 cm−1 derives from composition differences
of the tegument. In order to verify this explanatory approach, FT-NIR
analysis of different compartments was conducted. Subsequently, one
whole almond was blanched and the removed tegument as well as the
blanched almond were analyzed separately (see Fig. S2). In these
spectra, the intensity increase of the band from 4960 to 4510 cm−1 is
only observable in the tegument and the whole almond. While the te-
gument has fiber content of about 46 wt%, the whole almonds merely
exhibit 11–14 wt% fiber. The fiber fraction consists mainly of cellulose,
hemicellulose and lignin – biological macromolecules which all form
complex spectra independently – and might result in marked absor-
bance deviations due to the O–H bending and C–O stretching
M. Arndt, et al.
Fig. 1. MSC-corrected spectra of whole, bisected, ground, and freeze-dried al-
monds.
−1
combination in the range of 4960–4510 cm (Blackwell, 1977;
Ruggeri, Cappelloni, Gambelli, Nicoli, & Carnovale, 1998; Yada,
Lapsley, & Huang, 2011).
As previously mentioned, the whole almonds’ spectra exhibit overall
lower absorbance values caused by the smaller amount of reflected
light. This might be due to the fact that light can pass by the rounded
almond. Moreover, the porous structure of the tegument allows the
light to penetrate deeper and thus reduces the intensity of the reflected
light (Socias, Kodad, Alonso, & Gradziel, 2007).
While the median spectra absorbance values of bisected almonds are
the highest overall, the absorbance values show a relative decrease in a
range of 4370–4250 cm−1 compared to the spectra of homogenized
almonds. This could also be caused by the reflection of light since the
diameter of bisected almonds is usually slightly smaller than the light
beam width of the spectrometer (6 mm). A coverage of the light beam
by increasing the almonds surface can theoretically be achieved by
bisecting the almonds lengthways. Yet, this approach is less easy to
apply and may result in insufficiently thick and highly frangible almond
halves. Overall, the MSC-corrected spectra of the bisected almonds (see
Fig. 2. Median almond spectra after MSC and first derivative for (a) whole, (b) bisected, (c) ground, and (d) freeze-dried almonds.
4
Food Control 115 (2020) 107302
Fig. 1) are comparable to those of ground almonds, since in both cases
mainly the lipid-rich kernel is analyzed.
There are significant absorbance differences between MSC-cor-
rected spectra of the ground and freeze-dried samples – as expected –
regarding the water bands (about 6900 cm−1 and 5155 cm−1) since the
water content is reduced in the freeze-dried samples. The lipid-asso-
ciated bands of the freeze-dried and ground samples show clear simi-
larities since lipids are not strongly influenced by the additional lyo-
philization.
In conclusion, the four sample preparation techniques show com-
parable absorbance trends, however, the expression of the bands differs
in the recorded spectra. For this reason, the suitability regarding the
geographical origin determination for each preparation is discussed
separately in the following.
3.2. Suitability for the classification of geographical origin
Fig. 2 illustrates the derivative spectra (median over the countries of
origin, 9000–4000 cm−1, scatter corrected) for the four different
sample preparation techniques. Herein, intensity deviations with regard
to the geographical origin become apparent. The lipid associated band
at about 4330 cm−1, for example, shows clear intensity differences
depending on the sample's geographical origin. In numerous studies
lipids have already been mentioned as potential markers for the de-
termination of the geographical origin, as they can be influenced by
exogenous conditions (Amorello, Orecchio, Pace, & Barreca, 2016;
Klockmann, Reiner, Bachmann, Hackl, & Fischer, 2016). The extent of
these intensity differences varies greatly depending on the chosen form
of sample preparation. In order to depict these differences, the pre-
processed data (according to Section 2.4) were visualized utilizing a
principal component analysis (PCA). In Fig. 3, the first principal com-
ponent (PC1) is plotted against the second (PC2) for each preparation
technique. None of the shown PCAs allows for visual differentiation of
the individual countries entirely. For whole almonds, the first two PCs
explain 82.2% and 6.6% of the total variance (88.8% in total). In
contrast to the PCAs of the other sample preparations which represent
M. Arndt, et al.
Fig. 3. PCA score plots showing differentiation for (a) whole, (b) bisected, (c) ground, and (d) freeze-dried almonds.
only about 70% of the variance in the first two PCs, cluster trends are
observable slightly better in the whole almond PCA plot. However, the
Mediterranean almonds (Spain and Italy) form a cluster in all shown
PCAs and can thus be distinguished from other countries of origin. The
separation of the Mediterranean almonds is a relevant concern –
especially from an economic perspective – as these are the most ex-
pensive almonds compared to the almonds from the other origins (UN
Comtrade Database, 2018). Higher-order PCs may also contain in-
formation that contributes to the determination of geographical origin
(see Figs. S3–S6) since the explained variance increases up to 20 per-
centage points by adding the third and the fourth PC.
In order to compare the four sample preparation techniques quan-
titatively, a support vector machine (SVM) was used to classify the al-
monds regarding their geographical origin. A one-versus-one classifier
with a Gaussian radial basis function (RBF) kernel was trained via
LIBSVM (R package ‘e1071’) and validated using repeated nested cross-
validation which allows to optimize model parameters and obtain and
unbiased estimation of the generalization performance. The hy-
perparameter optimization was performed using a grid search in the
inner cross-validation loop (cost C from 10−5 to 105, gamma γ from
10−6 to 10−1).
5
Food Control 115 (2020) 107302
Further comparison of the classification results is possible since the
same almond samples were used for each sample preparation. As al-
ready presumed from the spectra, the validation accuracies (hereinafter
referred to as just classification accuracy) differ depending on the
chosen preparation (see Table 2). Comparatively, the freeze-dried al-
monds show the highest classification accuracy of 80.2% (±1.9%). The
superiority of this preparation is, most likely, due to an effective re-
moval of water which can cause unwanted signal overlay possibly re-
sulting in information loss. While the analysis of ground and bisected
almonds leads to accuracies of 71.9% (±3.5%) and 64.5% (±3.5%),
respectively, the most easily and quickly feasible analysis of whole al-
monds still achieves accuracies of 62.6% (±2.8%). The comparably low
accuracies of the whole and bisected almond analysis are most probably
explained by the insufficient coverage of the light beam resulting in
significant loss of information. Additionally, the classification accuracy
of whole almonds could be decreased by the influence of the tegument.
As the tegument is formed in an earlier growth phase, changes in ex-
ternal conditions have a greater impact on the inner kernel which grows
in a later phase. For example, water deficiency in a crucial growth
phase can lead to irregular kernel development (e.g. wrinkled almonds)
where only the tegument is fully developed (Hawker & Buttrose, 1980).
M. Arndt, et al. Food Control 115 (2020) 107302

Table 2 preparation, a comparability is preferred with samples of one harvest

Mean confusion matrix across all 20 repeats of the nested cross-validation of all period.
sample preparation techniques used. Confusion values are given as count va-
lues, classification accuracies as percentage.
3.3. Comprehensive comparison of the different sample preparation
predicted techniques
AU ES IR IT MA US
Apart from the classification accuracy, there remain other para-
actual whole (62.6%) AU 4.90 1.70 0.00 1.90 0.40 0.10 meters of fundamental importance for the proper selection of the most
ES 1.35 11.55 0.15 1.20 0.70 0.05 appropriate preparation technique. A summary of these parameters can
IR 0.85 1.55 1.45 0.00 0.70 1.45
be obtained from Table 3 which additionally presents the required work
IT 0.70 1.65 0.00 19.60 0.05 0.00
MA 0.05 2.95 1.10 0.10 0.55 0.25 time – both active and passive – and the required sample quantity.
US 1.55 1.80 1.65 0.00 0.00 2.00 As the feasibility, short processing time and low overall required
bisected (64.5%) AU 6.60 0.05 0.00 1.10 0.00 1.25 work effort are highly advantageous – especially from an economic
ES 1.65 7.55 0.00 5.75 0.00 0.05 point of view – the analysis of whole and bisected almonds prima facie
IR 0.05 2.00 2.90 0.20 0.00 0.85
IT 0.10 2.70 0.20 18.25 0.00 0.75
clearly stand out. However, this is not always the only relevant concern.
MA 0.00 0.00 0.15 0.00 4.70 0.15 For example, while ground and freeze-dried material from a sufficient
US 3.10 0.05 0.05 1.40 1.10 1.30 amount of almonds (here min. 100 g) might enable future precise
ground (71.9%) AU 6.65 0.10 0.00 0.00 0.55 1.70 analyses of almond admixtures, using whole or bisected almonds is less
ES 0.00 11.20 0.00 3.45 0.00 0.35
suitable for such purposes. Another benefit of grinding as sample pre-
IR 0.00 0.50 4.30 0.00 0.95 0.25
IT 0.00 3.05 0.00 18.95 0.00 0.00 paration is that the subsequent analysis is easily transferred to almond
MA 0.35 1.30 1.75 0.00 0.95 0.65 flour, which is being consumed more and more in recent years (FMI,
US 1.80 0.40 0.10 0.20 0.55 3.95 2018).
freeze-Dried (80.2%) AU 5.45 2.15 0.00 0.15 0.00 1.25 While freeze-drying of the almonds leads to the most accurate
ES 1.05 11.35 0.00 2.05 0.00 0.55
classification, it is also the most time-consuming sample preparation.
IR 0.05 0.00 5.70 0.00 0.25 0.00
IT 0.15 0.95 0.00 20.90 0.00 0.00 Nevertheless, the lyophilization time implemented in this study (48 h)
MA 0.00 0.00 0.10 0.00 4.90 0.00 could be reduced by using less than 60 g of sample material. If very
US 1.05 1.90 0.00 1.00 0.00 3.05 little time is available for the analysis, using whole almonds might be
the most reasonable method e.g. for inspecting incoming material.
Apart from lower accuracies, analysis of whole almonds has one addi-
In prior studies, the amount of analyzed almonds was evaluated in tional major setback: due to the naturally present water content in
order to exclude a potential influence of the basic population at this whole almonds, obtained spectra show broad absorbance bands at
point. There is no consensus on the required quantity of almonds re- wavenumbers of about 5155 cm−1 and 6880 cm−1. As these water-
garding the NIR screening of whole samples: in some cases one nut is related peaks could show different absorbance values depending on the
analyzed per sample (Moscetti et al., 2015), while in other studies five geographical origin (see Fig. 2), they might alter the obtained results as
nuts (Pannico et al., 2015) were included for different analytical ap- water content may vary within the same sample due to variety, storage
proaches. Analogous to Pannico et al., five almonds per sample were or transport. This issue could be mitigated by the exclusion of the water
measured for whole and bisected almonds, providing a good trade-off bands (6500–5500 cm−1) during the data processing according to Teye
between the required work effort and an accurate representation of the et al. (Teye, Huang, Dai, & Chen, 2013). Yet, the loss of information
analyzed almond population. reduces the obtained classification accuracy to around 54.6% (±3.5%)
In order to verify the determination of the almonds' origin via FT- and thus the sensitivity of the method (see Table S2).
NIR spectroscopy, the data set requires extension. As more samples per In conclusion, despite the high practical effort, the most promising
group increase the statistical power, a larger data set enhances the sample preparation technique for the determination of the geographical
model's robustness. Additionally, the influence of different harvest origin of almonds turned out to be freeze-drying subsequent to
years cannot be assessed based on the present data. Since the metabo- grinding. Classification of freeze-dried almonds is more robust, most
lome, which is closest to the phenotype, may vary due to different likely since varying water contents, e.g. caused by transport conditions,
climatic conditions, the harvest year has an influence on the non-tar- can no longer affect the spectra. Nonetheless, the analysis of whole or
geted FT-NIR analysis (Lee et al., 2010; Richter et al., 2019). However, bisected almonds are more suitable for an initial rapid screening. This
as the present study is focused on the optimization of the sample work unprecedentedly shows the significant impact of comprehensive

Table 3
Comprehensive comparison of the four sample preparations regarding work time – active and passive –, sample quantity and classification accuracy via support
vector machine (SVM).
whole bisected ground freeze-dried

estimated active work time (measurement and 10 min 10 min 30 min 50 min (inclusive treatments during freeze-
preparation) drying)
estimated passive work time (storage or freeze-drying) – – 24 h (for sublimation of dry ice) and min. 4 h 48 h (freeze- drying) and min. 4 h thawing
thawing
sample quantity 5 almonds 5 almonds min. 100 g min. 100 g
classification accuracy via SVM 62.6% 64.5% 71.9% 80.2%

6
M. Arndt, et al.
optimization of the sample preparation prior to FT-NIR screening.
In order to facilitate novel food profiling approaches like determi-
nation of the almond variety, new considerations and additional tests
are required. Since the composition – especially also the thickness – of
the tegument is strongly influenced by the genotype (Hawker &
Buttrose, 1980), it is conceivable that the analysis of whole almonds
would be more suitable in this case. However, as such applications are
yet to be developed, this work might serve as a starting point for pro-
mising analytical works in the future.
4. Conclusions
Four sample preparation techniques have been compared in terms
of their suitability for geographical origin determination of almonds. In
addition to the multivariate classification via SVM, the work time and
the mandatory sample size of each preparation are also discussed and
evaluated. Despite the high work and time involved, freeze-drying in
combination with prior grinding turned out to be the most reliable
preparation technique for origin determination of almonds.
Due to the highly similar composition of other nuts or seeds, this
procedure could possibly be transferred to these matrices after a man-
ageable adaptation and validation. The present study shows that FT-
NIR screening is suitable for an initial rapid determination of the origin
of almonds, for example during incoming goods inspections. In order to
improve the significance of the results, further investigations should be
carried out. In this context, further work would be important to shed
light on the influence of the harvest year and, in addition, other
countries of origin, e.g. France, Germany or China, would round off the
picture.
CRediT authorship contribution statement
Maike Arndt: Conceptualization, Investigation, Methodology,
Writing - original draft. Marc Rurik: Methodology, Software, Formal
analysis, Writing - original draft, Visualization. Alissa Drees:
Investigation, Writing - review & editing. Katharina Bigdowski:
Investigation, Writing - review & editing. Oliver Kohlbacher:
Conceptualization, Writing - review & editing, Supervision. Markus
Fischer: Conceptualization, Resources, Writing - review & editing,
Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgment
In particular, we would like to thank Marie Sophie Oberpottkamp,
Christian Ahlers and Kerstin Blum for their support in sample pre-
paration. We also want to thank Doreen Teske and Nils Neumann for
the help in sample acquisition. Additionally, we thank Bernadette
Richter, Torben Segelke and Tilman Eckert for providing expertise.
This work was funded by the University of Hamburg and was
strongly influenced by ideas developed within the scientific joint pro-
ject “Food Profiling” (funding code: 2816500914) funded since 2016 by
means of the Federal Ministry of Food and Agriculture (BMEL) by a
decision of the German Bundestag (parliament). Project support is
provided by the Federal Institute for Agriculture and Food (BLE) within
the scope of the program for promoting innovation. The focus of Food
Profiling lies on developments in the area of instrumental analysis for
the authentication of foodstuffs. Therefore, we would like to thank all
working groups involved in this project as well as the financial sup-
porter.
7
Food Control 115 (2020) 107302
Abbreviations
AU Australia
ES Spain
FT-NIR fourier transform near-infrared
IT Italy
IR Iran
MA Morocco
MSC multiplicative Scatter Correction
PC principal component
PCA principal component analysis
RBF radial basis function
sd standard deviation
SVM support vector machine
US or USA United States of America
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodcont.2020.107302.
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