Algerian bee pollen classification by Attenuated Total Reflectance Fourier

Transform Infrared Spectroscopy (ATR-FTIR) combined with

chemometrics analysis

Bachir Ben Seghir1, Hadia Hemmami2, Abdelkrim Rebiai2, Soumeia Zeghoud2,

Fatima Brahmia2, Djamel Ghernaout3,4, Ramzi Hadj Lajimi5, Noureddine
Elboughdiri3,6, Abdelfattah Amari7

1
Laboratory of Industrial Analysis and Materials Engineering (LAGIM), University 8 May 1945, P.O.
Box 401, Guelma 24000, Algeria
2
LaboratoryValorisation and Technology of Saharan Resources (VTRS), University of El-Oued, P.O.
Box 789, El-Oued 39000, Algeria
3
Chemical Engineering Department, College of Engineering, University of Ha’il, P.O. Box 2440, Ha'il
81441, Saudi Arabia
4
Chemical Engineering Department, Faculty of Engineering, University of Blida, P.O. Box 270, Blida
09000, Algeria
Department of Chemistry, College of Science, University of Ha’il, P.O. Box 2440, Ha'il 81441, Saudi
5

Arabia
6
Chemical Engineering Process Department, National School of Engineering Gabes, University of
Gabes, Gabes 6011, Tunisia
7
Chemical Engineering Department, College of Engineering, King Khalid University, Kingdom of
Saudi Arabia

Corresponding author*:
Phone: +213-

Abstract

Bee pollen is prepared themselves by pollens collecting from plants and has nutritive and
therapeutic properties that make it attractive for human health. It has a typical composition
related to the botanical origin and geographical location. This study aims to distinguish and
identify bee pollen belonging to different Algerian regions and different plants. A
methodology for the identification of pollen was developed based on Attenuated Total
Reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. This method is simple and
fast where samples are not destroyed, also unsupervised statistical methods principal
component analysis (PCA) and hierarchical clustering analysis (HCA) are performed.
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Seventy-two pollen samples were collected and the ATR-FTIR spectra were recorded without
processing the samples. ATR-FTIR spectra analysis allowed a reliable determination of the
components present in the different samples. Further, PCA and HCA were utilized to evaluate
the differences and similarities between the collected samples. Indeed, the PCA score plot and
HCA based on ATR-FTIR revealed the same discriminatory trend, where the samples were
divided into three main classes based on their total bee pollen. As a result, the PCA along with
the HCA was a good and consistent model for identifying and distinguishing pollen grains.
Keywords Bee pollen, ATR-FTIR Spectroscopy, Chemometrics, Multivariate data analysis

Abbreviations
ATR–FTIR Attenuated Total Reflectance Fourier Transform Infrared
PCA Principal Component Analysis
HCA Hierarchical Clustering Analysis
SIMCA Soft Independent Modeling by Class Analogy
PLS Partial Least Squares

Introduction
Bee pollen, usually referred to as the "life-giving dust", comes from the agglutination of
the salivary substances of floral pollen, nectar or honey, and bees [1]. Further, bee pollen has
been used in conventional medicine and supplemental foods for many years, as well as in
alternative diets, primarily thanks to its medicinal properties and health benefits [2],
producing protective effects on human health, and preventing prostate problems [3], allergy
desensitization [4], arteriosclerosis [5] and tumors [6]. Bee pollen has been reported to
increase mitotic rates, encourage tissue repair, enhance toxics removal, and reduce excessive
cholesterol levels [7]. Its radical scavenging operations have already been documented [8].

This material is also used as a food supplement because it contains the richest natural
source of carbohydrates, basic fibers, proteins, and lipids in amounts ranging from 13 to 55%,
0.3 to 20%, 10 to 40%, 1 to 10%, respectively. The minerals and trace elements, vitamins and
carotenoids, phenolic compounds, flavonoids, sterols, and terpenes are other minor
components. Flavonoids and phenolic acids have been suggested to be responsible for the
biological activities of these compounds [9]. Indeed, some nutritionists claim that only
consuming bee pollen will allow human beings to live properly [7].

Many analytical techniques such as spectroscopy (UV – Vis, FTIR, NMR, and MS) and
chromatography (TLC, HPLC, and GC) have been applied in the development of methods for

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natural products (as bee pollen) identification and discrimination. FTIR spectroscopy may be
an attractive choice among these techniques because it can meet the requirements of efficient
analysis (i.e., simple to use, effective, and inexpensive) [10]. While natural products can
contain a wide variety of chemical components, it is often found that their FTIR spectra vary
even for the same species [11]. FTIR has already been commonly used and is a well-
established method in various industries, including the herbal industry, for quality assurance
[12].

ATR-FTIR spectra include complex data describing the total chemical signal in a
sample. Changes in band location and strength in the FTIR spectra can be related to changes
in a sample's chemical composition. FTIR spectra may, therefore, be used to differentiate
between closely related species although the composition of a chemical compound is not
understood [12]. Obvious benefits of applying FTIR to differentiate between different
medicinal plants are not only successful and precise but also simple and non-separate.
Discrimination by visual inspection in the FTIR spectra is not simple because the FTIR
spectra pattern is very complex, and the assistance from chemometrics methods is necessary
to resolve this issue [13]. The benefit of using chemometrics to interpret FTIR is the ability to
relate the spectral pattern to secret information found in a sample [14]. FTIR spectral analysis
was widely used alone or in conjunction with chemometrics methods to classify species and
discriminate against some closely related species [15, 16].

Given its immense potential to cause allergic reactions in humans, rapid pollen
identification is still a daunting analytical task [17].There has been an increasing number of
experimental works in recent years devoted to an alternative and more objective method of
pollen identification focused on the vibrational spectra of pollen samples [18]. FTIR offers
chemical information rather than morphological information as it allows us to investigate the
vibrational dynamics of biochemical components such as lipids, peptides, proteins, nucleic
acids, and sugars [19].

FTIR spectroscopy has recently become an increasingly well-accepted analytical

method, owing to its versatility and cost-per-sample advantages. It achieves a high speed of
examination, which needs few to no planning of samples.

FTIR spectroscopy has been commonly used as an analytical method in numerous

laboratories and sectors, such as foodstuffs [20-22], pharmaceuticals [23, 24], and control of
quality [25, 26].

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 In this sense, using FTIR spectroscopy combined with multivariate statistical techniques
would allow large data sets to be analyzed simultaneously and patterns to be identified in the
samples or groups [27].

A number of studies on the usage of FTIR for the authentication, recognition or

classification of many agro-foods have been reported so far, notably honey [28-30], propolis
and bee pollen [31-33].
Using chemometrics together with classical methods for the classification of different
honey, propolis and pollen samples has been proposed in previous researches [18, 19, 27, 29,
31, 34, 35].
To our knowledge, there is no extensive study to classify bee pollen by geographical
region in Algeria that has been conducted on bee pollen by application of FTIR spectroscopy
in association with chemometrics treatments.

This work aimed (Fig. 1) to propose the model of classification of the Algerian pollen in
samples from thirteen different regions and different plants in a simple and fast manner
without destroying the sample, and this using ATR-FTIR spectroscopy combined with
unsupervised multivariate statistical methods (PCA and HCA).

Materials and Methods

Samples
Seventy-two samples of Algeria bee pollen from thirteen states were collected by
specialists in beekeeping, in a period ranging between 2016 and 2018. The detailed
information about the areas of study samples and their date of harvest is shown in Table 1.
The pollen was collected from the hives into small storage bottles and kept until the chemical
study period.

ATR-FTIR spectroscopy measurement

The samples of pollen were analyzed directly without treatment after crushing. ATR-
FTIR spectral analysis of untreated samples was performed using Nicolet iS5 ATR-FTIR
Spectrometer (Thermo Fisher Scientific, United States). The IR absorption spectra of all the
samples were recorded from in the region of 500-4000 cm-1 with 32 scans/min and resolution
of 16 cm-1.

ATR-FTIR spectral data pre-treatment

4
 The ATR-FTIR data sets (7261 x 72 datasets) from Thermo Scientific™ OMNIC™
Software Suite for ATR-FTIR data from normalized and smoothing were saved in file.csv and
copied manually to Microsoft Excel 2007 as two data sets (rows: samples; and columns:
wavenumber) for extracting their numerical values from spectra files. Then, the noise range
500-4000 cm-1 was cut off and the data were aligned in rows for samples and column for
wavenumber. All ATR-FTIR samples were subjected to unsupervised pattern recognition by
Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) which were
conducted by Orange3-3.13.0-Python36 Pro 2018 (University of Ljubljana, Slovenia).
For multivariate analysis, ATR-FTIR spectral data have been translated into an excel
sheet. Pre-treatment of spectral data is a major step preceding the chemometrics analysis to
reduce the effect of light dispersion, the variance of baseline, systematic noise. In this study,
baseline correction (pre-process spectra) pre-treatment was applied to remove scatter effects
from spectra by centering and scaling each spectrum.

Multivariate analysis

PCA is one of the multivariate data analysis techniques that support a complementary
role with HCA by both summing up data and minimizing data. These are quantitative methods
that are used to examine interrelations between a vast number of factors and describe them in
terms of their causes [36].
The statistical analysis of data was performed using the Orange data mining software on
the Windows platform. The standardization method was applied to the raw data analyzed,
followed by the chemometrics processing that included analysis of main components (PCA
and HCA. Further, PCA has been used as a data compression method by converting the
correlated data set into the uncorrelated main component collection. HCA helped identify the
samples examined into classes with similar characteristics [37].

Principal Component Analysis (PCA)

Principal Component Analysis (PCA) is one of the most popular chemometrics methods
[36]. PCA is an unsupervised method of clustering [38], an exploratory approach that
decreases the data matrix dimension and compresses the details into a few new variables
called key components, linear combinations of the original variables [39, 40]. In a two-
dimensional plot the PCA are displayed using the first two main components. The first major
variable, PC1, reflected the overall cumulative variance, the second, PC2 was orthogonal with
the first, covering as much of the residual variation as possible. The interrelationships among

5
different variables could be interpreted by plotting the PCs and analysing sample trends,
groupings, similarities, and differences[41].

Hierarchical Cluster Analysis (HCA)

Cluster analysis would aim to find the perfect clustering of multivariate observations in
the same sense that the clusters are different but the cluster observations are similar [42].
HCA has been used to organize the sample into groups using the cluster building the complete
correlation method was used, and the distance between clusters was calculated using the
Euclidean method [43] as a calculation of similarity.

Results and Discussion

In the current study, ATR-FTIR spectroscopy was used to analyze bee pollen samples
based on their spectral variations in the spectral range of 4000-500 cm-1. Fig. 2 shows
comparative infrared spectra of all samples in the 4000-500 cm-1 region. Table 2 shows the
band allocations in the ATR-FTIR continuum of bee pollen together with the corresponding
types of vibration.

Discrimination by ATR-FTIR spectral analysis

All pollens show broadband at ~3332cm-1 corresponding to O–H stretching vibration

from water [35], and two strong peaks at 2919 and 2858 cm-1 (with some difference for the
samples of El-Oued (P13) region which have an additional peak at 2777 cm-1), these peaks are
assigned to the C–H stretching vibrations of carbohydrates [31] and sugars [28], respectively.

All pollen samples show peaks or shoulders in the range 1870–1520 cm-1 is the carbonyl
area, and the double bond C=C [44], due to the bending vibrations of -OH from water, and
stretching vibrations of functional groups ketones C=O of fructose, and aldehyde CH=O of
glucose, is related to the stretching vibration of C=O functional group present in the acetic,
gluconic acids and originating from the fermentation of sugar [28] [see Table 2].

In region (1500-1800 cm-1), it can be pointed out that most of the samples show intense
peaks at 1646 cm-1 is inserted into the standard saccharide unit, steroid saponins and
flavonoid compounds, for example, C=C and C =O vibration[45] at 1630 cm-1 it may be
assigned to the carboxyl group in asymmetric stretching, while also important for the
identification of phenolic compounds, alcohols and carboxylic groups and also the hydrogen
vibration of amide N–H groups[31]. The broad peaks between 1440 and 1370 cm-1 result from

6
C–H deformation vibration of lipids and cellulose [46], -OH from water and stretching
vibrations of functional groups ketone C=O of fructose and aldehyde CH=O of glucose [28].
Those between 1355 and 1325 cm-1 can be assigned to the amide III (the in-phase
combination of N–H deformation and C–N stretching vibrations) [46], while a shoulder or a
convolution of peaks around 1340 and 1281 cm-1 can be assigned to the amide III (the in-
phase combination of N–H deformation and C–N stretching vibrations) [46].

In region (1300–500 cm-1) , are observed the characteristic patterns of most the samples
at about a peak at 1281 cm-1. The 1200–500 cm-1 range is the so-called fingerprint region,
where a strong peak (1281–900 cm-1) with shoulders at 1105, 1080, 1055, 1030, 1010 and 980
cm-1 is observed for all samples, corresponding to the C–H bending, C–O and C–C stretching
vibrations of sugar [47, 48]. In addition, in this region, all the samples show a peak at between
1170 cm-1 and1155 cm-1.Bands at 920, 865, 820, 780, and 700 cm-1 are characteristic for all
pollen samples and can be assigned to saccharides. Again, P13 samples show a typical peak at
617 cm-1 – 720 cm-1.

ATR-FTIR spectra analysis points out that the composition of saccharides, proteins,
lipids, and polyphenols is different for dissimilar bee pollens. With this perspective, the
spectral region 1800–650 cm-1 can be used to perform multivariate analysis, as already
reported [46].

Chemometrics analysis

To base classification and possible identification upon the whole chemical information
contained in the pollen, we used a multivariate method. For an evaluation of the overall
variance between different pollen, and to obtain an unbiased perspective on possible chemical
differences, we aim at a classification method that does not require the preselecting of
chemical parameters or weighting of input information. As a consequence, we chose to use
PCA and HCA as an unsupervised method. We applied PCA and HCA to a spectral data set
containing seventy-two spectra from different samples from thirteen regions different using
almost the full spectrum (500-4000 cm-1).

Principal component analysis (PCA)

Pre-processed spectra have been used to determine biochemical correlations between

pollen samples using PCA and HCA. Classification is unattended by PCA. Fig. 3 shows the
scatter plot for the first two principal components estimated with PCA, through which
visualization of the data structure is obtained. A natural separation of pollen samples into

7
three sets clear occurred in a 2-dimensional plot. The first principal component accounts for
70.2% of all the variances, while the second accounts for 20.9%, leading to a cumulative
proportion of 91.1% of all of the other variances that were considered significant. Thus, PC1
is more important than PC2 and further PC3 (3.3%) in this study. In general, the PCA was
able to discriminate pollen in three distinct groups based on similarity of chemical
components and geographical origin. In general, the PCA was able to discriminate pollen in
three distinct groups.

Hierarchical Cluster Analysis (HCA)

The resulting dendrogram reveals the formation of three clusters with a similarity level
of 70.4 % (see Fig. 4). This result is in accord with the result obtained using PCA. Therefore,
most of the pollen samples of the different species studied here can be distinguished based on
their ATR-FTIR spectra.

The results obtained from the unsupervised pattern recognition techniques (HCA and
PCA) combined by FTIR let us classify the samples into discriminate clusters in accordance
with their geographical origin, which reflect the notable similarity among the pollen samples
chemical composition. As a result, a more accurate segregation is demanded to be achieved
between the pollen samples from different Algerian regions. Consequently, must supervised
recognition techniques (SIMCA, PLS, etc.) were adopted to further ascertain the results
obtained from the unsupervised techniques.

The pace of analyzes, the abundance of Algeria bee pollen throughout the year, and the
viability of the procedure, among others, allow the chemometrics treatment of FTIR bee
pollen spectra to be a promising approach to bee pollen tree cultivar identification and
identifying.

Conclusion

According to the data hereby presented, it can be considered that the ATR-FTIR method
has an acceptable accuracy when applied in analysis of bee pollen samples. Furthermore, this
technique has a fast and more cost-effective for the correct characterization this product. It
could be concluded that authentication and classification of bee pollen samples sourced from
the Algerian regions could be effectively achieved using FTIR spectroscopic data in
combination with chemometrics. This undoubtedly could offer a credible simple model for the
quality control of bee pollen based upon their geographical origin and predominant chemical
constituents.

8
Acknowledgement

This research has been funded by the Research Deanship of University of Ha’il, Saudi
Arabia, through the Project RG-20 113

Compliance with ethical standards

Conflict of interest all authors declare no conflicts of interest.

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Figure captions

Fig. 1 Flowchart illustrates the steps of this work.

Fig. 2 ATR-FTIR spectra for the different samples.

Fig. 3 PCA score plot using the FTIR data matrix (500–4000 cm-1).

Fig. 4 A clustering dendrogram using the ATR-FTIR data matrix (500–4000 cm-1).

Figure 1

16
Figure 2

Figure 3

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Figure 4

Tables

Table 1 Areas of study samples and their date of harvest.

Code Region Date of harvest Source

A1 Acer negundo L
J1 Acer opalus subsp.
Bouira 7102 Anemonastrum narcissiflorum
O1
L
N1 Ajuga reptans L
J2 Ajuga reptans L
O2 Soybean
Mtija 7102
Js2 Spotted yellow loosestrife
B2 Red sand-spurrey
J3 Pink corydalis
O3 Pearly everlasting
Skikda 7102
B3 Caliculé Leatherleaf
V3 Canada fly honeysuckle
J4 Trembeling aspen
O4 Common storksbill
Constantine 7102
A4 Leatherleaf
Js4 Crocus sativus L
J5 Bitter Wintercress
Jo5 Birch
Js5 Common ragweed
O5 Tipaza 7102 Buckwheat
V5 European columbine
N5 Brunet’s milk-vetch
R5 Holly

18
 J6 Mexican dock
O6 Plantain lily
El-Bayadh 7102
Jo6 Meadow geranium
N6 Tatarian honeysuckle
J7 Common wormwood
O7 Everlasting pea
Js7 Garlic mustard
Tipaza 7102
N7 European columbine
R7 Bitter wintercress
Rs7 Round-leaved dogwood
J8 European bistort
O8 Basswood
Js8 Bouira- Wild sarsaparilla
7102
R8 Boumerdès Brunet’s milk-vetch
B8 Wild sarsaparilla
N8 Prostrate knotweed
J9 Creeping buttercup
O9 Broad fruited burred
B9 Laghouat, Blida Northern marsh yellowcress
7102
Bn9 and Médéa American beech
R9 Staghorn sumac
N9 Tall meadow-rue
J10 Bird’s-eye speedwell
O10 Agropyron caninum L
B10 Tizi-Ouzou 7102 Large flowered barrenwort
R10 Benoîte du Canada White avens
V10 Amélanchier Serviceberry
J11 Siberian pea shrub
O11 Pearly everlasting
Js11 Boumerdès 7102 Dill
R11 Spotted jewelweed
V11 Purslane speedwell
J12 Bitter wintercress
O12 Birch
Js12 Alder
Tizi-Ouzou 7102
B12 Black knapweed
R12 Creeping bugleweed
N12 Garlic mustard
Zygophyllum album L
A13
Genista saharae Coss & Dur
W13 Eucalyptus
Mathiolalivida DC
J13
EL-Oued Phoenix dactylifera L
O13 Anacyclus valentinus L
7102
Js13 Launaeare sedifolia O.K
Anacyclus valentinus L
B13
Launaeare sedifolia O.K
V13 Brassica oleracea var.viridis L
Brassica oleracea var.viridis L
Vs13
Mathiola livida DC

19
VIO13 Malcomia aegyptiaca spr
Retama raetam
R13 Eucalyptus
Genista saharae Coss & Dur
N13 Retama raetam

20
Table 2 Meaningful ATR-FTIR peaks of pollen samples: peak position in terms of
wavenumbers (cm-1), vibrational mode and chemical assignment.

Peak Position Biochemical

Vibrational Mode References
(cm-1) Assignments

~3332-3285 Stretching mode of OH Carbohydrates, Water [28]

The stretching vibration of

bonds Sugars, cellulose and
~2919, ~2858 lipids [28, 35]
C-H
N–H stretching vibrations

Stretching mode of the Carbonyl, alkyl-esters

~1877 [44]
carbonyl moiety

C=O stretching, C=O

Amide I, fatty acid,
stretching, N-H deformation,
amide II, cellulose or
~1720 C-N stretching vibrations, O-H [46]
water, and unsaturated
deformation vibration, and C=
lipids.
C stretching vibration

Stretching and bending modes

~1646 of Amide I of proteins [31]
peptide linkage

Amide I and II
~1540 Bending mode of CH2 moieties [46]
(shoulder)

Amide II
~1520 C=C stretching vibrations [49]

Lipids and cellulose,

C–H deformation vibration, -
water, functional
~1440–1370 OH stretching vibrations, C=O, [46, 50, 28]
groups ketone, fructose
CH=O
and aldehyde, glucose

N–H deformation and C–N Amide III

~1355–1325 [50]
stretching vibrations

In-phase combination of N–H

~1281 deformationand C–N stretching Amide III of proteins [46]
vibrations

21
 C–O, C–N, and C–C stretching
~1057 Sugar and proteins [51]
vibrations

~908–700 Vibrational of C–OH groups Saccharides [51]

~705 C–H bending Carbohydrates [52]

22