Journal of Food Composition and Analysis 104 (2021) 104126

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Journal of Food Composition and Analysis

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

Original Research Article

Classification of stingless bee honey based on species, dehumidification

process and geographical origins using physicochemical and ATR-FTIR
chemometric approach
Nur Faezah Ismail a, 1, M. Maulidiani b, 1, Syafizal Omar c, Muhammad Faiz Zulkifli a, c,
Mohd Naim Fadhli Mohd Radzi a, c, Norra Ismail d, Arif Zaidi Jusoh d, Suri Roowi d,
Wooi Meng Yew e, R. Rudiyanto f, Wan Iryani Wan Ismail a, g, *
a
Cell Signaling and Biotechnology Research Group (CeSBTech), Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, 21030, Kuala Nerus,
Terengganu, Malaysia
b
Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia
c
Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300, Bandar Puncak Alam, Selangor, Malaysia
d
Food Science and Technology Research Centre, Malaysian Agricultural Research and Development Institute (MARDI), 43400, Serdang, Selangor, Malaysia
e
Shimadzu Malaysia Sdn Bhd, No. 6 Lorong Teknologi 3/4 A, Nouvelle Industrial Park 2, Taman Sains Selangor 1, Kota Damansara, 47810, Petaling Jaya, Selangor,
Malaysia
f
Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia
g
Biological Security and Sustainability Research Group (BIOSES), Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, 21030, Kuala Nerus,
Terengganu, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: Stingless bee honey has been well recognized and consumed due to its nutritional and therapeutic values. This
Stingless bee honey study investigated the physicochemical properties and chemical profiles of Malaysian stingless bee honey from
Raw honey different species, dehumidification process, and geographical origins using chemometric techniques. In total, 122
Dehumidified honey
samples were collected from different species across different states in Malaysia. The range for each of the
Geographical origins
physicochemical properties in the studied samples was as stipulated by the Malaysian standard. However, a
Physicochemical properties
ATR-FTIR significantly different (p < 0.05) in the value of free acidity (FA), pH, and ash content (AC) was observed be­
Chemometrics tween honeys obtained from the species of Heterotrigona itama and Geniotrigona thoracica. The attenuated total
reflection Fourier–transform infrared (ATR-FTIR) chemometrics showed that the wavenumbers at 2934, 1400,
1040, and 1256 cm− 1 were more prominent in H. itama and G. thoracica, whereas the predominant wavenumbers
at 600 – 900 cm− 1 were observed in Tetrigona apicalis. The chemometric analyzes showed the reliability of
physicochemical properties and ATR-FTIR to differentiate the stingless bee honey on the basis of the dehu­
midification process and geographical origins but not by species. The findings indicate the potential use of the
ATR-FTIR-based chemometric approach in the classification and quality control of Malaysian stingless bee honey
besides the existing physicochemical analysis.

1. Introduction The beekeeping of stingless bee honey, also known as meliponiculture,

Malaysia is a country with a high diversity of stingless bees with 45
species have been recorded (Jaapar et al., 2016). Heterotrigona itama,
Geniotrigona thoracica, Lepidotrigona terminata, Tetrigona apicalis, and
Homotrigona alicae are among the common species found in Malaysia.
has been extensively promoted by the Ministry of Agriculture, Malaysia,
since 2014 (Ismail, 2016). The meliponiculture industry has served not
only to be a new source of wealth for Malaysia but also to meet up with
the increasing demand of the local market due to the dwindling of the
Apis mellifera honey industry (Mustafa et al., 2018).

* Corresponding author at: Cell Signaling and Biotechnology Research Group (CeSBTech), Faculty of Science and Marine Environment, Universiti Malaysia Ter­
engganu, 21030, Kuala Nerus, Terengganu, Malaysia.
E-mail address: waniryani@umt.edu.my (W.I.W. Ismail).
1
These authors equally contributed to this work.

https://doi.org/10.1016/j.jfca.2021.104126
Received 8 December 2020; Received in revised form 3 August 2021; Accepted 20 August 2021
Available online 23 August 2021
0889-1575/© 2021 Elsevier Inc. All rights reserved.
N.F. Ismail et al.
The Codex standard is currently used worldwide as the acceptable
standard for honey. Nevertheless, this standard was set on the basis of
the physicochemical properties of A. mellifera honey bee, and numerous
studies have shown that the Codex standard could not be used for
stingless bee honey (Nordin et al., 2018; Ismail, 2016). Presently, there
is no international standard for the regulation and quality control of
stingless bee honey. At the national level, the Department of Malaysian
Standards has proposed and published the Malaysian standard for
stingless bee honey (Kelulut) (MS2683:2017 Kelulut (Stingless bee)
honey—Specification) in 2017. The Malaysian standard was set for both
raw and dehumidified stingless bee honey (Malaysian Standards, 2017).
Physicochemical analysis including water content (WC), free acidity
(FA), pH, electrical conductivity (EC), insoluble matter (IM), hydrox­
ymethylfurfural (HMF), proline, ash content (AC), fructose, glucose,
maltose, and sucrose has been used for the criteria in the standardization
of Codex for honey (Codex Alimentarius Commission, 2019). Several
studies have reported the use of physicochemical properties in evalu­
ating the quality of Malaysian stingless bee honey (Abu Bakar et al.,
2017; Fatima et al., 2018; Kek et al., 2018; Omar et al., 2019; Shamsudin
et al., 2019; Ya’akob et al., 2019). However, the physicochemical
properties-based method has some limitations including high variability
between batches of honey samples, the need for a high amount of
samples for various parameters evaluation, and being time- and
cost-consuming (Razali et al., 2018). Hence, with the increase of adul­
terated honey in the local market, there is an urgent need for a reliable
but rapid and low-cost method for the evaluation and authentication of
stingless bee honey.
Fourier–transform infrared (FTIR) spectroscopy analysis has been
used to analyze the adulterants in stingless bee honey (Mail et al., 2019;
Se et al., 2018). One of the common sampling technologies for the FTIR
spectroscopy is attenuated total reflectance (ATR). The ATR is a
label-free, nondestructive analytical tool that could simplify the process
of spectra acquisition to study variable molecules in solid and liquid
samples (Anguebes et al., 2016; Glassford et al., 2013). Although
ATR-FTIR has some limitations in comparison with those commonly
spectroscopy methods, this method is more practical for research and
industrial purposes because it is a rapid and cheaper analytical tool than
the commonly used standard analytical methods including physico­
chemical analysis. Moreover, ATR-FTIR analysis utilizes less amount of
sample than does the physicochemical analysis. Therefore, the use of
ATR-FTIR could be considered as one of the standard methods for the
quality determination and control of Malaysian stingless bee honey.
Chemometrics is a multivariate data analysis method that has been
used for the fingerprint analysis and chemical profile of biological
samples (Karabagias et al., 2017; Se et al., 2018). The principal
component analysis (PCA) model is one of the most commonly used
chemometric techniques to reduce the dimensionality of datasets and to
increase interpretability but at the same time to minimize information
loss (Maulidiani et al., 2018). It is usually used for metabolite fingerprint
and classification purposes.
Previous studies have shown that different types of stingless bee
honey have their own distinctive characteristics that were reflected by
their physicochemical and chemical profiles (Abdullah et al., 2020;
Fatima et al., 2018; Omar et al., 2019). However, most of the studies on
the stingless bee honey in Malaysia were conducted using a small
number of samples that might not reflect the biological variation in the
country. Additionally, not all of the parameters for the physicochemical
properties were tested in the previous studies. To the extent of our
knowledge, this study is by far the biggest on Malaysian stingless bee
honey in terms of the number of species and samples used for the
analysis of physicochemical properties and chemical profiling. More
geographical origins across Malaysia have been included in this study
(13 states) in comparison with other similar studies using Malaysian
stingless bee honey (Abu Bakar et al., 2017; Fatima et al., 2018;
Shamsudin et al., 2019). Honeys from the wild species (H. alicae, T.
apicalis, L. conifrons, L. terminata, and T. binghami) and domesticated (H.
2
Journal of Food Composition and Analysis 104 (2021) 104126
itama and G. thoracica) were used in this study. The inclusion of the wild
species of Malaysian stingless bee honey may provide the data that will
help to accelerate the commercialization of these species in Malaysia
and other countries. Furthermore, the novelty of this study relied on the
comparative characterization of physicochemical and chemical profiles
between dehumidified and raw stingless bee honey samples, which have
not yet been extensively studied. Several past studies have used che­
mometric analysis to classify Malaysian stingless bee honey (Kek et al.,
2018; Shamsudin et al., 2019; Yap et al., 2019). However, to date, no
study has compared the reliability of physicochemical–chemometric to
ATR-FTIR chemometric approach.
This study was undertaken to evaluate the use of physicochemical
properties and chemical profiling (via ATR-FTIR) in combination with
the chemometric approach for the classification of Malaysian stingless
bee honey by their species, dehumidification process, and geographical
origins.
2. Materials and methods
2.1. Sample collection
A total of 122 samples of stingless bee honey obtained from the
identified species including H. alicae, H. itama, G. thoracica, T. apicalis, L.
conifrons, L. terminata, and T. binghami and unidentified species (un­
known) were harvested from 13 states across Malaysia from the period
of August until October 2016. The botanical origins of the samples were
varied with most of them were from multifloral species. The honeys
collected from the species of H. itama and G. thoracica were harvested
from domesticated beehives, whereas those from the species of H. alicae,
T. apicalis, L. conifrons, L. terminate, and T. binghami were harvested from
wild beehives. Matured, raw stingless bee honey samples (n = 88) were
harvested from sealed honeypots using a sterile syringe. Dehumidified
stingless bee honey samples (n = 34) were obtained from the beekeepers
after the honeys were dehydrated using a dehumidifier at a temperature
between 35℃ and 38℃. The authentication of the stingless bee species
was performed by a taxonomist during the sampling. A detailed
description of the samples is given in Table S1. All samples were stored
in amber bottles at a low temperature around 4℃ prior to analyzes.
2.2. Physicochemical analyzes
The physicochemical analyzes were conducted according to the
Codex Alimentarius International Food Standards for Honey (Codex
Alimentarius Commission, 2019) and Harmonised Methods of the In­
ternational Honey Commission (Bogdanov, 2009).
2.2.1. Water content (WC)
The WC for each honey sample was measured using MISCO BKPR-2
digital refractometer (MISCO refractometer, USA) at room temperature
according to the manufacturer. The unit for the WC was expressed in the
percentage of moisture of the honey.
2.2.2. Electrical conductivity (EC)
Twenty grams of honey was dissolved in distilled water, and the
solution was made up to 100 mL to prepare a 20 % (w v− 1) honey so­
lution before measurement using HI-98311 EC meter (Hanna In­
struments, USA).
2.2.3. Ash content (AC)
An ash dish was heated in a Phoenix electrical furnace (CEM, USA) at
600℃ before its weight was measured. Five grams of honey was added
to the ash dish and heated at 300℃ until it was completely carbonized.
The dish was further heated at higher temperatures up to 600℃ for 1 h.
The resulting ash was weighed and expressed as AC in g 100 g− 1 of
honey.
N.F. Ismail et al.
2.2.4. pH and free acidity (FA)
Ten grams of honey was dissolved in 75 mL of distilled water before
the pH was measured using a Delta 320 pH meter (Mettler Toledo,
Switzerland). The honey solution was titrated with 0.1 M sodium hy­
droxide (NaOH) until pH 8.3 to obtain its FA value. The FA is expressed
in the unit of milliequivalents acid per kilogram of honey (meq kg w
v− 1).
2.2.5. Hydroxymethylfurfural (HMF)
The HMF content of honey samples was determined spectrophoto­
metrically according to White (1979). Approximately 5 g of honey was
dissolved in 25 mL of distilled water before 0.5 mL of Carrez solution I
and II were added, respectively. The volume was then made up to 50 mL
with distilled water. The solution was filtered using 0.45 μm Whatman
filter paper and the first 10 mL of the filtrate was discarded. The
absorbance of 5 mL honey solution mixed with 5 mL of distilled water
was read against the reference solution of 5 mL honey solution mixed
with 5 mL of 0.2 % sodium bisulfite solution. The absorbance was
measured at 284 and 336 nm using Ultrospec 2100 Pro spectropho­
tometer (Amersham Biosciences, UK). If the absorbance at 284 nm ex­
ceeds 0.6, the honey solution will be diluted with water and the
reference solution will be diluted with 0.2 % sodium bisulfate with half
of the original concentration, respectively. The HMF content is
expressed in the unit of mg kg− 1 of honey and calculated as follows:
HMF = (A284 − A336) × 149.7 × 5 × D/W
where A284 = absorbance at 284 nm, A336 = absorbance at 336 nm, D =
dilution factor (if necessary), W = weight of honey in grams, the value of
149.7 is constant, and 5 is theoretical nominal sample weight.
2.2.6. Insoluble matter (IM)
IM was measured according to the Harmonised Methods of the In­
ternational Honey Commission (2009) with some modifications. Before
weighing, the 0.45 μm Whatman filter paper was dried in an oven and
left in an ambient temperature desiccator. Approximately 20 g of honey
was dissolved in 200 mL of water at 80℃ and then filtered using the
weighed filter paper. Next, the filter paper was dried at 100℃ for an
hour before it was cooled in the desiccator and weighed. The IM is
expressed in the unit of percentage by weight.
2.2.7. Proline
The honey sample was prepared by dissolving 5 g of honey in 50 mL
of distilled water before the volume was made up to 100 mL with
distilled water. Next, 0.5 mL of sample solution and 0.5 mL of water
(blank) were added to different tubes, whereas 0.5 mL of proline stan­
dard solution was added to three separate tubes. Next, 1 mL of formic
acid and 1 mL of ninhydrin solution were added to each tube. The tubes
were shaken vigorously for 15 min and boiled in a water bath for 15 min.
The tubes were transferred to a 70℃ water bath for 10 min before the
addition of 5 mL of 2-propanol-water-solution (50 % v v− 1) to each tube.
The tubes were left to cool for 45 min, and the absorbance was read at
510 nm.
2.2.8. Determination of sugars
The sugar profile (fructose, glucose, maltose, and sucrose) of each
sample was measured using a high-performance liquid chromatography
with a refractive index detector (Agilent, USA). Each of the sugar stan­
dards (2.00 g fructose, 1.50 g glucose, 0.15 g maltose, and 0.25 g su­
crose) and 5 g of honey were dissolved in 40 mL deionized water in
separate tubes before they were transferred into different volumetric
flasks containing 25 mL of methanol and topped up to 100 mL with
deionized water. The solutions were then filtered using a 0.45 μm
membrane filter and collected in glass vials. The samples and standards
were subjected to a cleanup procedure using an ISOLUTE C18 solid-
phase extraction column (Biotage, Sweden). The chromatographic
3
Journal of Food Composition and Analysis 104 (2021) 104126
analysis was performed using a mobile phase of acetonitrile water
(80:20, v v− 1), a flow rate of 1.3 mL min− 1 with an injection volume of
10 μL, and the column (ZORBAX NH2 4.6 × 250 mm, 5 microns, Agi­
lent), and detector temperature was kept at 30℃ during the whole run.
The unit was expressed as the percentage of sugar in honey. The in­
strument and column used in this study were verified according to the
manufacturer’s instructions.
2.3. Attenuated total reflection Fourier–transform infrared (ATR-FTIR)
analysis
The ATR-FTIR measurements were performed as previously
described in Goh et al. (2019). Infrared spectra were obtained from a
MIRacle ATR accessory (Pike Technologies, Germany) in an FTIR spec­
trometer (Shimadzu, IRTracer-100). Approximately 1 mL of the sample
was put in the ATR crystal center. Then, each sample’s spectrum was
collected in the wavelength range of 4000–600 cm− 1 with 40 in­
terferograms at a resolution of 4 cm− 1. Before each sample analysis, a
background spectrum of the ATR crystal was recorded using the same
instrumental conditions. The ATR plate was cleaned using a dust-free
tissue and acetone solution after each sample scan. All FTIR data were
converted to ASCII files and subjected to multivariate data analysis.
2.4. Preprocessing Fourier–transform infrared (FTIR) data
The spectra FTIR data for each sample were resulted-in from the
average values of three technical replicates. Following Goh et al. (2019,
2021), the spectra data were transformed from absorbance wave­
number, R, to the form of log (1/R). Subsequently, the transformed data
were filtered using the Savitzky–Golay (SG) smoothing method with a
window size of 21 and a polynomial of order 2. In the last pre-processing
data, the smoothed data then were normalized using Standard Normal
Variate (SNV) transformation. In these processing data, the SG algo­
rithm with polynomial regression was addressed to minimize instru­
mental noise in the spectra data and without changing phase shift of the
original data (Acharya et al., 2016); while SNV normalization was used
to remove the light scatter. The SG filtering was conducted in R software
using the prospectr R package (Stevens and Ramirez-Lopez, 2020).
2.5. Multivariate and statistical analysis
PCA was used in multivariate data analysis for discriminating
Malaysian stingless bee honey on the basis of (1) species and (2) origins
and (3) for discriminating between raw and dehumidified stingless bee
honey using physicochemical properties and FTIR data, respectively.
PCA was conducted using a built-in R function prcomp available via R
software (https://www.rdocumentation.org/packages/stats/versions/
3.6.2/topics/prcomp). Before the analysis using PCA, the data were
centered to 0 and scaled using a unit variance. R packages were used
including ggfortify (Tang et al., 2016; Horikoshi and Tang, 2020),
scatterplot3d (Ligges and Mächler, 2003), and factoextra (Kassambara
and Mundt, 2020). For the visualization of the results such as 2-D and
3-D score plots, biplot (overlay between 2-D score plots and loading
plots), and eigenvalues plots.
The data for physicochemical properties were subjected to statistical
tests using the GraphPad Prism software (Version 7, GraphPad Software,
Inc.), and results were expressed as mean ± standard deviation. The
normality of the data was assessed using the D’Agostino and Pearson
test, Shapiro–Wilk test, and Kolmogorov-Smirnov normality test. The
only sample group(s) with n ≥ 3 were included in the statistical analysis.
The statistical difference between the two groups was tested using the
unpaired t-test with Welch’s correction for normally distributed data,
Mann–Whitney U test, and Spearman r test for parameters with non­
normal distribution. The differences in the mean between more than two
groups were evaluated using the Kruskal–Wallis for nonnormal distri­
bution data. P-values of less than 0.05 and 0.01 were considered
N.F. Ismail et al.
significant. Outliers were included in the analysis unless mentioned
otherwise.
3. Results and discussion
3.1. Physicochemical analysis of Malaysian stingless bee honey
A total of 88 raw stingless bee honey samples used in this study were
evaluated for their physicochemical properties. The results for the
analysis are shown in Table S2.
Generally, we have observed an average WC (28.83 ± 1.03 %), FA
(233.60 ± 95.28 meq kg− 1), pH of (3.16 ± 0.15), EC (0.70 ± 0.23 mS
cm− 1), IM (0.28 ± 0.11 %), HMF (4.96 ± 13.81 mg kg− 1), proline (5.98
± 4.17 mg kg− 1), and AC (0.26 ± 0.13 %) in the samples from multiple
species of Malaysian stingless bee honey (Table S2). The sugar profile
was fructose (25.16 ± 9.10 %), glucose (20.61 ± 7.66 %), maltose (2.04
± 1.27 %), and sucrose (3.22 ± 1.87 %).
The WC of all the studied raw honey samples was recorded from
25.99 % to 30.00 %, which can be considered as high (Table S2). High
WC is one of the distinct characteristics of stingless bee honey in com­
parison to A. mellifera honey and mainly attributed to the weather,
namely, temperature and humidity, experienced by tropical countries to
which the stingless bees are native (Abdullah et al., 2020; Biluca et al.,
2016; Chuttong et al., 2016; Nordin et al., 2018). Additionally, har­
vesting season, honey maturity upon harvest, and storage conditions
could also influence the WC of stingless bee honey.
The samples analyzed in this study exhibited an acidic pH
(2.87–3.52, Table S2) and a broad range of FA (45.33–468.00 meq kg− 1)
(Table S2). The range for the pH is close to similar studies from Malaysia
(Fatima et al., 2018; Kek et al., 2018). Presently, only Chuttong et al.
(2016) from Thailand reported a huge range of FA up to 592.00 meq
kg− 1. The acidity of honey is contributed by the presence of a wide range
of organic acids introduced through plant nectars or produced during
storage via fermentation (Prica et al., 2014).
Our results revealed a wide range of HMF from 0.05 to 75.36 mg
kg− 1 in the studied samples (Table S2). A broad range of HMF was also
observed in other studies from Malaysia (0.02–48.68 mg kg− 1) (Ya’akob
et al., 2019) and Thailand (0.26–46.00 mg kg− 1) (Chuttong et al., 2016).
HMF is a product of sugar degradation produced through the Maillard
reaction during the processing and storage of honey (Shapla et al.,
2018). High HMF value could indicate poor handling and storage as well
as prolonged exposure to high temperatures (White, 1979; Fatima et al.,
2018; Shapla et al., 2018). In this study, all samples were stored at a low
temperature around 4℃ prior to analyzes. By contrast, Malaysian honey
stored at higher temperatures (25℃–30℃) for more than a year showed
a higher range of HMF (206.06–383.39 mg kg− 1) (Khalil et al., 2010).
Hence, the low HMF content observed in our study may indicate the
freshness of the stingless bee honey samples.
The Malaysian stingless bee honey samples displayed a range of EC
from 0.4 to 1.9 mS cm− 1 (Table S2). This is in accordance with the
previous report that Malaysian stingless bee honey has a broad range of
EC (Nordin et al., 2018). The EC of honey is influenced by several factors
such as salt content, the concentration of minerals, protein, organic
acids, floral sources, and storage time (Baloš et al., 2018; Nascimento
et al., 2015).
Moreover, the AC for the studied samples was between 0.04 and 0.63
g 100 g− 1 (Table S2), and this is close to the reported range of 0.14–0.89
g 100 g− 1 in Malaysian stingless bee honey (Majid et al., 2019). AC re­
flected the mineral present in the honey and is strongly associated with
the EC of the honey (Majid et al., 2019).
The values for the IM were observed at 0.28 ± 0.11 % (Table S2). To
the best of our knowledge, this study is the first to report the range of IM
in Malaysian stingless bee honey. However, De Almeida-Muradian et al.
(2013) reported higher IM in Brazilian stingless bee honey (32.49 ± 1.13
%). The honey IM is composed of debris, bee pollen, or filth particle,
which can be used as an indicator of the cleanness of the honey (De
4
Journal of Food Composition and Analysis 104 (2021) 104126
Almeida-Muradian et al., 2013).
Sugar is one of the main components of stingless bee honey with the
major ones are monosaccharides (fructose and glucose) and di­
saccharides (maltose and sucrose) (Nordin et al., 2018; Souza et al.,
2006). The range for the fructose and glucose in this study was 11.89 %–
59.05 % and 7.10 %–36.72 %, respectively (Table S2). The range for
sucrose and maltose in our study was 0.72 %–7.03 % and 0.53 %–5.22
%, respectively (Table S2). As far as we are aware, the results are the first
report on the sucrose and maltose content in Malaysian L. conifrons, T.
binghami, and H. alicae. Chuttong et al. (2016) reported a range of su­
crose content of 0.03–6.00 g 100 g− 1 and 37.00–53.00 g 100 g− 1 for
maltose in Thailand stingless bee honey.
Observing results for proline, a wide range from 0.33 to 16.53 mg
kg− 1 was recorded in the studied samples (Table S2). Proline is a major
component of amino acids that can be found in honey, which is related
to the floral sources and amount of pollen present (Kečkeš et al., 2013).
The proline can also be an indicator of honey ripeness since its content
decreases over time during storage (Czipa et al., 2019). Hence, the wide
range of proline observed might be attributed to the differences in the
botanical origins and handling and storage of the stingless bee honey
samples.
The sugar profile and proline in this study were in different ranges to
those reported by other similar studies from Malaysia (Fatima et al.,
2018; Se et al., 2018; Shamsudin et al., 2019). However, most of these
studies were conducted on a lower sample number (n < 30) in com­
parison with our study (n ≥ 80), which could account for the difference
observed in our study. Additionally, the difference could be contributed
by the botanical origins of the stingless bee honey. For example, the
samples in Shamsudin et al. (2019) were uniflora stingless bee honeys,
whereas most of our samples were multifloral stingless bee honeys.
We further compared the range of the physicochemical properties
found in this study to the Codex standard for honey (Codex Alimentarius
Commission, 2019) and the Malaysian standard for stingless bee honey
(Table 1). Generally, only EC, AC, HMF, and sucrose content were within
the threshold set forth by the Codex standard, whereas WC, FA, IM, and
proline were not (Table 1). The Codex standard does not set any limit for
pH and maltose content. By contrast, the range for physicochemical
properties, namely, WC, pH, HMF, AC, fructose, glucose, maltose, and
sucrose in both raw and dehumidified samples was as stipulated by the
Malaysian standard (Table 1). However, presently, the Malaysian stan­
dard does not set a limit for the EC, FA, IM, and proline.
Overall, our results further reiterate the need for a separate standard
for stingless bee honey from the A. mellifera honey due to huge differ­
ences in their physicochemical properties (Nordin et al., 2018). The
results could also be used as a basis to set up the revised standard for
Malaysian stingless bee honey with the inclusion of new physicochem­
ical parameters including EC, FA, IM, and proline for quality check
purposes.
3.2. Attenuated total reflection Fourier–transform infrared (ATR-FTIR)
analysis of Malaysian stingless bee honey
The chemical profile of the stingless bee honeys obtained from
different bee species were characterized using ATR-FTIR. The ATR-FTIR
analysis provides information on the chemical structure or specific
functional group of the sample. The analysis is simple, fast, nonde­
structive, and reagentless (Goh et al., 2019).
A strong and broad peak of the OH group with stretching vibration at
3242 cm− 1 was observed in the studied stingless bee honey samples. A
weak absorption band of the CH– group with stretching of carboxylic
acid and NH3 stretching of free amino acids was defined at 2934 cm− 1. A
weak absorption of the OH group with deformation vibration was
identified at 1641 cm− 1. Additionally, a lower energy region charac­
teristic of the combination of the O–H bending of the COH group and
CH–– bending of the alkenes was observed at the 1400 cm− 1 whereas
the O–H bending of the CO–H group was absorbed at 1348 cm− 1. The
N.F. Ismail et al. Journal of Food Composition and Analysis 104 (2021) 104126

Table 1
Physicochemical analysis data and sugar profiles of stingless bee honey in comparison with Malaysian Standard and International Codex Standard.
Stingless bee honey Honey

Analysis This study (Raw) Malaysian Standard This study (Dehumidified) Malaysian Standard International Codex
Mean ± SD Range (Raw) Mean ± SD Range (Processed) Standard

Water content (%) 28.83 ± 1.03 25.99 - < 35.00 20.31 ± 1.00 18.17 - < 22.00 < 20.00
30.00 22.53
Electrical conductivity (mS 0.70 ± 0.23 0.37 - 1.86 ND 0.56 ± 0.20 0.17 - 0.98 ND < 0.80
cm− 1)
Ash content (g 100g− 1) 0.26 ± 0.13 0.04 - 0.63 <1 0.31 ± 0.16 0.08 - 0.75 < 1.00 < 0.50
pH 3.16 ± 0.15 2.87 - 3.52 2.50 - 3.80 3.23 ± 0.15 2.90 - 3.56 2.50 - 3.80 ND
Free acidity (meq kg− 1) 233.60 ± 45.33 - ND 195.70 ± 44.00 - ND < 50.00
95.28 468.00 99.25 440.00
HMF (mg kg− 1) 4.96 ± 13.81 0.05 - 75.36 < 30.00 3.67 ± 10.70 0.06 - 51.59 < 30.00 < 80.00
Insoluble matter (%) 0.28 ± 0.11 0.02 - 0.60 ND 0.54 ± 0.14 0.26 - 0.82 ND < 0.10
Proline (mg kg− 1) 5.98 ± 4.17 0.33 - 16.53 ND 16.35 ± 6.79 4.55 - 39.50 ND > 8.00
Sum of fructose and glucose 44.66 ± 18.99 - < 85.00 % 28.15 ± 9.11 5.04 - 88.40 < 90.00 % > 60.00 %
(%) 15.40 95.77
Maltose (%) 2.04 ± 1.27 0.53 - 5.22 < 9.50 1.28 ± 0.61 0.31 - 2.28 < 10.00 ND
Sucrose (%) 3.22 ± 1.87 0.72 - 7.03 < 7.50 1.10 ± 0.67 0.20 - 2.59 < 8.00 < 5.00 %

The results are expressed as mean ± standard deviation. ND = not determined.

characteristics of carbohydrates at 1040 and 1256 cm− 1 (C–O stretch in
the COH group and CC stretch) and CH––– bending at 916 cm− 1 were
also identified. A strong and sharp peak at 1163 cm− 1 for the stretching
vibration indicated the presence of the C–O band of the COC––
linkage. The sweetness of the stingless bee honey that is attributed to the
chemical component of sucrose, glucose, and fructose can be observed at
the spectral region between 900 and 1500 cm− 1. The FTIR spectra
indicated the characteristic for saccharide configuration at 700–978
cm− 1. The ATR-FTIR spectra overlay of the stingless bee honey samples
is shown in Fig. S1.
3.3. Classification of Malaysian stingless bee honey based on species
The descriptive mean for the physicochemical properties of different
raw Malaysian stingless bee species, namely, H. alicae, H. itama, G.
thoracica, L. conifrons, L. terminata, T. apicalis, T. binghami, and uniden­
tified species were shown in Table S3. However, statistical analysis was
done by comparing the domesticated stingless bee species, H. itama and
G. thoracica, only due to the low number of samples (n ≤ 3) in other wild
species harvested in this study.
Generally, the WC, EC, IM, HMF, proline, fructose, glucose, maltose,
and sucrose were not statistically different between H. itama and G.
thoracica (Table S3, Fig. S2). By contrast, the mean for FA of H. itama
(220.60 ± 86.98) was significantly lower than G. thoracica (294.10 ±
105.00) (p = 0.01, Unpaired t-test). We have also observed a signifi­
cantly higher pH for H. itama (3.17 ± 0.14) than that for G. thoracica
(3.06 ± 3.06) (p = 0.02, Unpaired t-test). Additionally, H. itama showed
significantly higher AC in comparison to G. thoracica (0.28 ± 0.13, 0.17
± 0.12, respectively) (p < 0.01, Mann–Whitney test).
We applied PCA on all parameters of physicochemical properties
excluding EC, maltose, and AC due to data unavailability (Fig. 1A). The
PC1 and PC2 which attributed for 42 % of the total variance were
extracted to determine the best physicochemical properties that could
classify the stingless bee honey samples on the basis of their species. The
PCA biplot showed that G. thoracica and T. apicalis and the unknown
samples were clustered together in the upper left quadrant. However, no
specific cluster was examined for H. itama species as the samples were
spread all over the PCA biplot. Overall, the PCA biplot results indicate
that the samples from different species were not distinguishable based
on their physicochemical properties (Fig. 1A).
We then performed chemical profiling on the basis of ATR-FTIR
chemometrics to determine differences between the H. itama, G. thora­
cica, and T. apicalis. The PCA model showed the first principal compo­
nent (PC1) and the second principal component (PC2), which represent
37.18 % and 26.86 % of the total variance, respectively (Fig. 1B). The
observation of its score plot shows that most of the T. apicalis samples
were clustered in the lower left quadrant, whereas the G. thoracica
samples in the upper quadrant. The H. itama samples were mostly sit­
uated in the middle quadrant although some of them overlapped with
G. thoracica. A clearer overview of the chemical profile of stingless bee
honey from different species could be seen in the heatmap diagram
(Fig. 1C). The predominant characteristics wavenumbers of G. thoracica
were observed at 600–900 cm− 1, which were least observed in T. api­
calis. The wavenumbers at 2960, 1560, 1350, 1210, and 1080 cm− 1 were
more prominent in T. apicalis and H. itama but not in G. thoracica.
Differences in the physicochemical properties among different spe­
cies of stingless honey were observed in other similar studies from
Thailand and Malaysia (Chuttong et al., 2016; Fatima et al., 2018).
Different stingless bee species adapted to a different ecological system
that is the source for the floral during the foraging activities (Abdullah
et al., 2020). Therefore, the differences observed in some of the physi­
cochemical properties (FA, pH, and AC) between H. itama and G.
thoracica in this study might be attributed to the type of bee species that
produce different materials beehives and other possible environmental
variables.
Most of the current researches on physicochemical properties of
stingless bee honey were conducted on H. itama and G. thoracica species
thus making it difficult for us to compare our data of the T. apicalis in this
study. Honeys from H. itama and G. thoracica species are the most
domesticated and commercialized by the local beekeepers in Malaysia
(Jaapar et al., 2016; Kelly et al., 2014; Shamsudin et al., 2019). The log
hives of H. itama are easier to locate in the local forest and sustain in the
suburban area than those of the other stingless bee species (Abdullah
et al., 2020; Shamsudin et al., 2019). Given that the physicochemical
properties of T. apicalis were almost identical to the H. itama and G.
thoracica (based on the chemometric analysis), this indicates the po­
tential for domestication and commercialization of T. apicalis in
Malaysia.
Taken altogether, the physicochemical–chemometric analysis
showed the least variability between the H. itama, G. thoracica, and T.
apicalis, making it a less effective method for the classification by spe­
cies. By contrast, the ATR-FTIR in combination with chemometric
analysis could be used to distinguish the Malaysian stingless bee honey
by species.
3.4. Comparison between raw and dehumidified stingless bee honey
Some of the stingless bee honey found in the local Malaysian market
was dehumidified, and this trend is expected to be increased in the
future. Hence, we have compared the physicochemical properties and

5
N.F. Ismail et al. Journal of Food Composition and Analysis 104 (2021) 104126

Fig. 1. Chemometrics analysis of Malaysian stingless bee honey from different species. Principal component analysis (PCA) biplots of physicochemical properties (A).
Principal component analysis (PCA) score plot (B) and heat map analysis (C) based on ATR-FTIR data.

chemical profiles between raw and dehumidified Malaysian stingless
bee honey. To clarify, this study was not meant to track the changes
following dehumidification processes; rather, it was to observe the dif­
ferences between raw and dehumidified stingless bee honey. The anal­
ysis was performed using H. itama species only to avoid interspecies
variation. In total, 25 samples of dehumidified H. itama were compared
with 49 samples of raw H. itama.
The dehumidified H. itama exhibited significantly lower WC (p <
0.01, Mann–Whitney U), fructose (p < 0.01, unpaired t-test), and sucrose
(p < 0.01, unpaired t-test) when compared with raw H. itama (Fig. 2 and
Table 2). Dehumidified H. itama also has significantly higher EC (p <
0.05, Mann–Whitney test), IM content (p < 0.01, Mann–Whitney test),
and proline (p < 0.01) when compared with raw H. itama (Fig. 2 and
Table 2). However, a similar level of pH, FA, glucose, maltose, HMF, and
AC was observed between raw and dehumidified H. itama (Fig. 2 and
Table 2).
We further performed multivariate analysis to evaluate whether raw
and dehumidified are distinguishable based on their physicochemical
parameters. The data were presented in PCA biplot with the PC1 and the
PC2, which represent 23.22 % and 15.56 % of the total variance,
respectively (Fig. 3A). The data showed that the physicochemical pro­
files of dehumidified H. itama samples were distinctive from the
respective raw samples. The proline, IM, and AC may have contributed
to the PC2 variance with respect to the dehumidified samples. By
contrast, the glucose, fructose, sucrose, maltose, HMF, EC, WC, FA, and
pH were related to the PC1 shown by raw samples. The PCA model based
on the physicochemical properties of raw and dehumidified stingless bee
honey showed variances for PC1 and PC2 as 51.27 % and 31.77 %,

6
N.F. Ismail et al. Journal of Food Composition and Analysis 104 (2021) 104126

Fig. 2. Comparison of physicochemical properties between raw and dehumidified H. itama. Statistical significance was determined using the unpaired t-test with
Welch’s correction for normally distributed data (free acidity, pH, fructose and glucose) and Mann-Whitney U test for non-normal distribution data (WC, EC, IM,
HMF, AC, maltose, sucrose and proline). *p < 0.05 and **p < 0.01 indicated as significant.

respectively (Fig. 3B). A clear separation was observed in the PCA biplot
with the raw samples at the left side of the quadrant while dehumidified
samples at the right side.
The PCA plot for the chemical profile showed distinctive wave­
numbers in the raw honey when compared with the dehumidified
stingless bee honey (Fig. 3C). The wavenumbers at 3242 (OH stretch),
2934 (CH– stretching of carboxylic acids), and 1657 (OH deformation)
cm− 1 were the characteristics of raw samples. The wavenumbers at 1256
and 1040 cm− 1 corresponding to the C–O stretch in the COH group and
the CC–– stretch in the carbohydrate structure were also prominent in
raw samples. Conversely, the wavenumbers at 700–978 cm− 1 were
prominent in dehumidified samples representing the out-of-plane OH
deformation, C– – O in-plane deformation, and rocking vibrations of the
CH2 group of L-proline (Mary et al., 2009).
The main purpose of the dehumidification process is to remove the
WC to preserve the quality of honey (Aroucha et al., 2019; Carvalho
et al., 2009). Hence, the significantly lower WC observed in
dehumidified H. itama was as expected (Table 2). High WC could pro­
mote microbial activities and subsequent fermentation, which could
affect the taste, appearance, and quality of stingless bee honey (Singh
and Singh, 2018; Yap et al., 2019). Decreasing the WC may avoid
deterioration in the quality of stingless bee honey and lead to higher
shelf life (Aroucha et al., 2019).
It was reported that the decomposition of fructose is higher than
glucose in subcritical water conditions and with the presence of salt or
acid (Asghari and Yoshida, 2007; Kobayashi et al., 2016). The low pH
(acidic) and minerals that are naturally present in stingless bee honey
could trigger the decomposition of fructose in the dehumidified honey.
Thus, the reduced level of fructose observed in this study might be due to
the decomposition of the sugars to other compounds such as glyceral­
dehyde, dihydroxyacetone, HMF, and erythrose as a consequence of the
dehumidification process. A similar reason might be applicable in the
case of sucrose.
A significant decrease in EC was observed in dehumidified H. itama

7
N.F. Ismail et al. Journal of Food Composition and Analysis 104 (2021) 104126

Table 2 physicochemical and chemical profiles of the raw and dehumidified

Physicochemical properties of raw and dehumidified H. itama. Malaysian H. itama, separately. We have first clustered the raw H. itama
Analysis Raw Dehumidified from different states to their respective geographical regions in Penin­
sular Malaysia: Northern, East Coast, Southern, and West Coast regions
Mean ± SD 28.84 ± 0.91 20.22 ± 0.93*
Water Content (%)
n 46 24 (Fig. 4A). The Northern region was represented by Kedah and Pulau
Mean ± SD 220.60 ± 86.98 180.60 ± 90.02 Pinang states, whereas the East Coast region was represented by Ter­
Free Acidity (meq kg− 1)
n 47 25 engganu and Kelantan states (Fig. 4A). Johor state was classified as a
Mean ± SD 3.17 ± 0.14 3.22 ± 0.14 Southern region, whereas Selangor, Melaka, and Negeri Sembilan states
pH
n 46 21
Mean ± SD 0.72 ± 0.36 0.55 ± 0.20*
were representative of the West Coast. Meanwhile, the comparison for
EC (mS cm− 1) the dehumidified H. itama samples was done between samples harvested
n 49 23
Mean ± SD 0.28 ± 0.12 0.57 ± 0.14* from Peninsular Malaysia (as represented by Perak and Terengganu)
Insoluble Matter (%)
n 48 21 versus Borneo (which was represented by Sarawak).
Mean ± SD 2.33 ± 9.38 1.08 ± 1.92
HMF (mg kg− 1) Out of 12 physicochemical parameters examined, only proline con­
n 47 22
Mean ± SD 6.87 ± 4.25 16.57 ± 6.70* tent was significantly varied among the raw H. itama samples harvested
Proline (mg kg− 1) from different geographical origins (p < 0.05, Kruskal–Wallis test)
n 48 23

Ash Content (%)

Mean ± SD 0.28 ± 0.13 0.30 ± 0.12 (Table S5A, Fig. S3). The highest value for proline content was observed
n 46 23 in samples harvested from Southern (9.20 ± 3.93, n = 19) followed by
Mean ± SD 25.57 ± 8.06 15.29 ± 8.43*
Fructose (%)
n 49 25
West Coast (6.30 ± 3.40, n = 10), East Coast (5.73 ± 4.26, n = 6), and
Mean ± SD 20.62 ± 8.03 19.76 ± 13.41 lastly, Northern region (4.45 ± 4.36, n = 10). Conversely, dehumidified
Glucose (%)
n 49 25 H. itama honey showed significant differences in the content of fructose
Maltose (%)
Mean ± SD 1.96 ± 1.27 1.36 ± 0.66 (p < 0.01, unpaired t-test), IM (p < 0.01 Mann–Whitney test), and AC (p
n 45 22
< 0.01, Mann–Whitney test) between the samples harvested from
Mean ± SD 2.97 ± 1.92 1.09 ± 0.68
Sucrose (%)
n 49 23* Peninsular Malaysia and Borneo (Table S4B, Fig. S4). Other parameters
were observed at a similar level in samples from Peninsular Malaysia
The results are expressed as mean ± standard deviation. n = number of samples,
and Borneo (Table S4B).
ND = not determined. SD = standard deviation. Asterisk (*) signified significant
We further evaluated whether multivariate analysis of physico­
mean difference (p < 0.05).
chemical properties ATR-FTIR analysis could be used to discriminate the
raw Malaysian H. itama on the basis of their geographical origins. The
in this study, which is in agreement with the report by Carvalho et al.
chemometric analysis based on physicochemical properties showed a
(2009) (Table 2). However, it is currently unknown how dehumidifi­ 22.3 % variance of PC1 and a 19.2 % variance of PC2 (Fig. 4B). The PCA
cation could alter the EC of stingless bee honey.
biplot showed that the samples harvested from the Northern and East
The mean for proline of dehumidified honey (14.97 mg kg− 1) was Coast regions were situated in the upper side of the quadrant, and the
significantly higher than that of the raw honey (5.52 mg kg− 1) (Table 2).
Southern and West Coast samples, in the lower side of the quadrant. The
To the best of our knowledge, to date, no study has looked into the effect biplot further indicates that the H. itama honey from the Northern and
of dehumidification on the proline level in stingless bee honey.
East Coast regions have similar characteristics, whereas the H. itama
Our data showed no significant differences in the FA, HMF, and AC honey from the Southern Coast region shared similar features with that
between dehumidified and raw stingless bee honey (Table 2). By
in the West Coast region. The physicochemical properties such as sugars,
contrast, Carvalho et al. (2009) reported that dehumidification led to a EC, FA, and WC are the characteristics of the Northern and East Coast
significant reduction of the amount of free acids, higher HMF, and lower
regions, whereas proline and IM were attributed to the Southern and
AC in stingless bee honey. The differences observed between our study West Coast regions (Fig. 4B).
and Carvalho’s could be due to the dehumidification methods and spe­ Similar trends were also observed in ATR-FTIR chemometrics anal­
cies of stingless bee honey used. Carvalho’s studies used Brazilian ysis (Fig. 4C) and hierarchical cluster analysis (HCA) dendrogram
stingless bee honey (Melipona scutellaris and Melipona quadrifasciata) (Fig. 4D). The PCA model showed that the first two PCs cumulatively
that were dehumidified at 35.5℃ and were left in a controlled envi­ accounted for 59.0 % of the total variance in the original data. The H.
ronment for 5 days until the moisture content drop to approximately 17 itama honey from the Northern and East Coast regions were separated
%. We have used Malaysian H. itama subjected to a temperature between from the Southern and West Coast regions by PC1. The HCA calculated
35℃ and 38℃, and the average WC observed at the end of the dehu­ using Ward’s minimum variance method is a clustering analysis method
midification process was approximately 21 %. to evaluate the relationships among similar groups of samples (Mauli­
We have observed significantly higher IM in dehumidified H. itama diani et al., 2018). In this study, the HCA dendrogram showed two major
(0.57 ± 0.14 %) compared to raw H. itama (0.28 ± 0.12) (Table 2). This clusters, similar to the PCA result.
is in contrast with the report by Aroucha et al. (2019). In their study, the Our data are in agreement with those of other studies, which re­
dehydrated Brazilian stingless bee honey showed lower IM (0.62 %) ported that geographical origins have significant effects on the physi­
than did raw samples (1.05 %). As aforementioned above, variabilities cochemical properties of stingless bee honey (Abdullah et al., 2020;
in the species and methods used could account for the differences Fatima et al., 2018; Majid et al., 2019). For example, Fatima et al. (2018)
observed between our study and Aroucha’s. showed that WC, EC, AC, and HMF of H. itama and G. thoracica harvested
To summarize, dehumidification processes have led to the alteration from different states in Malaysia were associated with their geographical
of some physicochemical properties of stingless bee honey, namely, WC, origins. By contrast, the pH values of the Malaysian H. itama were sus­
fructose, sucrose, EC, IM, and proline, as well as their chemical profiles. tained regardless of their geographical origins.
The findings in ATR-FTIR chemometrics were in agreement with its Different geographical origins may attribute to differences in
phytochemical properties and showed the effectiveness of the PCA botanical sources during the foraging activities of stingless bee species
model in differentiating the raw and dehumidified samples. (Fatima et al., 2018; Majid et al., 2019). Shamsudin et al. (2019) re­
ported that botanical sources accounted for the differences in the
3.5. Classification of Malaysian stingless bee honey based on geographical physicochemical properties of Malaysian H. itama. In their study, H.
origins itama harvested from Meleleuca cajaputi (Gelam), Acacia mangium
(Acacia), and Averrhoa carambola L (starfruits) nectar across different
We next evaluated the effects of geographical origins on the states in Malaysia (Melaka, Johor, and Pahang, respectively) were

8
N.F. Ismail et al.
Fig. 3. Chemometrics analysis of raw and dehumidified H. itama. Principal component analysis (PCA) biplots of physicochemical properties (A). Principal
component analysis (PCA) score (B) and loading plots (C) of ATR-FTR data.
significantly varied in the moisture content, pH, and FA. However, we
cannot correlate our observation to the botanical origins because most of
the stingless bee honey used in this study came from multifloral samples.
Other environmental variabilities may also attribute to the differ­
ences observed among the samples of different geographical origins. For
instance, the states within the same region may experience similar
climate change in terms of temperature and humidity, thus similarity in
the pollination and stingless bee honey production. All the samples in
this study were harvested from August to October. This period is
considered as southwest monsoon season in Malaysia and is character­
ized as low precipitation and dry period (Chenoli et al., 2018). However,
there could be variation in the rainfall distribution between the states in
which the stingless bee honey samples were harvested. The states in
Peninsular Malaysia especially the East Coast zone including Kelantan
and Terengganu experience more often long-term heavy rainfall than do
the other states in Malaysia observed in this study (Othman et al., 2015;
Wong et al., 2016). Additionally, the soil content could affect the
physicochemical properties of stingless bee honey. For example, AC with
<0.5 g 100 g− 1 indicates free contamination related to the soil, in which
the nectar was collected during the bee foraging activities (Vit et al.,
2004; Moniruzzaman et al., 2014).
Taken altogether, chemometric analysis of physicochemical and
ATR-FTIR could be used to discriminate the Malaysian stingless bee
honey on the basis of their geographical origins. However, additional
9
Journal of Food Composition and Analysis 104 (2021) 104126
analysis is warranted to elucidate how geographical origins are attrib­
uted to the physicochemical and chemical profiles of Malaysian stingless
bee honey.
3.6. Comparison of PCA using physicochemical properties and FTIR data
In this study, six PCA models were developed for discriminating
Malaysian stingless bee honey by (1) species and (2) raw versus dehu­
midified stingless bee honey and (3) geographical origins on the basis of
physicochemical properties and ATR-FTIR data. Eigenvalues that
describe the explained variance by PCs for those six PCA models are
shown in Supplementary Materials (Fig. S5). Interestingly, different
patterns were observed in the eigenvalues of PCA models using physi­
cochemical properties and FTIR data. A total of eigenvalues from the
first two PCs of PCA models from FTIR data were larger than those from
PCA models from physicochemical properties data. The FTIR data
comprise 1718 variables, which are very large compared with physi­
cochemical properties data (only 11 variables); this indicates that FTIR
data contain more valuable information than physicochemical proper­
ties data. It also implies that the first two PCs of PCA models from FTIR
data could give better discrimination than those in PCA models from
physicochemical properties data (as shown by score plots in Fig. S5).
It is also found that after the first two PCs, the use of the three PCs in
the 2D score plots (i.e., PC1 vs. PC3 or PC2 vs. PC3) did not improve the
N.F. Ismail et al.
discrimination since the eigenvalues were relatively small (Fig. S6A).
However, the inclusion of the three PCs in the 3D score plots will slightly
improve the discrimination (Fig. S6B).
4. Limitations
We acknowledge that there were several limitations on the data
interpretation of this study. First, uneven sample size between the var­
iables was unavoidable due to resources (only H. itama and G. thoracica
are domesticated locally, and other stingless bee species were mostly
available in the wild) and financial, time, and logistical constraints
experienced during harvesting the stingless bee honey samples. Addi­
tionally, a smaller sample size of dehumidified stingless bee honey (34
samples) was used as most of the beekeepers do not have access to the
10
Journal of Food Composition and Analysis 104 (2021) 104126
Fig. 4. Comparison of physicochemical prop­
erties and chemical profile of H. itama harvested
from different geographical origins across
Malaysia. The Peninsular Malaysia map shows
the states in the different regions analysed in
this study; Brown colour represents North re­
gion, blue represents east region, yellow repre­
sents south region and magenta represents west
region (A). The Principal component analysis
(PCA) biplot of physicochemical properties (B).
Principal component analysis (PCA) score plot
(C) and hierarchical cluster analysis (HCA) (D)
generated using ATR-FTIR data. (For interpre­
tation of the references to colour in this figure
legend, the reader is referred to the web version
of this article).
costly dehumidifier. Second, outliers were included in all analyzes to
depict the data as a whole and to showcase the variations that normally
exist for honey samples in nature. Third, there could be other extrinsic
factors that play roles in the differences obtained when comparing the
species, the dehumidification process, and the geographical origins of
the stingless bee honey in this study. The possible factors include the
botanical sources, handling, and storage conditions of the stingless bee
honey. Proper records of these factors are warranted for a better indi­
cation and clarification of the data.
5. Conclusion
The chemometric analyzes of physicochemical and chemical profiles
based on ATR-FTIR were successfully applied to classify Malaysian
N.F. Ismail et al.
stingless bee honey to the respective species, dehumidification process,
and geographical origins. The FTIR chemometric result was in agree­
ment with its phytochemical properties indicating the effectiveness of
the PCA model in differentiating the raw and dehumidified samples.
Additionally, the PCA model successfully distinguished the H. itama on
the basis of their geographical origins. Similar trends were also observed
in ATR-FTIR chemometrics analysis and HCA dendrogram. Taken alto­
gether, the PCA models from FTIR data could give better discrimination
of Malaysian stingless bee honey on the basis of (1) species, (2) origins,
and (3) between raw and dehumidified stingless bee honey when
compared with PCA models from physicochemical properties data.
Thus, the ATR-FTIR-based chemometrics approach could be a potential
and reliable tool for the classification and quality control of the stingless
bee honey besides the analysis of physicochemical properties.
The findings from this study are crucial to providing further guidance
on the quality check purposes of stingless bee honey and possibly to
improve the current Malaysian standard of stingless bee honey for
commercialization drives in the future.
CRediT authorship contribution statement
Nur Faezah Ismail: Data curation, Writing - original draft. Mauli­
diani M.: Conceptualization, Methodology, Writing - review & editing.
Syafizal Omar: Investigation, Methodology, Data curation, Writing -
original draft. Muhammad Faiz Zulkifli: Investigation, Methodology,
Data curation, Writing - original draft. Mohd Naim Fadhli Mohd
Radzi: Investigation, Methodology, Data curation. Norra Ismail: Re­
sources, Methodology, Data curation, Validation, Writing - review &
editing. Arif Zaidi Jusoh: Resources, Methodology, Data curation,
Validation, Writing - review & editing. Suri Roowi: Resources, Meth­
odology, Data curation, Validation. Wooi Meng Yew: Resources,
Methodology. Rudiyanto R.: Data analysis, Validation. Wan Iryani
Wan Ismail: Project administration, Conceptualization, Methodology,
Supervision, funding acquisition, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research is supported by the PHC-Hibiscus grant (MyPAIR/1/
2020/WAB01/UMT//1) from Ministry of Higher Education Malaysia
and the special grant (vot: 53213) provided by the Malaysian Agricul­
tural Research and Development Institute (MARDI). We would like to
thank our respective universities for providing the facilities and support
throughout the study, as well as the beekeepers and MARDI that pro­
vided honey samples for the purpose of this research. We are also
thankful to Kok Suet Cheng (Faculty of Science and Marine Environ­
ment, Universiti Malaysia Terengganu) for her technical assistance in
ATR-FTIR analysis. This paper is dedicated to the memory of our student
the late Mr. Francis Kanyan Enchang, who was involved as one of the co-
authors to this article.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jfca.2021.104126.
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