Food Control 82 (2017) 145e153

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

Entomological authentication of stingless bee honey by 1H NMR-based

metabolomics approach
V. Zuccato a, C. Finotello a, I. Menegazzo a, G. Peccolo b, E. Schievano a, *
a
Department of Chemical Sciences, University of Padova, via Marzolo 1, 35131 Padova, Italy
b
Department of Veterinary Medical Sciences Alma Mater Studiorum, University of Bologna, Via Tolara di Sopra 50, I-40064, Ozzano Emilia, Bologna, Italy

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

Article history: The high value of stingless bee honey, better known as pot-honey, offers an incentive to fraud and re-
Received 3 March 2017 quires new analytical methods to guarantee the entomological origin. In this research, Geotrigona-Trig-
Received in revised form ona, Melipona, Scaptotrigona, and Apis mellifera Ecuadorian honeys were analyzed. Orthogonal projection
18 May 2017
to latent structure-discriminant analysis (OPLS-DA) built on 1H NMR data obtained by both water
Accepted 15 June 2017
Available online 18 June 2017
dilution and chloroform extracts spectra, conveyed to successful entomological discrimination. The
metabolic fingerprints of these different types of honeys are unique. The most important findings are
specific marker signals of entomological origin in chloroform extracts: metabolites deriving from the
Keywords:
Apis mellifera
cerumen secreted by the same bees are present and may be considered as the bee species signature on
Entomological origin honey. Furthermore, the endogenous diacylglyceryl ether is recognized as the key marker of pot-honey
Geotrigona-Trigona adulteration with Apis mellifera honey.
Honey © 2017 Elsevier Ltd. All rights reserved.
Melipona
Meliponini
1
H NMR-based metabolomics
Scaptotrigona
Cerumen
Waxes

1. Introduction
Bees of the world produce and store honey in beeswax combs
eApis species, and in cerumen pots eMeliponini species. Stingless
bees belong to the Meliponini Tribe (Michener, 2007) and thrive in
the tropical and subtropical regions of the world. Different evolu-
tionary factors caused a great biodiversity up to 500 species group
living in the Neotropics (Michener, 2013): an evidence is given by
the variety of materials and structures used to build their nests.
Wax secreted by stingless bees is mixed with plant resins to pro-
duce cerumen, and honey is stored in cerumen pots (Morais, Calaça,
& Rosa, 2013). Many species hollow in tree, some prefer other kinds
of cavities (between walls of buildings, for example), and others
nest in the ground. Consequently, the organoleptic and physico-
chemical characteristics of stingless bee honeys vary according to
the bee species, but they are completely different from that pro-
duced by the Apis genus bees (Souza et al., 2006). The main
* Corresponding author.
E-mail address: elisabetta.schievano@unipd.it (E. Schievano).
differences are in term of viscosity, taste, total acidity, diastase ac-
tivity, color, and water content (Chuttong, Chanbang, Sringarm, &
Burgett, 2016). The higher content of water in stingless bee honey
allows microorganisms to survive and to be active, so that it may
ferment naturally inside pots.
For all these reasons stingless bee honey carries a universe of
components that go well beyond the traditional product of
beekeeping. Indeed, it is not only directly consumed but also used
in traditional remedies to treat bruises, tumors, ocular cataracts,
pterygium, inflammation, infections, varicose veins, kidney dis-
eases, for wound healing, and as soothing balm before sleeping (Vit,
Vargas, Lo
pez, & Maza, 2015; Rao, Krishnan, Salleh, & Gan, 2016;
Guerrini et al., 2009).
Unfortunately, the production of this precious honey, when
compared to the production of Apis mellifera honey, is limited. The
honey is available in traditional markets and commands a signifi-
cantly higher price relative to A. mellifera honey (Fuenmayor, Díaz-
Moreno, Zuluaga-Domínguez, & Quicaza
n, 2013) encouraging the
appearance of honey falsification, putting the declared entomo-
logical origin at risk (Alves, 2013). Dilution with honey produced by
Apis bees, or even with various cheaper sweeteners or syrups,

http://dx.doi.org/10.1016/j.foodcont.2017.06.024
0956-7135/© 2017 Elsevier Ltd. All rights reserved.
146 V. Zuccato et al. / Food Control 82 (2017) 145e153
provide higher commercial profits.
For stingless bee honey, there are no international quality
standards. As the world's second most important honey producer,
after China (European Commission official website, 2017), the Eu-
ropean Union represents the greatest market for all apiculture
products. Despite this, the EU is a net importer of honey as domestic
production only covers around 60% of consumption. The main
supplier of honey imported into the EU is China, followed by
Ukraine and countries in Latin America. EU therefore represents a
potential, strongly interesting, expanding market for pot-honey
even taking into consideration its limited production and distri-
bution. However, this is contrary to the European legislation
currently in force (Directive 2001/110/EC of 20 December 2001)
that defines honey exclusively as “a natural sweet substance pro-
duced by Apis mellifera”. Consequently, the availability of relevant
methods to identify the entomological origin of honey could be a
first decisive step both to prevent fraud in producer countries and
to implement a regulatory reform allowing to open up EU market to
this product.
Numerous predictive methods have been studied. A multivar-
iate analysis approach with six quality factors of honey (free acidity,
moisture, sucrose, reducing sugars, HMF, and diastase activity) was
explored to discriminate the source bee of Venezuelan pot-honeys.
These preliminary findings need to be confirmed because the
botanical origin may play a role in determining some physico-
chemical differences, as well as in A. mellifera honey (Vit, Persano
Oddo, Marano, & Salas de Mejías, 1998; Sousa et al., 2016). The
phenolic fraction has also been compared between honeycomb and
pot-honey to estimate its value as an entomological predictor but it
seems to be more related to the geographical than the entomo-
logical origin (Vit, Soler, & Tom
as-Barbera
n, 1997). As major com-
ponents of honey, sugars were principally used to describe different
honey types (Bogdanov, Vit, & Kilchenmann, 1996; Vit, Fernandez-
Maeso, & Ortiz-Valbuena, 1998) and fructose, glucose, and maltose
contents (Vit, Fernandez-Maeso, et al., 1998) clusterized honey
types from Venezuela according to the source bee species. A recent
study on honeys of Apis mellifera, Melipona beecheii and Trigona
spp., collected in Yucatan, Mexico, suggested the use of the con-
ventional physicochemical parameters in combination with the
protein electrophoresis patterns as an alternative method for
entomological origin determination (Ramo
n-Sierra, Rui-Ruiz, &
Ortiz-Va
zquez, 2015). Again, Kek et al. (2017a) classified honeys of
common bees and stingless bees by coupling physicochemical
profiles with mineral and heavy metal content. More recently, a
DNA metabarcoding protocol has been developed to simulta-
neously deliver information on the botanical and entomological
origins of honey, but this method does not work with honeys
subjected to crystallization or rich in polyphenols (Prosser and
Hebert, 2017). Successful was the latest reported method, based
on a genetic identification of mitochondrial DNA sequences and
phylogenetic analysis by means of forensically informative nucle-
otide sequencing (FINS) technique (Kek, Chin, Tan, Yusof, & Chua,
2017b).
The multiple efforts of the various authors highlight the
importance of finding new discrimination methods, and in partic-
ular, a simple and reliable method is necessary, exclusively based
on entomological dependent parameters of the honey, rather than a
combination of factors depending also on the botanical and
geographical origin.
Preliminary reports suggested the potentiality of 1H NMR
spectroscopy coupled to multivariate analysis to discriminate
honeys from Australia, Bolivia, Brazil, Mexico and Venezuela, ac-
cording to their geographical and entomological origin (Schievano,
Mammi, & Menegazzo, 2013; Vit, Uddin, Zuccato, Maza, &
Schievano, 2015). Compared to other methods, with the NMR
technique all variables can be explored simultaneously, rather than
be obtained with more different tools, as it happens for the most
traditionally measured parameters. The coupling of NMR to
multivariate analysis has already been used in the past on other
kind of foods (Mannina, Sobolev, & Viel, 2012) and satisfactory
results have been recently published on botanical origin classifi-
cation on honey (Schievano, Finotello, Uddin, Mammi, & Piana,
2016).
The present work represents a depth of our researches and it is
focused on the entomological discrimination of genuine honeys. A
systematic study, both on water solutions and chloroform extracts,
was performed on honeys from Apis mellifera, Geotrigona-Trigona,
Melipona and Scaptotrigona bee species. NMR-based profiling was
applied to investigate the different compositions and to identify
potential markers, candidate for the source bee species authenti-
cation. It is here shown that 1H NMR-based analyses can provide
unequivocally the entomological origin and also monitor the
quality of honey.
2. Materials and methods
2.1. Chemicals and materials
Deuterated chloroform (CDCl3, 99.8%D), and deuterated water
(D2O,
99.97%D) were purchased from Sigma-Aldrich (Milan, Italy)
and from Euriso-Top (Gif sur Yvette, France) respectively. 5 mm
precision glass NMR tubes (535-pp, Wilmad) were purchased from
Sigma-Aldrich (Milan, Italy).
2.2. Honey and cerumen/wax samples
The 78 genuine Ecuadorian honeys were received from Patricia
Vit, who reports the following entomological and geographical
origins: 23 Apis mellifera (A) (Agua Blanca, Manabí province;
Cuenca, Azuay province; Chemico, Zamora Chinchipe province;
Loja, Loja province; Portoviejo, Manabí province; Mombaiza,
Morona Santiago province; Quevedo, Los Ríos province; Tablazo,
Esmeraldas province), 16 Geotrigona-Trigona (GT) (Agua Blanca,
Portoviejo and Quimís, Manabí province; Balsas and Los Ceibitos,
Santa Elena province; Camarones, Esmeraldas province; Alamor,
Cochas, Macara
and Zapotillo, Loja province), 15 Melipona (M)
(Chontayacu, Puyo, Pastaza province; La Chiquita, Esmeraldas;
Pindal, Loja province; La Moquillada, El Oro province; Tamiahurco,
Napo province; via Los Zorros, Orellana province), 24 Scaptotrigona
(S) (Calera Chica, Chiriboga, Machala, Moromoro, Piedras Blancas, El
Oro province; Denavip, El Trapiche, Potrerillos, San Pedro, Loja
province; Tamiahurco, Napo province; via Los Zorros, Orellana
province).
Stingless bees were collected in isopropyl alcohol when
possible, and sent dried for entomological identification to Dr.
S.R.M. Pedro, Biology Department, Faculty of Philosophy, Science
and Letters, Universidade de S~ ~o Preto, Brasil.
ao Paulo, Ribeira
Vouchers are deposited in the Camargo Collection, housed there.
Cerumen from stingless bees and wax from Apis mellifera were
collected and provided by P. Vit.
2.3. Sample preparation for NMR
2.3.1. Aqueous samples of honey
About 200 mg of honey were precisely weighted and dissolved
in D2O, to reach a total volume of 1.00 ± 0.025 mL. The solution
(650 mL) was transferred in a 5 mm precision glass NMR tube.
2.3.2. Chloroform extracts of honey
About 6 g of honey precisely weighed in a Teflon tube were
 V. Zuccato et al. / Food Control 82 (2017) 145e153 147

dissolved in 15 mL of deionized water; 15 mL of CHCl3 were added segmenting the organic extracts spectra (2.7e13.0 ppm) in 250
and the mixture was mechanically stirred for 10 min and then regular bins.
centrifuged at 10,000 rpm for 15 min at 4  C. The lower chloroform In each case, the solvent signal was deleted and the integral
phase was collected in a glass vial and the solvent was evaporated values were normalized by Total Sum Normalization. Calculations
under a gentle stream of nitrogen. The solid residue was dissolved were performed using the ACD program (ACD/Labs, Toronto, Can-
in 600 mL of CDCl3 and placed in an NMR tube for the analysis ada). The A, B, C matrices were exported to Microsoft Excel and
(Schievano, Peggion, & Mammi, 2010). transferred into SIMCA-Pþ software (13.0 Umetrics, Umea,
Sweden).
2.3.3. Waxes/cerumen samples
Waxes/cerumen were treated by dissolving about 0.5 g in 2 mL 2.5.2. Multivariate data analysis
of CDCl3 at 40  C and by filtering through a 0.45 mm membrane Statistical methods SIMCA-Pþ 13 (Umetrics, Umea, Sweden)
filter. was used for Principal Component Analysis (PCA), Projections to
Latent Structures-Discriminant Analysis (PLS-DA) and Orthogonal
2.4. Spectra acquisition and processing Projections to Latent Structures-Discriminant Analysis (OPLS-DA)
of the NMR data. The multivariate analysis was carried out onto
NMR experiments were recorded at 25  C (40  C for waxes mean-centered and “Pareto” scaled data. The quality of the models
dissolved in CDCl3) using a Bruker Avance DMX600 operating at was described by R2 and Q2 values. R2 is defined as the proportion
599.90 MHz for 1H and equipped with a 5 mm TXI triple gradient of variance in the data explained by the models and indicates the
probe. goodness of fit. Q2 is defined as the proportion of variance in the
Two spectra for each honey dissolved in D2O were acquired. For data predictable by the model and indicates predictability (Jung
major components analysis (principally sugars), the common one- et al., 2010). One vs others OPLS-DA models, where each class of
pulse sequence with water presaturation, 6 s relaxation time, 64 k honey was compared with the other classes considered together as
data points, and 32 transients, was used. For identification and a unique class, were used (Schievano, Stocchero, Morelato, Facchin,
quantification of minor components the spectrum was acquired by & Mammi, 2012). The resulting S-plot graph (Wiklund et al., 2008)
using a double pulsed field gradient spin echo (DPFGSE) sequence, was used to highlight the role of the variables in the model and to
modified by the addition of an inversion hard pulse after the first find the putative markers for each type of honey. The distance to
gradient in the G-S-G cluster to obtain a G-p-S-G block (G is a the model (DModX) test was applied to check for outliers. The
pulsed field gradient and S is a soft 180 pulse). The soft pulse was models were validated with the permutation test on the Y block to
an inversion Reburp sequence of 1.2 kHz sweep width and 3867 ms avoid randomness or overfitting to the model.
duration centered at 4.00 ppm (Schievano et al., 2015). All gradient
pulses were followed by a 100 ms recovery delay. This sequence
3. Results and discussion
drastically reduces the sugar signals allowing the use of higher
value of Receiver Gain (RG), which improves the digitalization of
The metabolic fingerprints of honey samples, both in water
the smaller signals.
dilution and in chloroform extracts, are unique. In a first stage of
For each chloroform extract, 256 transients were collected as
our study, the three explorative OPLS-DA models performed on the
64 k data points with a spectral width of 14 ppm, 2 s recovery delay,
acquisition time of 3.91 s, using a modified double-pulsed field
gradient spin echo sequence (Rastrelli, Schievano, Bagno, &
Mammi, 2009).
Line broadenings of 0.3 Hz and 0.5 Hz were applied before
Fourier transformation for D2O and chloroform solutions respec-
tively. Phase and baseline corrections were performed manually
and the spectra were calibrated on the aH1 proton of glucose at
5.22 ppm for aqueous samples, and on the residual chloroform
signal at 7.27 ppm for the organic extracts.
Water-soluble metabolites resonances assignments are based
on literature data.
The concentration of analytes, in aqueous solution, was deter-
mined relating the area of an appropriate analyte signal with that
one of 0.7 mM sodium 3-(trimethylsilyl)propionate-d4, used as an
internal standard.

2.5. Statistical analysis

2.5.1. Pre-statistical processing of NMR data

Three separate data matrices were obtained.
Matrix A (sugars signals): integration of the spectra in D2O ac-
quired with one-pulse sequence. The data reduction was performed
by segmenting 3.5e5.5 ppm spectral region in 87 bins by 0.03 ppm
intelligent bucketing.
Matrix B (minor components signals): integration of the spectra
in D2O acquired by using DPFGSE sequence. The data reduction was
obtained by segmenting the 0.7e3.0 ppm and 5.8e9.5 ppm spectral Fig. 1. OPLS-DA score plot derived from 1H NMR spectra of chloroform extracts ob-
regions in 191 bins by means of 0.03 ppm intelligent bucketing. tained by Geotrigona-Trigona (GT), Melipona (M), Scaptotrigona (S) and Apis mellifera (A)
Matrix C (signals from chloroform extracts) was obtained by honeys.
148 V. Zuccato et al. / Food Control 82 (2017) 145e153

Fig. 2. OPLS-DA score plots (on the left) and relative S-Plots (on the right) of the “Geotrigona-Trigona vs others” models. Panels A and B derive from matrix A, relative to sugar signals.
Panels C and D derive from matrix B, relative to water-soluble minor components signals. Panels E and F derive from matrix C, relative to chloroform extracts signals. The ellipse
represents the 95% confidence region for Hotelling's T2. Triangles for GT and circle for other honeys were used. The variables inside dotted rectangles represent the signals
responsible for differentiation of Geotrigona-Trigona from all other honey types; association of variables with assigned metabolites is given in the text.
 V. Zuccato et al. / Food Control 82 (2017) 145e153 149

Table 1
OPLS-DA parameters: R2 and Q2 of the model obtained by matrix A (sugars signals) matrix B (minor components signals) and matrix C (chloroform extracts signals).

Entomological Origin of Honey Matrix A Matrix B Matrix C

Sugars Minor components in D2O CDCl3 extracts

2 2 2 2 2 2
R X R Y Q R X R Y Q R2 X R2Y Q2

Geotrigona-Trigona (GT) 0.850 0.88 0.868 0.727 0.888 0.788 0.596 0.921 0.885
Scaptotrigona (S) 0.949 0.814 0.719 0.706 0.741 0.581 0.536 0.949 0.869
Melipona (M) 0.970 0.923 0.821 0.768 0.816 0.545 0.526 0.945 0.775
Apis mellifera (A) 0.944 0.854 0.768 0.742 0.943 0.862 0.51 0.954 0.901

Fig. 3. 1H NMR spectra of the aqueous honey samples: superposition of anomeric regions (5.50e4.90 ppm). From top to bottom: (GT) Geotrigona-Trigona, (S) Scaptotrigona, (A) Apis
mellifera and (M) Melipona. Sugars responsible of the entomological classification are indicated with abbreviations: kjb, kojibiose; mlz, melezitose; trn, turanose; mlt, maltulose; raf,
rafinose; imt, isomaltose; plt, palatinose; im3, isomaltotriose; mlb, melibiose; glu, glucose.

four bees classes (16 Geotrigona-Trigona, GT; 15 Melipona, M; 24
Scaptotrigona, S; 23 Apis mellifera, A) well anticipated the clustering
tendency of the NMR data. As an example, Fig. 1 reports the score
plot of OPLS-DA performed on the organic extracts matrix.
To emphasize the class discrimination and facilitate the identi-
fication of putative biomarkers, one vs others OPLS-DA was
analyzed. Precisely, for each of the four honey typologies, three one
vs others OPLS-DA models were built derived from the three
following data matrices: sugar matrix (matrix A), water diluted
minor components matrix (matrix B) and organic extracts matrix
(matrix C) respectively. Fig. 2 reports, as an example, the three score
plots and the relative S-plots of the GT vs others OPLS-DA models. A
good entomological separation is evident.
All the 12 OPLS-DA models (Fig. 1S) demonstrated high
explained variation (0.94 < R2 > 0.75) and predictive ability
(0.95 < Q2 > 0.5) as shown in Table 1 (maximum and minimum
tabulated values). Permutation tests of corresponding PLS-DA
models exhibit valid models (Eriksson, Byrne, Johansson, Trygg, &
Wilkstro€ m, 2013). The S-plot of all the one vs others OPLS-DA
models were analyzed to highlight the discriminatory variables
corresponding to the signals of the putative markers for the indi-
vidual classes of honey.

3.1. Metabolomics analysis on water diluted honey

In water dilution, more similarities are visible between Apis

Fig. 4. 1H NMR spectra acquired with DPFGSA sequence to optimize the digitalization
of minor compounds present in the aqueous honey samples. Overlaid fingerprint
spectral region: 2.5e0.75 ppm. From top to bottom: (GT) Geotrigona-Trigona, (S)
Scaptotrigona (M) Melipona and (A) Apis mellifera aqueous honey samples. Abbrevia-
tions: ala, alanine; pro, proline; ile, isoleucine; leu, leucine; u, unassigned peaks.
mellifera, Melipona and Scaptotrigona, compared to Geotrigona-
Trigona pot-honey spectra. This is in agreement with the different
chemical and sensory properties found for GT honey, which pre-
sents the highest moisture and free acidity, perceived with thin
150 V. Zuccato et al. / Food Control 82 (2017) 145e153
Fig. 5. 1H NMR spectra of chloroform extracts of honeys. Overlaid fingerprint spectral regions: 5.65e2.20 ppm and 6.95e7.70 ppm. From top to bottom: (GT) Geotrigona-Trigona, (S)
Scaptotrigona, (M) Melipona and (A) Apis mellifera. The resonances of marker compounds for different entomological honeys (*) are highlighted. In red, signals also present in waxes
(w) are evident, while the yellow color indicates the diacylglycerylether molecule signals. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
visual consistency and sour taste (Unpublished Data).
The diagnostic sugars for entomological classification (matrix A)
are visualized in Fig. 3. The discrimination is based on several
mono-, di- and tri-saccharides present in each honey but in
different relative amounts. GT honeys show the relatively lowest
quantities of fructose and glucose and the highest content of di-
and tri-saccharides (raffinose, isomaltose, isomaltotriose, melibiose
and palatinose) as demonstrated by the high loading value of res-
onances in the 3.70e3.90 and 4.85e5.01 ppm regions (Fig. 2B, and
Fig. 3). Melipona pot-honeys contain relatively low quantities of
turanose (5.28 ppm) and of di- or tri-saccharides in respect to the
other honeys (Fig. 3). The sugar signals responsible for the Scap-
totrigona classification are in the 5.37e5.45 ppm spectral region,
assigned to protons of melezitose and kojbiose (Consonni, Cagliani,
& Cogliati, 2012). The sugar composition of Apis mellifera honey,
compared to the pot-honey, is characterized by the higher contents
of glucose and fructose. This finding is in agreement with the data
reported by other authors (Ramo
n-Sierra, Rui-Ruiz, & Ortiz-
V
azquez, 2015; Chuttong et al., 2016).
Concerning to the minor compounds present in water solutions,
the signals responsible for the honeys entomological classification
were found in the aliphatic region (0.5e3.0 ppm, shown in Fig. 4).
The aromatic region (6.0e8.0 ppm) was not diagnostic for this
purpose because the aromatic amino acids, phenylalanine and
tyrosine, are present in a too variable concentration in all the
analyzed samples. GT honey differs from other honey types in the
content of 3-hydroxy-2-butanone, known as acetoin, 2,3-
butanediol, and other not identified compounds. The diagnostic
resonances of these metabolites are indicated in Fig. 4. These me-
tabolites are fermentation products of saccharides by microorgan-
isms, like yeast and bacteria, naturally present in pot honey
(Menezes, Vollet-Neto, Leo
n Contrera, Venturieri, & Imperatriz-
Fonseca, 2013). We estimated an average concentration of
7.2 ± 3.4 g/100 g and 0.4 ± 0.3 g/100 g for acetoin and 2,3-buta-
nediol respectively. In Melipona and Scaptotrigona honeys, these
compounds are found in an average concentration of 0.5 ± 0.6 g/
100 g and 0.1 ± 0.1 g, while in the Apis mellifera honeys, analyzed in
this study, they are under the detection limit. The much low levels
of fermentation products in A honey (<1 mg/kg) are also reported
in literature (Piasenzotto, Gracco, & Conte, 2003). Moreover,
discriminant metabolites from the A vs others S-plot are proline and
alanine in Apis genus honey (Fig. 4).
The one vs other OPLS-DA models obtained from matrix B for
both Melipona and Scaptotrigona honeys showed the lowest Q2
value (Table 1). Indeed, they presented only the 87% and 97% of
prediction capability, in respect to the others that have the 100%. To
emphasize the discrimination of these two classes, pairwise com-
parison M vs S honeys by OPLS-DA was made and the model
presents 100% of correctly classified samples. The most significant
loadings to discriminate Scaptotrigona and Melipona are relative to
amino acids content as proline, leucine and isoleucine, lower in M
(Fig. 4).
3.2. Metabolomics analysis of chloroform extracts
The peculiarity of chloroform extracts is that they contain
marker compounds of entomological origin. Indeed, while in water
dilution the classification is reached thank to a different content of
the same metabolites, in chloroform extract it is reached for the
presence of specific compounds for each type of honey. The S-Plots
of OPLS-DA models obtained by chloroform extracts data in matrix
C highlights these signals (Figs. in SI), most of them in the aliphatic
region of the spectra.
For GT pot-honey (Fig. 2, panel F), the spectral intervals
responsible of its classification are 4.24e4.32, 4.57e4.61 and
5.09e5.12 ppm. Diagnostic resonances of M pot-honey in chloro-
form extracts are in the 3.60e3.90 ppm region. In addition, aro-
matic signals seem to be important for the Melipona differentiation
(7.24 and 7.33 ppm). Marker protons of S honey resonate in the
5.34e5.42, 4.68e4.72 and 2.75e2.94 ppm spectral ranges, while
the signals responsible of A honey classification are in the
2.23e2.42, 3.42e3.66, 4.03e4.17, 4.30e4.34, 4.88e4.91,
5.14e5.22 ppm ranges. Overlaid spectral fingerprint regions in
Fig. 5 visualize the discriminating resonances (indicated with
asterisks).
Moreover, a comparison between chloroform extracts of honey
and wax/cerumen NMR spectra gave interesting information for a
deeper understanding of these results.
Honey chloroform spectra contain signals coming from flowers
visited by bees but also from waxes or cerumen pots, naturally
contained in honey in traces, and from other endogenous com-
pounds. In particular, wax is secreted by the bees in wax glands,
specialized cells near the surface of abdomen. Honeybees use the
wax to build honeycomb, which must have suitable chemical and
physical properties depending on the climatic conditions or on its
different function. Therefore, it is not hard to imagine that different
bees may secret different waxes. For example, the nest of the
Geotrigona-Trigona bees is usually under the ground and it is of
course different in comparison to the other type of nests, built on
the ground. Furthermore, wax secreted by stingless bees is mixed
with plant resins to produce cerumen pots (Morais et al., 2013). The
NMR spectra of chloroform extracts of cerumen and waxes from the
GT, S, M and A bees confirm the different composition. Interest-
ingly, some specific compounds from cerumen or, more generally,
from waxes, are also found in chloroform extracts of honey pro-
duced by the same bee species. Fig. 6 reports the NMR spectra of
 V. Zuccato et al. / Food Control 82 (2017) 145e153 151

Fig. 6. Comparison between 1H NMR spectra of chloroform extracts of honey (above) and of cerumen/waxes (below) by Geotrigona-Trigona (GT), Scaptotrigona (S), Melipona (M) and
Apis mellifera (A). The cerumen or waxes signals marked with w have high loading value in the S-Plots built from OPLS-DA models.
152 V. Zuccato et al. / Food Control 82 (2017) 145e153
both honey and cerumen/waxes chloroform extracts of GT, S, M and
A, respectively. In Fig. 5, the signals indicated with *w highlight the
waxes resonances having also high value of loading in the S-Plots.
These findings allow us to state that some of entomological
markers for pot honey come from their waxes. The chemical
structures of all these molecules have not been identified yet.
In the case of Apis mellifera the statistically important signals at
4.04 ppm and in the 2.20e2.40 ppm range derived from its waxes
and are identified as long chains of carboxylic esters. The other A
characterizing resonances (5.21,4,35, 4.18 and 3.56 ppm) are rela-
tive to a diacylglyceryl ether containing two oleic acid chains in
positions 2 and 3, and to a saturated hydrocarbon C18 chain in
position 1 (Schievano, Morelato, Facchin, & Mammi, 2013). This
compound had already been isolated from honeydew, where it is
relatively more abundant, but it is always present in honey. Our
recent study on more than 1000 chloroform extracts of Apis melli-
fera honeys (Schievano et al., 2016) allows us to confirm its ubiq-
uitary presence.
4. Conclusions
In this study, the great potential of NMR spectroscopy coupled
with chemometrics for the traceability of Melipona, Geotrigona-
Trigona and Scaptotrigona stingless bee pot honeys and of Apis
mellifera honey was demonstrated. Three different matrix data set
were used in order to give a complete overview of the discriminant
composition of these four types of honeys. The one vs others OPLS-
DA models showed high accuracy to recognize their entomological
origin. A first attempt of interpreting the observed differences in
terms of significant spectral buckets, by means of the inspection of
S-plots, was made. In water dilution, the discrimination is reached
because of different content of sugars, amino acids and fermenta-
tion indicators. In addition, markers of entomological origin are
present in chloroform extracts. Some diagnostic entomological
markers derived from specific waxes compounds, naturally present
also in honey. Furthermore, the diacylglycerylether, already iden-
tified as an endogenous compound of Apis mellifera honey, is here
recognized as a distinctive marker to discover fraud of mixtures
labeled as a pure pot-honey.
These are very important and useful discoveries. If diac-
ylglycerylether is present in a honey, the meliponine entomological
origin of pot-honey can be discarded. The possibility to authenti-
cate the stingless bee pot honey in producer countries, allows to
maintain consistency between the high market price and the great
value of this honey. In the same way, the presence of specific waxes
compounds in a honey, even in traces, can be considered the
signature of the producer bee, as guarantee of the more precise
entomological origin.
Definitely, the metabolomics discrimination approach reported
in this work leads now to the development of an innovative method
to recognize both frauds and entomological origin, because a quick
1
H NMR spectrum on the chloroform extract of a honey is enough
to show the entomological related marker compounds. Finally, the
implementation of the regulatory norms to open up EU market to
this ancient health product seems to be a very close chance.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
The authors wish to thank all the persons, particularly Patricia
Vit, who collected the honey samples, and provided their ento-
mological origin (more details in SI).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.foodcont.2017.06.024.
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