Food Chemistry 183 (2015) 43–48

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Electrophoresis characterisation of protein as a method to establish the

entomological origin of stingless bee honeys
Jesús Manuel Ramón-Sierra, Jorge Carlos Ruiz-Ruiz ⇑, Elizabeth de la Luz Ortiz-Vázquez
Departamento de Ingeniería Química-Bioquímica, Instituto Tecnológico de Mérida, Av. Tecnológico km. 4.5 S/N, C.P. 97118 Mérida, Yucatán, Mexico

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

Article history: Increasing production of stingless-bee honey and the prospect of broader marker for natural and organic
Received 27 May 2014 products indicate the need to establish parameters to determinate the entomological origin and authen-
Received in revised form 5 March 2015 ticity of honey. In this research, honeys of Apis mellifera, Melipona beecheii and Trigona spp. were collected
Accepted 7 March 2015
in Yucatan, Mexico. Stingless-bee honeys contained more water and less total sugars and reducing sugars.
Available online 16 March 2015
SDS–PAGE patterns show distinctive bands for each kind of honey. The SDS–PAGE pattern of A. mellifera
proteins honey showed three bands with molecular weights between 10.2 and 74.8 kDa, there were five
Keywords:
proteins bands in M. beecheii honey with molecular weights between 6.1 and 97.0 kDa and nine for
Honey
Stingless bees
Trigona spp. proteins between 9.3 and 86.7 kDa. Conventional physicochemical parameters along with
Physicochemical parameters electrophoresis profiles of stingless-bee honeys proteins could be an alternative for determination of
Electrophoresis entomological origin.
Entomological origin Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction combs made of cerumen (a mixture of propolis and beeswax) for

Stingless bees (Meliponini) are a large monophyletic group of
highly eusocial bees found in abundance in warm humid forests
around the globe. They are indispensable pollinators within tropi-
cal ecosystems (Roubik, Segura, & Camargo, 1997), and vary widely
in both individual and colony size. They share the presence of a
corbicula, a pollen-carrying structure on the hind legs, with the
other corbiculate bees, which include the highly eusocial honey
bees (Apini), primitively eusocial bumble bees (Bombini), and the
mostly solitary orchid bees (Euglossini) (Rasmussen & Cameron,
2010). Although stingless bees and honey bees both exhibit highly
eusocial behaviour, including perennial colonies of workers and a
single queen, the two tribes have likely evolved their particular
kind of sociality independently (Whitfield, Cameron, Huson, &
Steel, 2008). Beekeeping with honey bees belonging to the genus
Apis is more widespread than meliponiculture – beekeeping with
stingless bees (e.g., Melipona spp.) – which is a well-known tradi-
tion in tropical countries. Both groups of bees belong to the family
Apidae, order Hymenoptera. They are differentiated at the subfam-
ily level, Apinae for honey bees and Meliponinae for stingless bees.
The main structural differences between honey bees and stingless
bees is nest construction: stingless bees construct horizontal
⇑ Corresponding author. Tel.: +52 (999) 9645000/9645001; fax: +52 (999)
9448181.
E-mail address: jcruiz_ruiz@hotmail.com (J.C. Ruiz-Ruiz).
their nests, and honey pots instead of honeycombs for storing
honey (Vit, Medina, & Enríquez, 2004). There is a large size varia-
tion within the Meliponinae. For instance, Melipona fuliginosa, the
largest stingless bee, is more than 13 mm long, whereas a dwarf
species like Trigonisca duckei measures only about 2 mm. The size
of the colonies varies from a few hundred individuals in some
Melipona species to densely populated nests with tens of thou-
sands bees in species of the genus Trigona (Roubik, 1989).
There are reports of the existence of more than 500 species of
Meliponinae (classified in 50 genres), which are distributed in
the tropical and subtropical regions of the world. In Latin
America, these bees were kept by ancient pre-Columbian cultures,
such as the Mayas and Nahuatls (Vit et al., 2004). At least 46 spe-
cies of stingless bees inhabit Mexico, 16 of which are in the
Yucatan Peninsula (Kylea, Clarke, Oldroyd, Quezada-Euán, &
Rinderer, 2001). In the Yucatan Peninsula, stingless bee culture
dates from pre-Hispanic times. At the height of the Mayan civil-
isation, cultivation of stingless bees reached a level of sophistica-
tion comparable to the cultivation of Apis mellifera in Europe at
the same time (De la Rúa, May-Itzá, Serrano, & Quezada-Euán,
2007).
A wide range of attributes may suggest that stingless bee honey
enhances several systems to control digestive, respiratory, female
fertility, skin and visual disorders. Melipona beecheii is the most
important stingless bee species in Mexico and Guatemala due to
the honey yields and reported medicinal properties (Vit et al.,

http://dx.doi.org/10.1016/j.foodchem.2015.03.015
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
44 J.M. Ramón-Sierra et al. / Food Chemistry 183 (2015) 43–48
2004). Pollen and cerumen from the nests are also used in local
therapies, and the larvae of Melipona and Trigona species are
included in local diets (Ramos-Elorduy & Moreno, 2002). Trigona
(Tetragonisca) angustula is the most frequently reported species
for cataract treatment in Guatemala, Mexico and Venezuela.
However, these healing properties of honey and resin collected
by stingless bees have only recently begun to study and evaluate
outside Mayan peasant communities, so that there is a significant
economic potential in this area (Quezada-Euán, May-Itzá, &
González-Acereto, 2001).
Stingless bee honey is completely different from that produced
by the bees of the genus Apis although the former product reaches
a higher price in the market compared with the traditional honey
(Vit et al., 2009). Although several Apis species and stingless bees
produce honey widely relished by humans as a food, the official
definition of ‘honey’ is restricted to A. mellifera by the Codex
Alimentarius Commission. Imitations of Meliponinae honey have
been found in local markets in Mexico (Medina & Ortiz, 1999).
Honey standards have been modified according to the botanical
origin but not according to species origin. It is important that these
factors are taken into account and quality standards set for other
types of stingless-bee honey (Almeida-Muradian & Hitomi-
Matsuda, 2007). Numerous methods have been introduced for
establish quality control parameters and standardisation of honey,
e.g., measuring acidity, ash, electrical conductivity, diastase activ-
ity, hydroxymethylfurfural, invertase activity, nitrogen, reducing
sugars, sucrose and water (Vit, Persano-Oddo, Marano, & Salas de
Mejias, 1998).
Some secondary metabolites like phenols and flavonoids have
also been compared between honeys of A. mellifera and stingless
bees to estimate their value as entomological predictor based on
differences in bee diets. However, metabolites are related to the
geographical origin of the honeys rather than entomological origin
(Vit, Soler, & Tomás-Barberán, 1997). Vit, Fernández-Maeso, and
Ortiz-Valbuena (1998) extracted stingless bee honeys from 27
nests in the West and South of Venezuela, and analysed the sugar
content by HPLC. The concentration and ratios of the three fre-
quently occurring sugars, fructose, glucose and maltose, were
related to the entomological origin of the samples. Three maltose
ratios were calculated and all of them were distinctive for
Meliponini, Trigonini and the intermediate Scaptotrigona spp. hon-
eys. The authors proposed the hexoses-to-maltose ratio as predic-
tor of origin because differences at the tribe level are easily
observed without further statistical analysis; in the present set of
honeys, Scaptotrigona spp. presented values between 11 and 12,
other Trigonini were lower than 3 and Meliponini were higher than
15. In other study, H-nuclear magnetic resonance (NMR) spec-
troscopy was used to analyse pot honey samples of different geo-
graphical and entomological origin. An NMR-based metabolomic
approach, applied to 67 samples, tested and confirmed the validity
of the multivariate statistical analysis in the discrimination. NMR is
an efficient tool to differentiate honeys by their geographical ori-
gin; within limited geographical areas, it is possible to distinguish
honey samples in terms of bee species. Furthermore, the structural
identification of a geographical marker compound was achieved,
and a specific region in the NMR spectrum was found to be respon-
sible for the entomological separation (Schievano, Stocchero,
Morelato, Facchin, & Mammi, 2012).
The identification of proteins in honey by SDS–PAGE has been
considered less as an index of quality control of honey, and there
is a little information regarding markers of entomological origin
using this method. A subtropical region of Mexico like Yucatan
Peninsula has a huge potential for production and export of sting-
less-bee products. The increasing stingless-bee honey production
and the perspective of a broader market for natural and organic
products suggests a need to establish parameters to determinate
the entomological origin of honey to protect the consumer against
fraud. The aim of this work was to determine some conventional
physicochemical parameters generally used to analyse A. mellifera
honeys to analyse samples of stingless bee honeys and evaluate the
SDS–PAGE as a method to establish the entomological origin of A.
mellifera, M. beecheii and Trigona spp. honeys.
2. Materials and methods
2.1. Honey and insect samples
Honey samples of A. mellifera, M. beecheii and Trigona spp. were
collected in the municipalities of Cantamayec, Mani and Tizimín
(Yucatán), and Calkiní (Campeche) in México during the months
from April to May of 2009, 2011, and 2013. Samples were also col-
lected during the months from October to November of 2012. The
collections coincided with periods of maximum flowering of some
species associated with the production of honey. A. mellifera insects
were taken from healthy hives established in beekeeping units. M.
beecheii insects were taken from healthy hives established in
cooperatives of Mayan stingless beekeeping. Trigona spp. insects
were taken from wild hives. Three samples for each type of honey
were collected. Honey samples were preserved in glass bottles and
refrigerated (5 °C) until analysis. Insect samples were preserved in
polyethylene bags and frozen ( 20 °C) until analysis. Unless other-
wise stated, all reagents used in this study were reagent grade.
2.2. Physicochemical analysis
Water content, total acidity, total and reducing sugars were
determined following official Mexican methods (NMX-F-036-
NORMEX, 2006).
2.2.1. Water content
Water content was determined by refractometry, measuring the
refractive index (RI) using a standard model Abbe type refractome-
ter at 25 °C. Water content (%) was then obtained from the
Chataway table. Measurements were made in triplicate.
2.2.2. Total acidity
Total acidity was determined by a titrimetric method: 10 g
honey sample was dissolved in 75 mL, CO2 free water in a
250 mL beaker. The electrode of the pH metre was immersed in
the solution, which was aggitated with a magnetic stirrer and
titrated again 0.05 N NaOH to pH 8.5 (free acidity). Once complete,
10 mL of 0.05 N NaOH were added without delay and back-titrated
against 0.05 N HCl to pH 8.30 (lactone acidity). Total acidity was
calculated by adding together free- and lactone acidities. The
results were expressed as miliequivalentes/kg (meq/kg).
Determinations were made in triplicate.
2.2.3. Total sugars, reducing sugars and non-reducing sugars
2 g of sample were transferred to 200 mL volumetric flask. An
aliquot of 25 mL of the sample were pipetted into a conical flask
and 25 mL Fehling’s solution added. The mixture was heated to
boiling while being stirred, and maintained at boiling for 2 min
before adding methylene-blue indicator solution. The titration
was completed within the total boiling time of 3 min by drop-wise
addition of standard glucose solution from the burette. For inver-
sion, 50 mL aliquot was transferred to a 100 mL volumetric flask,
10 mL of HCl (1:1) were added. Then, the sample was heated at
70 °C for 1 h. Determination of sugar was made in triplicate.
Non-reducing sugars were estimated as the difference between
the total sugar content and reducing sugar content on subtraction
(total sugar-reducing sugar).
 J.M. Ramón-Sierra et al. / Food Chemistry 183 (2015) 43–48
2.3. Protein extraction
2.3.1. Bees and stingless bees
The bees and stingless bees used in the experiment were cap-
tured from colonies of Yucatan, Mexico. Twenty midguts from A.
mellifera, M. beecheii and Trigona spp., were dissected in ice cold
0.9% NaCl, and stored at 20 °C for protein quantification and poly-
acrylamide gel electrophoresis. The midgut extracts were obtained
by homogenising the midguts, and taking to a centrifuge at
l0.000 rpm during 5 min. The supernatant was dissolved in
100 lL of distilled water. Aliquots of these extracts were used for
total protein quantification by the method of Bradford (1976) using
bovine serum albumin as standard.
2.3.2. Bee honey and stingless bee honeys
For the analysis twenty-five grams of honey were used. In order
to maintain the stability of the proteins in the samples 2.5 mL of
acetate buffer (1.59 M, pH 5.3) and 1.5 mL of sodium chloride
(0.5 M) were added. Finally distilled water was added to achieve
a final volume of 50 mL (Ureña-Varela, Arrieta-Bolaños, Umaña,
& Zamora, 2007). The protein extraction was done using the
method reported by Peterson (1997) with some modifications.
Briefly, 1.0 mL of honey sample was mixed with 0.1 mL of 0.15%
deoxycholate and allowed to stand for 10 min at room temperature
(25 °C). After 0.1 mL of 72% trichloroacetic acid was added and cen-
trifuged at 1900g for 30 min at room temperature (25 °C). After the
centrifugation the supernatant was discarded and the pellet was
washed three times with two volumes of acetone. Finally the pellet
was allowed to dry at room temperature (25 °C), then dissolved in
150 ll of distilled water and stored at 4 °C for later analysis (Bollag,
Rozycki, & Edelstein, 1996).
2.4. Protein quantification
The protein quantification was done according to Bradford
(1976). A standard curve was performed with bovine serum albu-
min (BSA) at concentrations of 1, 2.5, 5, 10, 15 and 20 lg/lL. In the
case of the samples 10 lL of protein extract was diluted in 90 lL of
distilled water then 1.0 mL of Bradford reactive was added. The
samples were incubated for 5 min at room temperature (25 °C).
Finally standards and samples were read at a wavelength of
595 nm in a UV/VIS spectrophotometer (Perkin Elmer Lambda
XLS).
2.5. Determination of protein profile and molecular weight
Denaturing electrophoresis (SDS–PAGE) was performed accord-
ing to Bizani et al. (2005). A discontinuous system was used con-
sisting of two acrylamide gels, a separating gel (15% acrylamide)
and a stacking gel (4% acrylamide), and a running buffer (Tris–
Glycine–SDS). For the preparation of the samples 5 lg of each
extract were taken and diluted in 6 ll of loading buffer 5(5) dis-
tilled water was added to achieve a final volume of 50 m. The sam-
ples were incubated for 5 min at 95 °C in order to favour the
denaturation of proteins. Gels were run at 100 V for 2 h at room
temperature. The gels were incubated for 2 h in a solution of 25%
methanol, 10% acetic acid and 0.03% formaldehyde to fix the pro-
teins. Then the gels were washed 3 times for 20 min with 30% etha-
nol. The washed gels were incubated in dark for 1 min in a solution
of 0.01% sodium thiosulfate. To remove the remaining sodium thio-
sulfate, the gel is rinsed three times in distilled water and incu-
bated for 20 min in darkness in a solution of 0.2% of silver nitrate
and 0.14% formaldehyde; the silver solution was removed with
three washings of distilled water. The development was conducted
with 6.0% sodium carbonate, 0.03% formaldehyde and 0.03%
sodium thiosulfate; the gel was stirred until banding watch it,
45
the reaction is stopped with a solution of 25% methanol and 10%
acetic acid. The relative molecular weight of the polypeptides of
honey samples was determined by plotting a standard curve of
Rf vs. log Mw for known samples (ladder), and read off the
log Mw of the sample after measuring distance migrated on the
same gel.
2.6. Statistical analysis
All results were analysed using descriptive statistics with a
central tendency and dispersion measures. One-way ANOVAs
were run to evaluate physicochemical characteristics and content
of protein nutritional contribution. All analyses were done
according to Montgomery (2004) and processed with the
Statgraphics Plus version 5.1 software. Jaccard index (J) was used
to establish similitude between insects and honeys. Jaccard
index was calculated according to Ludarig and Reynold (1988).
J = a/a + b + c; where a = bands shared between two samples;
b = total number of bands in sample 1; c = total number of bands
in sample 2.
3. Results and discussion
3.1. Physicochemical parameters
Since stingless bee honeys have a different composition than A.
mellifera honey, some physicochemical parameters, such as water
content, total sugars, reducing sugars, sucrose, acidity, ash, hydrox-
ymethylfurfural (HMF) and diastase activity have been proposed to
differ between genuine A. mellifera honey and honeys from sting-
less bees (Vit et al., 2004). Of the eight parameters examined, only
four were significantly different, namely water content, total acid-
ity, total sugars and reducing sugars, and were chosen to examine
differences between A. mellifera honey and honey from M. beecheii
and Trigona spp. (Table 1). There was a significant (p P 0.05) differ-
ence between honey of different entomological origin in water
content. Trigona ssp. honey contained the most moisture (25.0 g/
100 g). Total acidity was higher in honey from M. beecheii
(38.6 meq/100 g) than honeys from A. mellifera and Trigona spp.
Total and reducing sugars were determined following the volumet-
ric Lane and Eynon method. The higher values were exhibit by
honey from A. mellifera followed by honeys from Trigona ssp. and
M. beecheii.
There were significant (p P 0.05) differences between honey of
different entomological origins in water content. Trigona ssp.
honey contained the highest amount of moisture (25.0 g/100 g)
compared to A. mellifera and M. beecheii honeys. Water content is
a quality parameter important for honey shelf life and is critical
to prevent microbiological spoilage; according to Fredes and
Montenegro (2006) honey containing lower moisture content will
have a longer shelf life. Water content also affects some physical
properties of honey, such as viscosity and glucose crystallisation
(Bogdanov, Ruoff, & Oddo, 2004). The occurrence of watery honey
in stingless bees may be related to the humid tropical
Table 1
Physicochemical parameters of A. mellifera, M. beecheii and Trigona spp. honeys.
Parameter A. mellifera M. beecheii Trigona spp.
Water content (g/100 g) 19.0a 22.8b 25.0c
Total acidity (meq/100 g) 37.3a 38.6b 36.6a
Total sugars (g/100 g) 77.3c 61.4a 70.6b
Reducing sugars (g/100 g) 69.2c 53.1a 63.7b
Non-reducing sugars (g/100 g) 8.1a 8.3a 6.9b
Superscripts indicate significant statistical difference.
46 J.M. Ramón-Sierra et al. / Food Chemistry 183 (2015) 43–48

environment, in which it is difficult to extract nectar with low 3.2. Protein profile and molecular weight
water content. There is a risk of spoilage when the honey contains
significant amounts of water. However, honeys from stingless bees Fig. 1 shows the SDS–PAGE pattern of honey protein in samples
do not spoil more readily than honeys containing less water. of different entomological origin. Three distinctive bands were
Possibly, in processing nectar into honey, the stingless bees add detected in the separating gel for A. mellifera honey. The molecular
enzymes or other substances that reduce microbiological activity weight of the proteins was found to be between 10.2 and 74.8 kDa.
or act as a preservative. This hypothesis would also support the For M. beecheii honey, five distinctive bands were detected in the
claims for the medicinal activity of these honeys (Torres, separating gel; the molecular weight of the proteins was between
Garedew, Schmolz, & Lamprecht, 2004). The acidity results 6.1 and 95.0 kDa. Finally, for Trigona ssp. Honey, nine distinctive
obtained in this work are in accordance with Mexican legislation bands were detected in the separating gel; the molecular weight
(NMX-F-036-NORMEX, 2006) for A. mellifera honey (the maximum of the proteins was between 9.3 and 86.7 kDa (Table 3). It was
value is 40 meq/100 g). Higher acidity values may be due to fer- observed that A. mellifera and M. beecheii honey samples did not
mentation of sugars to alcohol by microorganisms and further oxi- shared bands with similar molecular weight. On the other hand,
dation to carboxylic acids (Schneider, Horn, & Hammes, 2003). A. mellifera and Trigona ssp. honey samples shared one band of
Acidity values of honey evaluated in this study confirm the absence similar molecular weight (74.8 kDa).
of deterioration. There were significant (p P 0.05) differences Fig. 2 shows the SDS–PAGE protein patterns from midgut sam-
between honey of different entomological origins in total and ples of different entomological origin. Ten distinctive bands were
reducing sugars content. A. mellifera honey contained the highest detected in the separating gel for A. mellifera midgut. The molecu-
amount of both parameters (77.3 and 69.2 g/100 g) compared to lar weight of the proteins was found to be between 7.9 and
M. beecheii and Trigona ssp. honeys. The results obtained for reduc- 99.2 kDa. For M. beecheii midgut, 17 distinctive bands were
ing sugars content in this work are not in accordance with Mexican detected in the separating gel; the molecular weight of the pro-
legislation (NMX-F-036-NORMEX, 2006) for A. mellifera honey (the teins was between 7.8 and 136.3 kDa. Finally, for Trigona ssp. mid-
minimum value is 63.8 g/100 g). However, official methods for gut, 14 distinctive bands were detected in the separating gel; the
honey quality control have been developed specifically for A. mel- molecular weight of the proteins was between 9.2 and 128.0 kDa
lifera honey and, as already stated, the honey from stingless bees (Table 3). A. mellifera and M. beecheii midgut samples shared bands
have no standards. According to Vit et al. (2004) standards for of similar molecular weight of 7.8, 9.4, 54.2 kDa. Whilst A. mellifera
stingless bee honeys are different and propose a minimum value
of 50 g/100 g. The values obtained in the present study to honeys
of M. beecheii and Trigona ssp. are similar to those proposed by
Table 2
Vit et al. (2004). The results obtained in this study indicate that Molecular weight of proteins extracted from honeys and midguts of A. mellifera, M.
physicochemical parameters, such as water content, total acidity, beecheii and Trigona spp.
total sugars, reducing sugars and non-reducing sugars, could be
Apis mellifera Mellipona beecheii Trigona spp.
used for the quality control for stingless bee honeys.
Honey Midgut Honey Midgut Honey Midgut
74.8 99.2 97.0 136.3 86.7 128.0
33.5 75.1 27.1 124.6 74.8 115.0
10.2 63.9 16.2 106.1 64.5 98.9
54.2 7.6 102.4 49.0 92.8
45.8 6.1 97.3 31.1 87.1
31.7 79.0 20.5 75.1
26.3 61.0 17.0 66.3
23.9 54.9 14.8 58.3
9.9 48.6 9.3 48.7
7.9 33.2 41.2
28.8 14.6
24.5 13.1
15.8 11.6
14.1 9.6
12.9
9.4
7.8

Table 3
Jaccard index of similarity between honeys and midguts of A. mellifera, M. beecheii and
Trigona spp.

Entomological origin Jaccard index

Honey Insect midgut Honey/insect
extract midgut
extract
Apis mellifera/Melipona beecheii 0.00 0.21 0.05
Apis mellifera/Trigona ssp. 0.14 0.17 0.06
Mellipona beecheii/Apis mellifera 0.07 0.18 0.06
Mellipona beecheii/Trigona ssp. 0.05
Trigona ssp./Apis mellifera 0.17
Trigona ssp./Mellipona beecheii 0.13

Fig. 1. SDS–PAGE patterns of honey proteins of different entomological origin. Lane Jaccard index (J) as calculated according to Ludarig and Reynold (1988). J = a/
1, Molecular marker. Lane 2, Apis mellifera. Lane 3, Mellipona beecheii. Lane 4, Trigona a + b + c; where a = bands shared between two samples; b = total number of bands
ssp. in sample 1; c = total number of bands in sample 2.
 J.M. Ramón-Sierra et al. / Food Chemistry 183 (2015) 43–48
Fig. 2. SDS–PAGE patterns of midgut proteins of different entomological origin.
Lane 1, Molecular marker. Lane 2, Apis mellifera. Lane 3, Mellipona beecheii. Lane 4,
Trigona ssp.
and Trigona ssp. midgut samples shared bands of similar molecular
weight of 9.2, 75.1, and 98.8 kDa.
Table 2 shows the Molecular weight of proteins extracted from
honeys and midguts of A. mellifera, M. beecheii and Trigona spp.
Honey and midgut from A. mellifera were characterised by the
presence of two bands with approximate molecular weights of
10 and 75 kDa. Honey and midgut samples from M. beecheii were
characterised by the presence of four bands with approximate
molecular weights of 8, 16, 29 and 95 kDa. Finally, honey and mid-
gut samples from Trigona ssp. were characterised by the presence
of five bands with approximate molecular weights of 9, 14, 49,
75 and 87 kDa.
The molecular weights of proteins from insect midguts were
between 7.8 and 136.3 kDa. Table 2 shows the major constituents,
in order of their relative molecular weights (Mr). Midgut samples
from A. mellifera, M. beecheii and Trigona ssp. shared three bands
with approximate molecular weights of 47, 67, and 99 kDa.
According to Abd El Aal, Zalat, Abouzeid, and Ibrahim (2002) the
99 kDa band corresponded to the enzyme phosphorylase, the
67 kDa band could be catalase and the 47 kDa band is character-
istically ovalbumin. Midgut samples from A. mellifera and M. bee-
cheii shared one band with approximate molecular weight of
24 kDa. According to Mohammed and Babiker (2009), the 24 kDa
band corresponded to the enzyme lysophospholipase. Finally, mid-
gut samples from M. beecheii and Trigona ssp. shared one band with
approximate molecular weight of 14 kDa. According to
Mohammed and Babiker (2009), the 14 kDa band corresponded
to lysozyme. The bands identified as intestinal enzymes in extracts
of A. mellifera were not identified in the honey from this insect.
Two bands identified as the intestinal enzymes phosphorylase
(99 kDa) and lysophospholipase (24 kDa) in extracts of M. beecheii
were identified in the honey from this insect. Similar behaviour
was observed for Trigona ssp. extracts and honey which shared
two bands identified as the intestinal enzymes catalase (64 kDa)
and lysozyme (14 kDa). The Jaccard similarity index shown in
47
Table 3 was used to compare protein bands from the honeys and
midgut extracts. It was found that the highest similarity in honeys
(0.14) occurred between A. mellifera and Trigona ssp. honeys. And,
the lowest similarity (0.0) occurred between A. mellifera and M.
beecheii honeys.
For midgut extracts, it was found that the highest similarity in
extracts (0.21) occurred between A. mellifera and M. beecheii midgut
extracts. And, the lowest similarity (0.0) occurred between A. mel-
lifera and Trigona ssp. midgut extracts. In making the comparison
between distinctive patterns of honey with midgut extracts from
different species, it was found the highest similarity (0.17) occurred
between Trigona ssp. honey and A. mellifera midgut extract. And, the
lowest similarity (0.05) occurred between A. mellifera honey and M.
beecheii midgut extract, and between M. beecheii honey and Trigona
ssp. midgut extract. Honeys and midgut extracts have distinctive
bands that accurately distinguish the entomological origin of the
sample. The pattern of bands could be regarded as protein markers
for each type of honey. SDS–PAGE is an analytical method that
could be used to determine the authenticity of entomological origin
of different types of honeys.
4. Conclusion
A sub-tropical region, such as the Yucatan Peninsula in Mexico,
has huge potential for production and export of stingless bee prod-
ucts. Increasing stingless bee honey production and the prospec-
tive of a broader market for natural and organic products mean
parameters to determinate the entomological origin of honey and
protect the consumer against adulteration are needed. In this
study, were evaluated physicochemical parameters and used
SDS–PAGE to differentiate between honeys produced by A. mel-
lifera and stingless bees. Compared with physicochemical parame-
ters of A. mellifera honey, the most relevant differences with M.
beecheii and Trigona ssp. honeys were higher water content and
lower total sugars and reducing sugars. SDS–PAGE of protein hon-
eys and bee midgut extracts had distinct bands, which accurately
distinguish the entomological origin of the sample. These differ-
ences observed between physicochemical parameters and elec-
trophoretic patterns could result from physiological differences
between A. mellifera and stingless bees (M. beecheii and Trigona
spp.). The physicochemical parameters of stingless honey and its
protein electrophoretic patterns could be used to establish both
its quality control and its entomological origin.
Acknowledgment
This research was funded by the Dirección General de
Educación Superior Tecnológica.
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