INTRODUCTION
Honey is a
popular natural food produced by honey bees (Apis
mellifera), that is known to be an energetic and
health beneficial nutritional option (Vanhanen et al.,
2011).
Its
complex composition, due mainly to the presence of organic and inorganic compounds,
is strongly influenced by both natural and anthropogenic factors, which vary
based on its geographical origins. Alves (2008), Pereira
(2008) and Mendes et al. (2009) classified two types of sources that may promote
the contamination of honey: primary sources from flower resources, water, air,
soil, etc., when bees absorb unwanted elements; and secondary sources, such as
procedures for bee management, harvesting, processing and
storage of honey, practiced by beekeepers unintentionally. These
conditions expose honey to physical, chemical and biological agents (Sebrae, 2009; Lorenzon et al.,
2017). The actions of these related agents contaminate honeys silently. When the level of contamination is high, it
is a significant indicator of a deficiency in bee health and of the state of
the environment (Snowdon and Cliver,
1996).
In order to yield medicinal effects, and to be used as a natural
sweetener, honey should be free of any contaminants, using also analyses of the
types of elements for evaluating honey
quality. Nowadays, there is an increasing interest in the study of biological
systems, however this is not a trivial task, because the behavior
of the biological system, biological objects and their reaction to human
intervention is unpredictable (Mckee, 2003; Zacepins and Stalidzans,
2012). Currently,
searches for trace element content of honey is more related to non-natural
sources (eg Vincevica-Gaile
et al., 2011; Alqarni et al., 2012; Solayman et al., 2016; Siliciet al., 2016), other studies pointed
out some minerals associated with the soil where melliferous
flora grows (eg Fernández-Torres
et al., 2005, Ariyama et al., 2006; Baroni et al., 2009, Batista et al., 2012; Yücel and Sultanoğlu, 2013). However,
the presence of biological elements should also be evaluated searching
for signals of synergism they may show with the environment.
Due to the quick
development of beekeeping in many countries, while preventing the honey
quality, this study is aimedat presenting types of
elements and residues in honey and also at evaluating its quality focused on
microbiological and morphological methods.
METHODOLOGY
A set of 187 varieties of natural honeys were
obtained by donation and purchase in stores and apiaries. The samples were gathered
from 51 counties in the state of Rio de Janeiro, which is
an important commercial center and the second largest economy in Brazil. Some beekeepers and entrepreneurs reported that they usually sell
local and imported honeys in the domestic market.
Samples
were prepared following Louveaux et al. (1978)
methodology, using 10 grames of honey per
sample. Light microscopy of 40x and 100x magnification was used to check the
presence of trace elements, and to count and identify the pollen types. Acetolysis was excluded to prevent destruction of
morphological components and to preserve honey quality. During the count of
pollen grains, the presence of yeast, bacteria, crystal, fungi, starch grains,
soot, raphides, vegetal tissue, honeydew and other
components were registered and photographed by a digital
camera. These elements were classified and their frequencies (%) were
calculated.
RESULTS AND
DISCUSSION
Two criteria for classification of elements were proposed: (a) biological: they help to evaluate the environment; and (b) dirtiness: they are strange and can be easy to find out. These criteria were adopted considering the variety of elements that occurred in the honey (Table 1) and based on the following reviewers. Barbieri et al. (2001) suggested qualifying the elements in honeys in organic or inorganic, live or inert, harmful or harmless. Ziobro (2000) considered dirtiness as heavy, light and easily sifted elements in honeys. Additionally, according to the Brazilian legislation (Brasil, 2000; 2010), elements of any nature that compromise the quality and identity of honey should be named as strange substances, such as insect body parts, larvae, sand and others.
Biological elements originated by plants occur in honeys when honey bees forage on flowers or other organs of the plants searching for trophic resources. These are elements found in honey analysis: (1) pollen grains, mainly associated with body hairs of bees; (2) fragments of vegetal tissue, such as raphides, parts of cell walls and sclerenchymatic cells; (3) grainy mass originated from leaf surfaces that can be found in a diffused form or as a compact and agglomerated form; (4) amyloplasts originated from broken pollen grains or from the air (atmospheric) sediments. In air sediments, starch grains are always larger than pollen amyloplasts. In honeys, starch grains are rare and can be identified as elements in the shape of a cross-malt (Barth personal observation; (5) algae from water; (6) oil, commonly from visited flowers of Malpighiaceae and Solanaceae; (7) fruit sediments (Fig. 1).
The microorganisms
that occur in several places visited by bees are discharged as biological
elements. They are also part of the industrial processes of honey, whose level
can affect the honey’s safety. These biological elements found in honeys
included: (1) yeast, fungi, fungi spores and hyphae of fungi, (2) mites and (3)
bacteria (Fig. 2).
Dirtiness
or undesirable materials are non-components of honey. These residues appear
under inappropriate sanitary conditions that can result in different levels of
contamination. The presence of these elements in honeys make them a vehicle to
detect pollutants, as follows: (1) synthetic fibers; (2) starch grains; (3)
sand grains and crystals; (4) soot (caused by fumigation of beehives or
industrial residues); (5) parts of equipment and materials; (6) fragments of
insects (Fig.3).
The frequency of dirtiness in our honey samples is high (32%), highlighting the presence of fragments of insect bodies, starch grains and soot, this last residue being the most frequent one (24%). Mallman (2010) noticed the presence of dirtiness and other matters ranging between 29% and 71%, whose average level was high. Honey samples that came from the Brazilian northeast, which were registered by Sousa and Carneiro (2008), showed a level of 65% in dirtiness, putting these samples out of the Brazilian standards, thus disqualifying such honey. Lima Silva et al. (2008), Lieven et al. (2009) and Sereia et al. (2011) reported the level of contaminants and dirtiness higher than 50% affects the purity of honeys. Cordeiro et al. (2012) reported no impurities or other elements in honeys from the Brazilian northeast under microscopy analysis. Previous studies showed the most frequent dirtiness in honey samples: Mallman (2010) fragments of insect bodies and pieces of wood; Sousa and Carneiro (2008) mites, fragments of insect bodies and larvae; Martins et al. (2014) high level of fragments of insect bodies (68%).
In some honeydew samples,
grainy mass, soot particles, spores, hyphae of fungi and frequent pollen grains
from anemophilous plants, according to Barth (1989),
were also found. However, the level of these elements in honeydew is relatively
low (13%) and follows the standards of honey from other Brazilian regions (Barth,
1970; Freitas, 1991; Aires and Freitas,
2001).
When analyzing all honey
samples presenting microorganisms and dirtiness, the level of elements rises to
58% (n=109), which requires careful assessment. As stated in the Brazilian
legislation (Brasil, 2000; 2001), the honey must be
free from contamination of chemical products, as well as from solid particles
transported by the air, objects from manipulators and processing, other strange
substances and strict occurrence of microorganisms.
The presence of yeast,
fungi, spores, hyphae, bacteria and mites in honey samples in the present study
is alarming as well, and reached the level of 26%. Some bee diseases caused by
fungi have not yet been notified by law in Brazil, and the proposed analysis
may offer an important signal. Researches conducted
by Pacheco et al. (2009) and Keller
et al. (2014) have shown the occurrence of the disease known as Brazilian sac brood
(BSB), which is due to some pathogenic fungi and their mycotoxins.
These authors recorded unusual frequency of fungal spores and hyphae in honeys,
although honeys did not have structure that favors the growth and maintenance
of fungi. Thus, the presence of this element in honeys could be an important
indicator of high incidence of fungi in honey bee hives.
Previous researchers
are warning about the increase of the biota in honeys. Lima Silva et al. (2008) warned that honey samples from Brazilian
southeast were above the recommended limits of microorganisms; Sereia et al.
(2011) in the south of Brazil, Lieven et al. (2009)
and Sodré et al. (2007) in the Brazilian northeast
reported similar results as well. Ananias (2010) reported the level of 33%
(n=66) of colonies of fungi and yeasts from Brazilian central region. Santos et
al. (2010) and Gois et al. (2015) also noticed
similar levels in honeys from Brazilian northeast as well. Otherwise, the
organic honey samples from some regions of southern Brazil were within the limits
of safe food, according to Alves et al. (2009). As
noted above, most of authors found microbes as such level that make them
primarily responsible for the contamination of Brazilian honeys, indicating a
deficiency in the application of good practices in beekeeping. Rosa et al. (2006) warned the presence of fungal
filaments in foods, which can lead to deterioration by the
action of certain enzymes and toxic metabolites.
The results of our study point out that the Melissopalinology
analysis (as microscope analysis of foods) is an important instrument for
evaluating certain kinds of adulteration of honeys, as also emphasized by Russman (1998) and Barbieri et
al. (2001).
The complexity
structure of honeys, providing nutritional and
therapeutic values, the processes in the production cycle, from the region of
honey production until its storage, including the consumers home, deserve more
cautiousness. Likewise, the quality control of honeys should be strengthened
following the current legislation in order to guarantee credibility and the consumer’s
appreciation of the product.
Competing interests
We showed the need and the
relevance to adapt the bee products to the market reaching
the best quality required by the legislation and to enhance the technical
services. Thus, our team will be
assisting producers who must achieve more competitive prices in the market of
bee products.
Authors' contributions
M Lorenzon, S Linhares – They carefully organized the database and tables, reviewed and wrote the manuscript.
O Barth – She performed in the pollen and residues analysis of the experiment, made the scientific figures and reviewed the manuscript.
Acknowledgements
The authors are thankful to Emanoela Muniz and Érika Sales
for supporting laboratorial analyses. They are also thankful to the National
Council of Scientific and Technological Development (CNPq)
for having financially supported the research (process number 578134/2009), with
scholarships to Ortrud Monika Barth (process number
304067/2013-0) and to Susana Linhares Haidamus (Animal production post-graduate program, UFRRJ].
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