Diversity of allochtonous substances detected in bee pollen pellets

Bee pollen comes from different flower types and is primarily collected by the Apis mellifera honey bee to feed its larvae in their first stage of growth (Estevinho et al., 2012). Pollen pellets have long been recognised as beneficial in medical therapy and nutrient supplements (Linskens and Jorde, 1997; Villanueva et al., 2002) and medical research has confirmed bee pollen is valuable in the following human health aspects; (i) allergy desensitisation (ii) tumour presence (iii) prostate problems and (iv) arteriosclerosis. It has also proven beneficial in tissue repair, promoting toxic elimination, rapidly decreasing excessive cholesterol and radical scavenging activity (Estevinho et al., 2012; Nogueira et al., 2012; Linskens and Jorde, 1997).


Introduction
Bee pollen comes from different flower types and is primarily collected by the Apis mellifera honey bee to feed its larvae in their first stage of growth (Estevinho et al., 2012).Pollen pellets have long been recognised as beneficial in medical therapy and nutrient supplements (Linskens and Jorde, 1997;Villanueva et al., 2002) and medical research has confirmed bee pollen is valuable in the following human health aspects; (i) allergy desensitisation (ii) tumour presence (iii) prostate problems and (iv) arteriosclerosis.It has also proven beneficial in tissue repair, promoting toxic elimination, rapidly decreasing excessive cholesterol and radical scavenging activity (Estevinho et al., 2012;Nogueira et al., 2012;Linskens and Jorde, 1997).
Recent publications on pollen pellets lack detailed information on allochthonous inorganic substances.These have relatively small size and specific chemical and physical composition with differing positive and negative effects on human health, and they are often relicts of conventional technological procedures that are very difficult to effectively rectify (Linskens and Jorde, 1997).
district in south-western Slovakia, and then discusses their potential impacts in further applications.

Provenance and characterization of pollen grains
Pelletised pollen grains were purchased from a local beekeeper from the Želiezovce (Levice) S-W Slovak district.This region is located in the 'Pannonian flora area ' (Futák, 1984) between the Ipeľsko-Rimavská furrow and Danubian lowland phytogeographic districts (Fig. 1).
Flower pollen samples were collected in mid-flowering season and technologically modified by certified procedures (Almeida-Muradian et al., 2005;Schulte et al., 2008).Botanical-palynological identification of pollen grains was settled in the following steps (i) evaluation of general grain morphology; (ii) measurement and calculation of pollen grain uniformity and size by software ImageJ (https://imagej.net/)(iii) detection of specific structures such as surface apertures and (iv) each flower pollen class was finally classified to accommodate "species-specific" features including structure and ornamentation (Almeida-Muradian et al., 2005, Klimko et al., 2000;Lin et al., 2013;Punt et al., 2007;Schulz et al., 2000).

Physical-chemical determination of flower pollen with natural and artificial allochthonous substances
Scanning electron microscopy (SEM) and chemical composition determined by energy-dispersive X-ray spectroscopy (EDX, of EDS analysis) (QUANTA 450 FEG, FEI Company, USA) were used according to measurements published by Konvičková et al. (2016), andKořenková et al. (2017).This analysis provided (i) surface flower pollen grain visualisation for identification, characterisation and classification (ii) physical-chemical description of allochthonous substances including their size, morphology, surface and chemical nature and (iii) mineral soil aggregates and residues from flower pollen preparation and modification.A thin layer of gold was dispersed on all sample surfaces to ensure the best conduction.
Microscopic observation identified the presence of fragments of bees, plants and harvesting process materials in the pollen pellets (Campos et al., 2008).
Results confirmed pollen species, branched hairs from the bee body surfaces (Fig. 3) and microscopic dead fungal biomass; after some difficulty identified as imperfect fungi of phylum Deuteromycota (Fig. 4).However, fungal hyphae occurrence was rare compared to bulk pollen materials.The hyphae presence could be due to (i) accidental bee transfer to the pollen or (ii) microbial contamination hazardous to human health which appeared at high moisture levels and was often accompanied by other pathogenic microorganisms such as gram positive and negative bacterial spores, yeasts and acarid mites (Campos et al., 2008;Nogueira et al., 2012) -although evidence of these pathogens was not Oliveira, 2016).Furthermore, these additives can be incorporated in pellets and produce negative effects in biological, pharmacological and human organoleptic sensory pathways (Campos et al., 2008).
Our results confirmed that not all natural and artificial allochtonous particles can have their effects nullified or be completely removed from pellets, particularly those produced in technological processes.Others are small with non-respirable size (Fig. 5-8) and have been incorporated in situ during pellet formation; especially in rain and wind-driven meteorological conditions (Knox et al., 1997;Ormstad et al., 1998;Campos et al., 2008).Pollen species also depend on individual levels of adherence to inorganic mineral surfaces.This depends on pollenkit content; the viscous organic substances on the pollen surface which generate strong energy forces between close molecules; such as Van der Wall attraction (Lin et al., 2013).Although the pollen surface is responsible for initial inorganic particle-surface attachment, it is also able to resist attachment during technologically allochtonous material purification and elimination processes with the aid of specially constructed purifiers based on bulk density separation.These inorganic materials have absolute resistant properties in freezing, drying at 40 °C and lyophilisation, even when they are slightly modified (Campos et al., 2008).
Our results in the accompanying figures reveal a wide range of inorganic materials associated with different pollen species: A/ Figure 5   Scanning electron micrograph depicts 10 to 12 μm sized particle with square-shape background and no other symmetric-facet morphology; Inset: Energy-dispersive X-ray spectrum analysis reveal O and Fe chemical elements corresponding to mineral Fe oxide

Figure 6
Scanning electron microscopy (SEM) of heterogeneous 15μm soil aggregate lacking defined morphology; but with mineral different mineral types, mainly alumo-silicates, associated with pollen from Brassica napus (Oilseed rape, Brassicaceae).Inset: Energy-dispersive X-ray spectrum showing Si, Al, O, Mg, K, Fe and Ti elements confirming non-homogenous soil aggregate confirmed in our samples.Negative synergic effects of microbial contamination could decrease pollen quality or introduce harmful human health reactions such as allergies (Kačániová et al., 2004;Kačániová et al., 2011) and therefore mycogenic analysis is beneficial in evaluating presence and activity of microscopic fungi, and preclude any of the abundant mycotoxins (Linskens and Jorde, 1997;Kačániová et al., 2011).
In addition, inorganic materials in aerodispersive environments can influence pollen pellet quality parameters.They include agricultural fertilizers, pesticides, veterinary antibiotic residues and organic molecules from other sources (Chauzat et al., 2006;de Acta fytotechn zootechn, 20, 2017(3): 60-65 http://www.acta.fapz.uniag.skFoods with adsorbed soil particles introduce dangers in oral ingestion and subsequent gastrointestinal problems.This was identified in Hooda et al's.( 2004) 'human health risk assessment' research.The bioavailability of soil particle macro-, micro and trace element mineral nutrients for human and animals is most closely related to increased mobility and potential toxicity and directly dependent on combinations of individual characteristics.The major criteria here include soil material type, dose, timeexposure dependency, particle size and morphology.
The chemical and physical attributes include crystallinity, leaching and release ability, reactivity and inertia (Ruby et al., 1999;Hooda et al., 2004).Our results detected inorganic substances and minerals in pollen pellets which maintain significant chemical and physical resistance in a wide range of environments, and Ruby et al. (1999) and Hooda et al. ( 2004) add that human absorption is independent of metal biological availability.

Conclusions
Pollen pellets obtained from the Levice district in the Slovak Republic contained the following four pollen species; Trifolium pratense (Red clover, Fabaceae), Brassica napus (Rape Oilseed, Brassicaceae), Helianthus annuus (Sunflower, Asteraceae) and Taraxacum officinale (Dandelion, Asteraceae).Interestingly, microscopic inspection of the resultant pollen pellets revealed one Ti-Mn-Fe grain, but this is considered a residue from technological processes or harvesting treatments The pellets also contained attached branched hairs from bee body surfaces, dead fungal hyphae biomass, fine soil mineral particles and aggregates including quartz (SiO2), Fe oxide and alumosilicate.However, these foreign substances occur rarely compared to bulk pollen materials and do not lower food quality or pose particularly harmful effects to human health.Finally, pollen pellets are widely recognised as one of the most excellent nutritional food sources, and this has inspired our new research into the pollen content of protein, lipid, ash and other organic substances; with publication of results in the near future.technological processes because similar material types have been identified as light alloys or multilayer metal products with excellent thermal, electrical, and mechanical properties (Kim et al., 2013).These and similar high-content artificial materials cause problems when combined with pollen because they accelerate harmful human health effects (Linskens and Jorde, 1995;Ruby et al., 1999).
The 0.05 to 0.01 mm size-dependency of all evaluated particles enables the general classification of 'coarse dust' .This is contrasted with the less than 10 μm particle size of dangerous human-inhalable particles, which normally have particular morphology and surface-tovolume ratio.Moreover, Ormstad et al. ( 1998) assert that it is inhalable fractions smaller than 2.5 μm which create the most harmful human effects.

Figure 1
Figure 1Flower pollen harvested from the Želiezovce, Levice district in SW Slovakia where land cover is dominated by arable land with minor broadleaves forests(Miklós et al., 2002)

Figure 2 Figure 3 Figure 4
Figure 2Scanning electron microscopy of Trifolium pratense pollen (Red clover, Fabaceae).Inset: energy-dispersive X-ray spectroscopy determined pollen pellet chemical composition, with C, O, P, S, K and Ca the main element components depicts the Fe oxide solid-particle soil component which acts as geochemical barrier to absorb the hazardous heavy metal elements such as Pb, Cd, As (Čurlík, 2011).B/ Figure 6 shows the occurrence of non-homogenous soil aggregates containing Si, Al, O, Mg, K, Fe and Ti elements.Si, Al, K, Mg in addition, Ti and Fe are in oxide form and silicates and alumosilicates.The oxides, silicates and alumosilicates are natural polen pellet contaminants distributed by air from soil systems (Knox et al., 1997; Ormstad et al., 1998).C/ Figure 7 highlights that quartz (SiO 2 ) is the most resistant and abundant soil and sediment material in the sample area.Its multiple re-deposition and transport is deduced from fragment morphology and size (Čurlík, 2011).D/ Figure 8 reveals the sole particle with Ti -Mn -Fe chemical composition, no natural facet morphology and lacking correspondence to naturally occurring mineralogical groups.Chemical analytic determination of its origin in Figure 8 (inset) proved difficult because the absence of oxygen precludes classified as oxides or sulphates.The most logical deduction is that this particle originates from artificial

Figure 5
Figure 5Scanning electron micrograph depicts 10 to 12 μm sized particle with square-shape background and no other symmetric-facet morphology; Inset: Energy-dispersive X-ray spectrum analysis reveal O and Fe chemical elements corresponding to mineral Fe oxide

Figure 7
Figure 7SEM of 9-12μm angular quartz grain fragments associated with pollen from Helianthus annuus (Sunflower, Asteraceae) Inset: Energy-dispersive X-ray spectrum with Si and O elements confirming quartz mineral