Nebojsa Nedic

Ultrahigh‐performance Liquid Chromatography and Mass Spectrometry (UHPLC–LTQ/Orbitrap/MS/MS) Study of Phenolic Profile of Serbian Poplar Type Propolis

INTRODUCTION Propolis is a resinous natural substance collected by honeybees from different plant sources. Due to the presence of various phytochemicals, this bee-product exhibits numerous biological activities, including anti-bacterial, anti-viral, anti-inflammatory, anti-oxidant, immunostimulating and anti-tumour effects. As the chemical composition and biological activity of propolis depend on its botanical and geographical origin, searching for new bioactive substances in various types of propolis from unexplored regions is of great importance. OBJECTIVE The aim of this study is the evaluation of the phenolic profile of poplar propolis samples in order to characterise Serbian propolis, to identify possible new constituents and to specify the phenolic components relevant for differentiation of poplar propolis samples into two subgroups through simultaneous analysis of poplar bud extracts. METHODS Ethanolic extracts of propolis and poplar buds were comprehensively analysed using ultrahigh-performance liquid chromatography coupled with hybrid mass spectrometry, which combines the linear trap quadrupole and Orbitrap MS/MS mass analyser together with chemometric methods. RESULTS Extensive fingerprint analysis of Serbian propolis was achieved for the first time. Seventy-five phenolic compounds were detected. Eight of them were identified in propolis for the first time. Pattern-recognition methods applied to the content of ten quantified phenolics verified the existence of two subgroups of propolis, with galangin, chrysin and pinocembrin as the most influential distinguishing factors. CONCLUSION The phenolic composition of the analysed propolis samples confirm their affiliation to the European poplar type propolis and the existence of two subgroups according to botanical origin.

Detecting population admixture in honey bees of Serbia

Summary Honey bee workers were sampled across the Serbian territory during 2009–2010 from mostly non-migratory apiaries to determine the population structure of these bees using morphometric, genetic, and spatial information. A total of 134 bees were sampled, of which 77 were analysed using classical wing morphometrics and 122 bees were successfully analysed using 24 DNA microsatellite markers. A combination of methods including multivariate statistics and assignment tests (frequency-based and Bayesian) revealed the honey bees of this region to resemble the subspecies Apis mellifera macedonica, Apis mellifera carnica or hybrids of these two subspecies. Based on Bayesian assignment (‘Structure’) and spatial PCA, honey bees within the Serbian territory were composed of 56%-58% A. m. carnica and 42%-44% A. m. macedonica. Spatial analysis showed the existence of a north-west to south-east cline in genetic differentiation. The bees in the north-west resemble A. m. carnica,while the bees in the south-east of the country are more similar to A. m. macedonica. Thus, the extent of A. m. macedonica within Serbia was greater than previously estimated. We define a line of hybridisation between A. m. carnica and A. m. macedonica within our study area. The cline of differentiation was still evident using a combination of genetic and spatial information, in spite of beekeeping activities including transhumance and breeding efforts.

Influence of frost damage on the sugars and sugar alcohol composition in quince (Cydonia oblonga Mill.) floral nectar

Cold stress adversely affects growth and productivity, and triggers a series of morphological, physiological, biochemical and molecular changes in plants. Since sugars are present in all floral nectars in greater amounts than any other constituent, the aim of this study was to examine how frost exposure changes sugar metabolism and how it affects on the content of sugar components in the nectar of quince. Three quince cultivars (‘Vranjska’, ‘Triumph’ and ‘Leskovačka’) were investigated in this study. The contents of sugars (glucose, fructose, sucrose, trehalose, maltose, isomaltose, rhamnose, arabinose, ribose, melezitose, raffinose, and panose) and sugar alcohols (sorbitol, erythritol, mannitol and galactitol) were analyzed by high performance anion exchange chromatography (HPAEC) with amperometric detection. The results showed that after late spring frosts and irreversible damage of flower parts, the nectar of the three quince cultivars contained elevated levels of fructose, trehalose, arabinose, ribose, rhamnose, raffinose, galactitol and mannitol, indicating an impairment of central carbohydrate metabolism. The ratios between individual sugars, such as the glucose/fructose ratio, were changed in the nectar of damaged flowers in all three quince cultivars. The examined cultivars showed similar sugar response to cold stress. The only exception was ‘Leskovačka’ for the glucose and melezitose pathway, which means that composition of those two sugars changed significantly according to the genotype. The larger are the carbohydrates reserves in different parts of a fruit tree, the higher is the tolerance to any form of frost damage, the results of this study could help in the understanding of how different quince cultivars react to this kind of stress and how they modulate their sugar metabolism.

Mold/aflatoxin contamination of honey bee collected pollen from different Serbian regions

Assessment of microbiological quality of bee collected pollen is very important, because of its use as a supplement in the human diet. In this study, 26 samples collected from different location in Serbia were tested for the presence of mold through mycologial analysis. The presence of aflatoxin B1, one of the most dangerous and the most widespread mycotoxin was also determined. It was established that 10 of the investigated samples were contaminated with some genera or species of mold, but all of the investigated samples were contaminated with aflatoxin B1. Considering that there is no unique and official procedure for mycological analysis of bee collected pollen, these findings suggest the need for their establishment. Mycological analysis should be followed by mycotoxicological analysis since the absence of mold does not confirm the absence of aflatoxin B1 in bee pollen.

Natural mycobiota and aflatoxin B1 presence in bee pollen collected in Serbia.

Total fungal count, incidence of fungi and aflatoxin B1 (AFB1) concentration were studied in 33 samples of bee pollen randomly collected from beekeepers in Serbia. The total number of fungi was determined by dilution method whereas AFB1 was detected using the Enzyme-Linked Immuno-Sorbent Assay (ELISA). The mycological estimation showed the presence of nine genera of fungi as followed: Acremonium, Alternaria, Aspergillus, Cladosporium, Epiccocum, Fusarium, Mucor, Penicillium and Rhizopus, with total number ranging from 1 x 103 to 1 x 105 CFU g-1. The results have shown the predominance of the fungi from the genera Aspergillus and Alternaria. Among Aspergillus species it was observed that the most frequent species was A. flavus with incidence of 27.27 %. Mycotoxin AFB1 was detected as 100% positive in all samples (100%) with an average concentration of 8.61 μg kg-1. The obtained results indicated that honey bee pollen must be strictly controlled during its manipulation in the harvesting and manufacturing. Therefore, the implementation of good manufacturing (beekeeping) practice to define procedures for honeybee products could be crucial to reduce the risk of possible contamination and provide natural and safety product without risk on the human health. [Projekat Ministarstva nauke Republike Srbije, br. 46008, br. 46009 i br. 46010]

Physicochemical parameters and microbiological status of honey produced in an urban environment in Serbia

Honey is a natural substance produced by honey bees (the genus Apis) enjoyed by people due to its unique nutritional and medicinal properties. The aim of this study was to determine the physicochemical parameters (moisture, ash, water-insoluble content, reducing sugars, sucrose, free acidity, diastase activity, hydroxymethylfurfural, and electrical conductivity) and microbiological status (total number of aerobic mesophilic bacteria, total number of sulfite-reducing clostridia, the presence of Salmonella spp., total numbers of fungi and yeasts and the presence of Clostridium botulinum) in honey (honeydew, blossom, sunflower, acacia, and linden) produced in an urban environment in Serbia. We analyzed 19 apiary samples of honey, collected during the 2011 harvesting season, by using recommendation methods. Physicochemical parameters of the examined honey produced in the urban environment indicated the honeys were of acceptable quality. Bacillus spp. were detected in four honeys, yeasts were detected in three honeys, and Clostridium botulinum type E was detected in one honey using PCR. The current study also showed the presence of diverse honey varieties in Serbia.

Colony strength in the spring inspection and its impact on the amount of foraged pollen at the time of red clover pollination.

In this study, the impact of honey bee colony strength in the spring inspection on the colony strength at the time of pollination, the amount of foraged pollen and on the colony strength in autumn was observed. The honey bee colonies were, after the spring inspection, divided into two groups, based on the amount of bees. The weak colonies, in spring inspection, had an average up to 4 frames occupied by bees and the strong colonies, in the spring inspection, had more than 6 frames occupied by bees. In addition to the amount of bees, the amount of brood and food supplies were assessed in the inspections. It was determined that the stronger colonies had more pollen foragers in all three year of observation. The quantity of foraged pollen, in addition to strength in the spring inspection, was influenced by year. In two years (first and third) more pollen and larger quantity of red clover pollen was collected by the strong colonies, while in the second year, more pollen and a large quantity of red clover pollen was collected by weak colonies. In the fall inspections was found that the strong colonies still had more bees and brood, more pollen and, also, more honey in relation to the weak colonies. [Projekat Ministarstva nauke Republike Srbije, br. TR31057]

CORRELATION OF MORPHOLOGIC AND PRODUCTION TRAITS OF HONEY BEE COLONIES FROM SERBIA

State of mutual or reciprocal relationship between traits is determined by the correlation coefficients. Correlation of morphological and production traits was determined in two generations of honey bee colonies. In the first generation, mother queen bees were studied, and in the second generation, their offspring. The study of one generation lasted two years. Morphological traits were determined in the beginning of the study and production traits were determined in the spring and autumn inspections of every year. In the first generation, the positive and mostly high correlation between morphological traits, and honey yield and hygienic behaviour was determined. Correlation of morphological traits, colony strength and food supplies per colony ranged from highly positive to slightly negative values. It was found that there was a high correlation between some production traits (colony strength in the spring and autumn inspections and honey yield per colony). In the analysis of offspring (second generation), most traits that were positively correlated in mother generation now, also, showed a similar relationship. The only difference, in contrast to mother generation, was that, for most traits, the intensity of interaction increased.

Urban honey - the aspects of its safety

Abstract To contribute to the development of urban beekeeping, we designed this study to obtain more information about the contamination of urban bee products with toxic metals, polycyclic aromatic hydrocarbons (PAHs), and pesticides. The samples of honey (N=23), pollen (N=13), and floral nectar (N=6) were collected from the experimental stationary apiary of the Belgrade University Faculty of Agriculture located in centre of Zemun (a municipality of the Belgrade metropolitan area) in 2015 and 2016. Metals (Pb, Cd, As, Cu, Zn, Fe, Mn, Ni, Cr, and Hg) were determined with inductively coupled plasma quadrupole mass spectrometry (ICP-QMS). Polycyclic aromatic hydrocarbons (PAHs) were analysed with high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Pesticides were analysed with gas chromatography - mass spectrometry (GC-MS). The honey samples were generally within the European and Serbian regulatory limits. The levels of all the 123 analysed pesticides were below the limit of quantification (LOQ). Regarding PAH levels in honey, the highest content was found for naphthalene. The elevated levels of Hg and Cr and of PAHs in the pollen samples indicated air pollution. Pesticide residues in pollen, however, were below the LOQ. In nectar, metal levels were relatively similar to those in honey. Our results suggest that the investigated urban honey meets the regulatory requirements for metals, PAHs, and pesticides and is therefore safe for consumption.

Characterization of sugar and polyphenolic diversity in floral nectar of different ‘Oblačinska’ sour cherry clones

‘Oblačinska’ sour cherry, an autochthonous cultivar, is the most planted cultivar in Serbian orchards. Since fruit trees in temperate zone reward insects by producing nectar which ‘quality’ affects the efficiency of insect pollination, the aim of this study was analyzing of sugars and polyphenolics in floral nectar of 16 ‘Oblačinska’ sour cherry clones with different yielding potential. The contents of sugars and sugar alcohols were analyzed by ion chromatography, while polyphenolic profile was established using liquid chromatography/mass spectrometry technique. Fourteen sugars and six sugar alcohols were detected in nectar samples and the most abundant were fructose, glucose, and sucrose. Eleven polyphenols were quantified using available standards, while another 17 were identified according to their exact masses and characteristic fragmentations. Among quantified polyphenols, rutin, naringenin, and chrysin were the most abundant in nectar. Principal component analysis showed that some polyphenol components (naringin, naringenin, and rutin) together with sugars had high impact of spatial distribution of nectar samples on score plot.