Phytochemical profiling of red raspberry (Rubus idaeus L.) honey and investigation of compounds related to its pollen occurrence.

BACKGROUND: Red raspberry (Rubus idaeus L.) is an important nectar source for honey production in some specific habitats as well as an important crop, so the definition of the features of this kind of honey is noteworthy. However, due to its rarity on the market, red raspberry honey is poorly characterized. The aim of this work was the phytochemical characterization of honey containing red raspberry from different geographical origins, through melissopalynological analyses concurrently with untargeted metabolomics achieved with different chromatographic techniques coupled to mass spectrometry: solid-phase micro-extraction/gas chromatography/mass spectrometry (SPME-GC-MS) and high-performance liquid chromatography/Orbitrap mass spectrometry (HPLC-Orbitrap). RESULTS: Only 4 out of the 12 samples involved in the study contained raspberry pollen as dominant pollen, although these honeys did not group in the hierarchical cluster analysis nor in the classical multidimensional scaling analyses used for data evaluation. The first result was the detection of mislabelling in two samples, which contained raspberry pollen only as minor or important minor pollen. Of the 188 compounds identified by HPLC-Orbitrap and of the 260 identified by SPME-GC-MS, 87 and 31 compounds were present in all samples, respectively. The structurally related compounds nicotinaldehyde and nicotinamide, nicotinic acid and nicotinyl alcohol were present in 100% of the samples and correlated with R. idaeus pollen count (r > 0.60, Pearson's correlation analysis). CONCLUSION: This study reveals important aspects about the characterization of red raspberry honey and could give new insights on bee diet and preferences, since niacin compounds resulted interestingly to be related to the presence of red raspberry pollen. © 2024 Society of Chemical Industry.

Fingerprinting Chemical Markers in the Mediterranean Orange Blossom Honey: UHPLC-HRMS Metabolomics Study Integrating Melissopalynological Analysis, GC-MS and HPLC-PDA-ESI/MS.

(1) Background: Citrus honey constitutes a unique monofloral honey characterized by a distinctive aroma and unique taste. The non-targeted chemical analysis can provide pivotal information on chemical markers that differentiate honey based on its geographical and botanical origin. (2) Methods: Within the PRIMA project "PLANT-B", a metabolomics workflow was established to unveil potential chemical markers of orange blossom honey produced in case study areas of Egypt, Italy, and Greece. In some of these areas, aromatic medicinal plants were cultivated to enhance biodiversity and attract pollinators. The non-targeted chemical analysis and metabolomics were conducted using ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS). (3) Results: Forty compounds were disclosed as potential chemical markers, enabling the differentiation of the three orange blossom honeys according to geographical origin. Italian honey showed a preponderance of flavonoids, while in Greek honey, terpenoids and iridoids were more abundant than flavonoids, except for hesperidin. In Egyptian honey, suberic acid and a fatty acid ester derivative emerged as chemical markers. New, for honey, furan derivatives were identified using GC-MS in Greek samples. (4) Conclusions: The application of UHPLC-HRMS metabolomics combined with an elaborate melissopalynological analysis managed to unveil several potential markers of Mediterranean citrus honey potentially associated with citrus crop varieties and the local indigenous flora.

Mint (Mentha spp.) Honey: Analysis of the Phenolic Profile and Antioxidant Activity.

Research background: The composition of honey is influenced by the botanical source and geographical area of the nectar from which it is derived. Unifloral honeys reach higher market value than multifloral honeys due to their specific aromas, which result from volatile and phenolic compounds. Experimental approach: The aim of our study is to characterize the phenolic composition of a rare unifloral variety of honey - mint (Mentha spp.) honey. For this purpose, we performed standard physicochemical analyses, pollen analysis, determined total phenolic and flavonoid content, analysed antioxidant activity and performed qualitative and quantitative analyses of phenolic compounds for five mint honeys. Results and conclusions: Our results indicate that mint honey samples have high phenolic content, expressed in gallic acid equivalents, from (76.7±0.6) to (90.1±1.1) mg/100 g, and flavonoid content, expressed as quercetin equivalents, from (6.7±0.6) to (12.5±0.8) mg/100 g. These honey samples also exhibit strong antioxidant activity, expressed as Trolox equivalents, from (33.6±2.8) to (51.3±1.2) mg/100 g and from (14.4±0.8) to (55.1±2.4) mg/100 g when analysed with DPPH and ABTS assays, respectively. Quantitative LC-MS/MS analysis revealed that the most abundant phenols in all samples were chrysin, apigenin and p-coumaric acid. Qualitative LC-MS/MS analysis identified the presence of kaempferide, diosmetin, acacetin and several caffeic acid derivatives. Novelty and scientific contribution: Our study indicates that mint honey contains unique phenolic profiles, which likely contribute to its distinctive aroma and strong antioxidant activity. A detailed description of the rare honey varieties gives beekeepers greater visibility and easier access to the demanding natural product market.

Quality Evaluation of Light- and Dark-Colored Hungarian Honeys, Focusing on Botanical Origin, Antioxidant Capacity and Mineral Content.

Melissopalynology, antioxidant capacity and mineral and toxic element contents were analyzed in eight types of Hungarian honeys. Based on color, two groups were distinguished: light honeys comprised acacia, amorpha, phacelia and linden honeys; while dark honeys included sunflower, chestnut, fennel and sage honeys, with 100 to 300 and 700 to 1500 mAU, respectively. The unifloral origin of each sample was supported using pollen analysis. The absorbance of honey correlated positively with antioxidant capacity determined by three different methods (TRC, DPPH, ORAC), and also with mineral content. The exception was the light amber linden honey with significantly higher K content and antiradical activity than other light honeys. The Mn, Zn and Fe contents were the highest in chestnut, sunflower and fennel honeys, respectively. The black meadow sage honey performed best regarding the content of other elements and antioxidant activity. The concentrations of several toxic elements were below the detection limit in the samples, indicating their good quality. The principal component analysis (PCA) revealed correlations between different antioxidant assays and minerals, and furthermore, confirmed the botanical authentication of the honeys based on the studied parameters. To our best knowledge, the present study is the first to provide a complex analysis of quality parameters of eight unifloral Hungarian honeys.

Origin Identification of Hungarian Honey Using Melissopalynology, Physicochemical Analysis, and Near Infrared Spectroscopy.

The objective of the study was to check the authenticity of Hungarian honey using physicochemical analysis, near infrared spectroscopy, and melissopalynology. In the study, 87 samples from different botanical origins such as acacia, bastard indigo, rape, sunflower, linden, honeydew, milkweed, and sweet chestnut were collected. The samples were analyzed by physicochemical methods (pH, electrical conductivity, and moisture), melissopalynology (300 pollen grains counted), and near infrared spectroscopy (NIRS:740-1700 nm). During the evaluation of the data PCA-LDA models were built for the classification of different botanical and geographical origins, using the methods separately, and in combination (low-level data fusion). PC number optimization and external validation were applied for all the models. Botanical origin classification models were >90% and >55% accurate in the case of the pollen and NIR methods. Improved results were obtained with the combination of the physicochemical, melissopalynology, and NIRS techniques, which provided >99% and >81% accuracy for botanical and geographical origin classification models, respectively. The combination of these methods could be a promising tool for origin identification of honey.

DNA barcoding detects floral origin of Indian honey samples.

The unique medicinal and nutritional properties of honey are determined by its chemical composition. To evaluate the quality of honey, it is essential to study the surrounding vegetation where honeybees forage. In this study we used conventional melissopalynological and DNA barcoding techniques to determine the floral source of honey samples collected from different districts of the state of Mizoram, India. Pollen grains were isolated and genomic DNA was extracted from the honey samples. PCR amplification was carried out using universal barcode candidates ITS2 and rbcL to identify the plant species. Furthermore, TA cloning was carried out to screen the PCR amplicon libraries to identify the presence of multiple plant species. Results from both the melissopalynological and DNA barcoding analyses identified almost exactly the same 22 species, suggesting that both methods are suitable for analysis. However, DNA barcoding is easier and widely practiced. Hence, it can be concluded that DNA barcoding is a useful tool in determining the medicinal and commercial value of honey.

Allergenicity of Gramineae bee-collected pollen is proportional to its mass but is highly variable and depends on the members of the Gramineae family.

BACKGROUND: Gramineae bee-collected pollen is identified as being at the origin of allergic accidents but the biological potency of Gramineae bee-collected pollen is not well known. Cereal grasses (e.g., Zea) and European wild forage grasses (FG) are contained in bee-collected pollen. METHOD: In this experiment, Zea-mass and FG-mass were identified in bee pollen mass and the proportion of Zea and of FG was calculated using the bee pollen melissopalynology spectrum. Skin reactivity to Zea and to FG were assessed by measuring wheal diameters (W) from skin prick tests using three serial dilutions of bee-collected pollen on 10 allergic patients to Gramineae, in order to calculate the relationship between Zea mass (Masszea) or FG mass (MassFG) in bee pollen and skin reactivity. RESULTS: The linear function Log10(WFG)=0.24(Log10(MassFG))+0.33 (R=0.99) was established using a bee pollen sample with 0.168mg of FG pollen per mg. The linear function Log10(Wzea)=0.23(Log10(Masszea))+0.14 (R=0.99) was established using a bee pollen sample with 0.983mg of Zea pollen per mg. Gramineae allergens seem to be little altered by bee secretions. Gramineae bee pollen retains its allergenic capacity but it depends on the members of the Gramineae family. CONCLUSIONS: To our knowledge this is the first time it has been shown that skin reactivity to Gramineae is proportional to the absolute Gramineae mass contained in the bee-collected pollen and that it depends on the members of the Gramineae family.

Allergenicity of Artemisia contained in bee pollen is proportional to its mass.

Bee product mugwort is identified as being at the origin of allergic accidents but the biological potency of Artemisia contained in bee pollen is not well known. In this experiment, Artemisia mass was identified in bee pollen mass and after having calculated the proportion of Artemisia using the bee pollen melissopalynology spectrum. Skin reactivity to Artemisia was assessed by measuring wheal diameters (W) from skin prick tests using three serial dilutions of bee pollen on 11 allergic patients to Artemisia, in order to calculate the relationship between Artemisia mass (Massartemisia) in bee pollen and skin reactivity. The dose-response power regression curve (Wartemisia)=3.328 (Massartemisia)0.297 (R2=0.9947) and the linear function Log10 (Wartemisia)=0.297 (Log10 (Massartemisia)+0.520 (R=0.9974)) were established using a bee pollen sample with 0.246 mg of Artemisia pollen per mg. Mugwort allergens seem to be little or not altered by bee secretions and bee pollen retains its allergenic capacity. To our knowledge this is the first time it has been shown that skin reactivity of patients allergic to mugwort is proportional to the absolute mugwort mass contained in the bee pollen.

Nutritional position of managed honey bees during pollination of native plants by the melissopalynology method.

Pollination services are crucial for maintaining ecological stability and ensuring food security for humans. Managed honey bees, which are economically valuable and are experiencing population growth due to the increasing demand for their products, play a significant role in pollination. To produce high-quality honey, beekeepers often choose natural high meadows, characterized by high plant species richness, for their apiaries. This practice, in turn, may contribute to the pollination of native plants, as managed honey bees are likely to forage on diverse floral resources within these meadows In this study, we investigated the nutritional position of managed bees in the pollination of native plants in Iran using the melissopalynology method to determine the extent of their contribution to the pollination of native plants. Ninety-four honey samples were collected from beekeepers located in the natural pastures of two biodiversity hotspots in Iran (Zagros and Alborz). Then, plant pollens were extracted from the honey and photographed by scanning electron microscopy. In the next step, plant species were identified, and their abundance was calculated. The results showed that managed bees visited 54 plant genera, seven of which were non-native plants. Additionally, more plant species and the highest abundance of pollen were observed at altitudes ranging from 1000 to 3000 m. Therefore, beekeepers set up their hives in this altitude range to obtain high-quality honey. In general, in this study, the results of melissopalynological analysis, involving the identification of plant genera and pollen counts, revealed that managed honey bees likely contributed less than 3% to the pollination of native plant species in Iran.

Foraging of Honeybees from Different Ecological Areas Determined through Melissopalynological Analysis and DNA Metabarcoding.

The environment significantly impacts the lives of bees and their feeding. This study aimed to investigate bee foraging using melissopalynological analysis and DNA metabarcoding in intensive farming, reserved, and urbanized areas. The highest alpha diversity was observed in the reserved and intensive farming areas. The urbanized area had less diversity. In the intensive farming area, Sinapis, Helianthus, and Fagopyrum predominated; in the reserved area, Melilotus, Helianthus, and Brassica predominated. In the urbanized area, garden plants, namely radish (Raphanus sativus) and cucumber (Cucumis hystrix), and agricultural plants, namely soybean (Glycine max) and melon (Cucumis melo), were often found. The most significant agreement was between the rbcL and the melissopalynological analysis. The ITS2 revealed equal matches with both rbcL and melissopalynology, but this marker missed or underestimated some genera. Trifolium pretense and Brassica nigra were identified simultaneously by the melissopalinology method and two genetic markers in DNA metabarcoding. The species Convolvulus arvensis, Melilotus officinalis, Echium vulgare, Brassica rapa, Helianthus divaricatus, and Onobrychis viciifolia were found in all ecological areas. Imperfect databases impose some limits in the identification of some taxa using metabarcoding. The further research and expansion of plant databases is needed. Studying the food preferences of bees in different environmental conditions and landscapes is necessary to develop measures to preserve their populations.

Melissopalynological Analysis of Honey from French Guiana.

Beekeeping directly depends on the floral biodiversity available to honey bees. In tropical regions, where nectar and pollen resources are numerous, the botanical origin of some honey is still under discussion. A precise knowledge of plants foraged by honey bees is useful to understand and certify the botanical origin of honey. In this study, attention was paid to honey samples from the French Guiana Atlantic coast where beekeepers generally place their hives in four types of biotopes: seaside vegetation, mangrove, savannah, and secondary forest. Pollen analysis of 87 honey samples enabled the identification of major plants visited by Africanized honey bees during the dry season (approximately from July to January). Through melissopalynologic analysis, 51 pollen types were identified and classified according to their relative presence. Frequently observed pollens (with relative presence > 50%) in French Guiana kinds of honey were those from Mimosa pudica, Cocos sp., Rhyncospora sp., Avicennia germinans, Paspalum sp., Spermacoce verticillata, Tapirira guianensis, Cecropia sp., Myrtaceae sp., Mauritia flexuosa sp., Solanum sp., and Protium sp. In many honeys, only M. pudica was over-represented (relative frequency > 90%). Color and electrical conductivity in French Guiana honeys exhibit significant variations, with color ranging from 27 mm to 110 mm Pfund, and electrical conductivity ranging from 0.35 to 1.22 mS/cm.

Elemental composition and evaluation of noncarcinogenic risks of bee pollen from different Turkish areas.

Bee pollen is a complex compound formed by the honey bee through a mixture pollen, nectar, and bee saliva. It contains many elements that have importance for the human biochemical process. However, when the content of minerals in the pollen exceeds the biological limit, it can be toxic to health. This study aimed to assess and identify the presence of 16 essential (potassium [K], phosphorus [P], calcium [Ca], magnesium [Mg], sodium [Na], iron [Fe], silicon [Si], manganese [Mn], zinc [Zn], boron [B], copper [Cu], molybdenum [Mo], nickel [Ni], chromium [Cr], selenium [Se], and cobalt [Co]) and 16 nonessential elements (aluminum [Al], beryllium [Be], barium [Ba], arsenic [As], cadmium [Cd], mercury [Hg], thallium [Tl], lithium [Li], antimony [Sb], vanadium [V], lead [Pb], rubidium [Rb], strontium [Sr], cesium [Cs], titanium [Ti], and uranium [U]) in bee pollen samples from different floral sources in Turkey, while also evaluating the noncarcinogenic risks associated with bee pollen. A melissopalynological investigation was conducted to identify the plant origins of the bee pollen samples. Thereafter, the levels of 32 elements in bee pollen samples were quantified using inductively coupled plasma-mass spectrometry (ICP-MS). Finally, calculations were performed to determine the recommended dietary allowance percentage (RDA%), estimated daily intake (EDI), target hazard quotient (THQ), and hazard index (HI). The bee pollen samples had varying levels of macro, trace, and ultratrace elements, with K, P, Ca, Mg, and Na being the most abundant macro elements and Ti, Ba, Ni, Cr, and V being the least abundant ultratrace elements. The RDA% values for essential elements in bee pollen were found to vary, with Cu, Zn, Fe, and Cr having the highest levels. The results of the THQ and HI calculations demonstrated that consuming bee pollen at recommended rates did not pose a risk to the health of adults or children. Integr Environ Assess Manag 2024;20:1575-1585. © 2023 SETAC.

The Contribution of Botanical Origin to the Physicochemical and Antioxidant Properties of Algerian Honeys.

Honeys from different regions of Algeria were analyzed to determine their pollen characteristics and physicochemical properties (humidity, pH, electrical conductivity, diastase content, color, phenols, flavonoids and antioxidant activity). The antioxidant activity was investigated using the free radical scavenging and Ferric reducing/antioxidant power assays. The melissopalynological analysis revealed 129 pollen types from 53 botanical families. The pollen types found as dominant were Coriandrum, Bupleurum, Brassica napus type, Hedysarum coronarium, Ceratonia siliqua, Eucalyptus, Peganum harmala, Ziziphus lotus and Tamarix. Principal component analysis and cluster analysis were used to analyze significant relationships between the physicochemical variables and the botanical origin of the honeys and establish groupings based on the similarities of their physicochemical and antioxidant properties. The results showed that Ceratonia siliqua, Eucalyptus, Arbutus and honeydew honeys had a higher antioxidant contribution and higher phenolic and flavonoid contents than the rest of the honeys. In addition, the contributions of Mediterranean vegetation such as Myrtus and Phyllirea angustifolia were significant in this honey group. This paper demonstrates the diverse botanical variability for honey production in Algeria. However, there is a gap in its characterization based on its botanical origin. Therefore, these studies contribute positively to the needs of the beekeeping sector and the commercial valorization of the country's honey.

Validating a multi-locus metabarcoding approach for characterizing mixed-pollen samples.

BACKGROUND: The mutualistic interaction between entomophilous plants and pollinators is fundamental to the structure of most terrestrial ecosystems. The sensitive nature of this relationship has been disrupted by anthropogenic modifications to natural landscapes, warranting development of new methods for exploring this trophic interaction. Characterizing the composition of pollen collected by pollinators, e.g. Apis mellifera, is a common means of exploring this relationship, but traditional methods of microscopic pollen assessment are laborious and limited in their scope. The development of pollen metabarcoding as a method of rapidly characterizing the abundance and diversity of pollen within mixed samples presents a new frontier for this type of work, but metabarcoding may have limitations, and validation is warranted before any suite of primers can be confidently used in a research program. We set out to evaluate the utility of an integrative approach, using a set of established primers (ITS2 and rbcL) versus melissopalynological analysis for characterizing 27 mixed-pollen samples from agricultural sites across Canada. RESULTS: Both individual markers performed well relative to melissopalynology at the family level with decreases in the strength of correlation and linear model fits at the genus level. Integrating data from both markers together via a multi-locus approach provided the best rank-based correlation between metagenetic and melissopalynological data at both the genus (ρ = 0.659; p < 0.001) and family level (ρ = 0.830; p < 0.001). Species accumulation curves indicated that, after controlling for sampling effort, melissopalynological characterization provides similar or higher species richness estimates than either marker. The higher number of plant species discovered via the metabarcoding approach simply reflects the vastly greater sampling effort in comparison to melissopalynology. CONCLUSIONS: Pollen metabarcoding performed well at characterizing the composition of mixed pollen samples relative to a traditional melissopalynological approach. Limitations to the quantitative application of this method can be addressed by adopting a multi-locus approach that integrates information from multiple markers.

Multivariate Statistical Approach for the Discrimination of Honey Samples from Galicia (NW Spain) Using Physicochemical and Pollen Parameters.

Raw honey is a food with a close relation to the territory in which it is produced because of factors such as soil conditions, weather patterns, and plant communities living in the area together. Furthermore, beekeeping management affects the properties of honey. Protected Geographical Indication Miel de Galicia protects the honey produced in Galicia (Northwest Spain). Various types of honeys (362 samples) from this geographical area were analyzed using chemometric techniques. Principal component analysis was favorable to analyzing the physicochemical and pollen variables with the greatest weight in the differentiation of honey. The linear discriminant analysis correctly classified 89.8% of the samples according to the botanical origin using main pollen spectra and physicochemical attributes (moisture, pH, electrical conductivity, diastase content, phenols, flavonoids, and color). Regarding unifloral honey, blackberry, eucalyptus, and heather honeys were correctly grouped, while five chestnut honeys and fourteen samples of honeydew honeys were misclassified. The chestnut and honeydew honeys have similar physicochemical properties and frequently similar pollen spectra profiles complicating the differentiation. Experimental evidence suggests the potential of multivariate statistics in the characterization of honey of the same geographical origin. Therefore, the classification results were good, with electrical conductivity, total phenol content, total flavonoid content and dominant pollens Eucalyptus, Erica, Rubus and Castanea sativa as the variables of higher importance in the differentiation of botanical origin of honeys.

Phenolic compounds in Hungarian acacia, linden, milkweed and goldenrod honeys.

Honey is a valuable source of nutrients, minerals and phenolic compounds. Phenolic acids and flavonoids are associated with health benefits of honey and can serve as markers for distinguishing honey types. This study aimed at determining the phenolic profile of four Hungarian unifloral honeys that were not analyzed previously. After verifying their botanical origin with melissopalynological analysis, total reducing capacity was determined with Folin-Ciocalteau method, and phenolic composition was analyzed with HPLC-DAD-MS. From the 25 phenolic substances examined, pinobanksin was the most abundant, followed by chrysin, p-hydroxybenzoic acid and galangin. Quercetin and p-syringaldehyde were detected only in acacia honey, which contained higher levels of chrysin and hesperetin compared to the other three honeys. Milkweed and linden honeys displayed higher levels of caffeic, chlorogenic, ferulic and p-coumaric acids compared to acacia and goldenrod honeys. Taxifolin may serve as a unique marker compound of milkweed honey. Goldenrod honey contained the highest level of syringic acid. Principal component analysis supported the indicator role of polyphenols in honey identification, discriminating clearly the four unifloral honeys. Our results suggest that phenolic profiles may be useful to find markers of honey's floral origin, but geographical origin can strongly influence the composition of characteristic compounds.

Quality of Commercially Available Manuka Honey Expressed by Pollen Composition, Diastase Activity, and Hydroxymethylfurfural Content.

Manuka honey plays a significant role in modern medical applications as an antibacterial, antiviral, and antibiotic agent. However, although the importance of manuka honey is well documented in the literature, information regarding its physicochemical characteristics remains limited. Moreover, so far, only a few papers address this issue in conjunction with the examination of the pollen composition of manuka honey samples. Therefore, in this study, two parameters crucial for honey quality control-the diastase number (DN) and the hydroxymethylfurfural (HMF) content-as well as the melissopalynological analysis of manuka honey, were examined. The research found a large variation in the percentage of Leptospermum scoparium pollen in honeys labeled and sold as manuka honeys. Furthermore, a significant proportion of these honeys was characterized by a low DN. However, since low diastase activity was not associated with low HMF content, manuka honey should not be considered as a honey with naturally low enzymatic activity. Overall, the DN and HMF content results indicate that the quality of commercially available manuka honey is questionable.

Soybean is a Common Nectar Source for Honey Bees (Hymenoptera: Apidae) in a Midwestern Agricultural Landscape.

Large-scale soybean [Glycine max (L.) Merr.] cultivation has substantially transformed the Midwestern landscape in recent decades. Floral nectar produced by immense fields of soybeans has the potential to influence foraging ecology and resource accumulation of honey bee (Apis mellifera L.) colonies. In this study, we combined microscopic and molecular pollen analysis of honey samples with waggle dance inference of spatial foraging patterns to demonstrate that honey bees routinely forage on soybeans in Ohio. In analyzing honey samples from across the state, we found ubiquitous presence of soybean pollen in honey collected from agricultural lands during soybean bloom. The abundance of soybean pollen in honey increased with the amount of soybean fields surrounding the apiaries. Honey bee waggle dances recorded during soybean bloom revealed that honey bees preferred soybean fields for foraging over other habitat types. With these results, future research efforts aimed at enhancing mutual interactions between soybeans and honey bees may represent an unexplored pathway for increasing soybean production while supporting honey bees and other pollinators in the surrounding landscape.

Comb honey and processed honey of Croton macrostachyus and Schefflera abyssinica honey differentiated by enzymes and antioxidant properties, and botanical origin.

In this study, comb and industrially processed honey samples collected from Bonga forest were investigated in relation to Melissopalynology and enzyme content, antioxidant and physicochemical properties. Melissopalynology categorized honey samples as Croton macrostachyus and Schefflera abyssinica honey. The physicochemical properties of the honey were determined using the Association of Official Analytical Chemists (AOAC) and International Honey Commission (IHC) methods. The enzymatic and antioxidant properties of honey were evaluated using the spectrophotometric method. The highest enzyme activity was observed in Croton macrostachyus comb honey (diastase = 7.44 ± 0.13°Schade and invertase = 13.97 ± 0.2 Invertase number (IN). Croton macrostachyus processed honey exhibited the highest values in flavonoids (83.36 ± 1.65 mg Catechin equivalents (CEQ)/100g)), Ferric reducing antioxidant power (FRAP) (69.94 ± 1.0 mg Ascorbic acid equivalents (AAE)/100g), and Inhibitory Concentration (IC50) (136.3 ± 0.00 mg/ml), while Schefflera abyssinica comb honey had stronger 1,1-diphenyl-2-picrylhydrazyl radical scavenging (DPPH) (49.47 ± 0.00%) activity. The principal component analysis revealed that enzymes can be associated with comb honey, and antioxidants with processed honey. Thus, comb and processed honey can be differentiated based on the enzyme level, and Croton macrostachyus and Schefflera abyssinica honey can be identified using pollen analysis.

Pollen and Chemical Content of Beebreads from Serpentine Areas in Albania and Bulgaria.

Beebread from serpentine localities in Albania and Bulgaria were characterized based on their pollen and chemical element content (macroelements K, Ca, Mg, P and microelements Cd, Co, Cr, Cu, Fe, Mn, Na, Ni, Pb, Zn) aiming to (1) evaluate the effect of serpentine soil on the quality of beebread; (2) compare elemental concentrations in samples from serpentine areas in Albania and Bulgaria; and (3) compare the differences in pollen spectra. Chemical element content was determined using microwave digestion of beebread samples followed by ICP-OES measurements. The analytical procedure developed was validated by added/found method. Analytical figures of merit of analytical method proposed were presented. The melissopalynological analysis was applied for pollen characterization. The results demonstrate clear difference in the pollen spectra between the two sets of samples, confirming differences in local serpentine flora in both countries, but specific pollen type is difficult to be suggested. The pollen content is related to the flowering period, climatic conditions, and bees forage preferences. The samples vary in their elemental concentrations depending on the pollen type and year of collection. The highest average concentrations found for K, Ca, Mg, and P are close to values reported in the literature. However, elevated concentrations observed for Ni, Cr, Mn, and Fe in beebread, especially from Albania, are in line with the serpentine characteristics of studied areas. The concentrations of Cd and Pb for all beebread samples are below permissible limits. The results should be taken into consideration in future specific food safety regulations at national and international level.