Search

Determining the levels of agrochemicals, such as pesticides, that honey bees are exposed to is critical for understanding what stress factors may be contributing to colony declines. Although several pesticide detection methods are available for honey, limited work has been conducted to adapt these methods for pollen. Here, we address this gap by modifying the Dutch mini-Luke extraction method (NL method) for pesticide analysis in honey and pollen from throughout the island of Ireland. The NL method was modified to enable detection in small-sized samples and validated for both pollen and honey matrices. The modified NL method combined with liquid and gas chromatography-tandem mass spectrometry gave consistent results in terms of accuracy and precision measured by recovery experiments and was successfully applied in the analysis of a range of pesticide residues. The modified NL method developed here provides a key tool for detecting pesticides in honey bee colony resources and the environment more broadly.

Beside honey, honeybees ( Apis mellifera L.) are able to produce many byproducts, including bee pollen, propolis, bee bread, royal jelly, and beeswax. Even if the medicinal properties of these byproducts have been recognized for thousands of years by the ancient civilizations, in the modern era, they have a limited use, essentially as nutritional supplements or health products. However, these natural products are excellent sources of bioactive compounds, macro- and micronutrients, that, in a synergistic way, confer multiple biological activities to these byproducts, such as, for example, antimicrobial, antioxidant, and anti-inflammatory properties. This work aims to update the chemical and phytochemical composition of bee pollen, propolis, bee bread, royal jelly, and beeswax and to summarize the main effects exerted by these byproducts on human health, from the anticancer and immune-modulatory activities to the antidiabetic, hypolipidemic, hypotensive, and anti-allergic properties.

Pollen is a source of nutrients for honeybees ( Apis mellifera L.) and suitable for human consumption as well. In our research, a multiresidue method for pesticide determination was developed and validated for the bee pollen matrix. 247 components met the validation criteria for limit of detection, limit of quantification, linearity, and interday repeatability. Average recoveries varied between 70 and 120% except for 14 analytes, which were corrected during on-going validation. The matrix effect was strong for certain analytes, which required the use of matrix-matched calibration. The pesticide residue profiles of 21 pollen samples of different botanical origins were identified by the developed method. The most common active substances were chlorpyrifos, thiacloprid, and acetamiprid. Some products contained pesticides that are already banned. According to our estimates, the tested samples do not pose an acute risk on honeybees, although the combination of pesticides may cause synergistic toxicity.

Bee pollen (BP) shows profound gut-protecting potentials. BP lipids (BPLs) mainly composed by phospholipids and polyunsaturated fatty acids might be one of the important contributors, while how BPL exerts gut-protecting effects and is transported through intestinal cell monolayers need to be investigated. Here, we exploited a strategy that combines an UPLC-Q-exactive orbitrap/MS-based lipidomics approach with a human intestinal cell (Caco-2) monolayer transport model, to determine the transepithelial transportation of BPL from Camellia sinensis L. (BPL-Cs), in pathological conditions. The results showed that BPL-Cs protected Caco-2 cells against dextran sulfate sodium (DSS)-induced intestinal barrier dysfunction by improving cell viability, maintaining membrane integrity, increasing tight junctions (ZO-1 and Claudin-1), and eliciting the expressions of antioxidative-related genes ( NQO1 , Nrf2 , Txnrd1 , and GSTA1 ). Lipidomics analysis revealed that DSS suppressed the transport and uptake of most of BPL-Cs including glycerophospholipids, sphingomyelins, and glycosylsphingolipids. Pretreatment with BPL-Cs significantly regulated glycerophospholipid and sphingolipid metabolisms, potentially involved in building permeability barriers and alleviating intestinal oxidative stress. Finally, eight classes of lipids were identified as the potential biomarkers for evaluating DSS-induced Caco-2 cell dysfunctions and BPL-intervened modulation. These findings shed light on the development of BPL as gastrointestinal protective food supplements in the future.

Tyrosinase inhibitors are important in cosmetic, medical, and food industries due to their regulation of melanin production. A tyrosinase inhibitor was purified from Camellia pollen using high-speed countercurrent chromatography and preparative high-performance liquid chromatography and was identified as caffeine by NMR and mass spectrometry. It showed strong mushroom tyrosinase inhibitory activity with an IC 50 of 18.5 ± 2.31 μg/mL in a noncompetitive model. The caffeine did not interact with copper ions in the active center of the enzyme but could quench fluorescence intensity and change the secondary conformation of this tyrosinase. A molecular dynamics simulation showed that caffeine bound this tyrosinase via Lys379, Lys 376, Asp357, Glu356, Thr308, Gln307, Asp312, and Trp358, thus changing the binding sites of l-tyrosine and the loop conformation adjacent to the active center. In vitro cell model analysis revealed that caffeine exhibited significant inhibitory effects on both intracellular tyrosinase activity and melanin production of B16-F10 melanoma cells in a concentration-dependent manner. These comprehensive results suggest that caffeine is a strong tyrosinase inhibitor that has the potential to be developed as skin-whitening agents in the cosmetics and pharmaceutical industries or as antibrowning agents in the food industry.

Protein-based nanoparticles (NPs) with favorable properties including enhanced absorptivity and low toxicity still suffer a major challenge for rapid nutraceutical or drug release after oral administration. Hence, we introduced a secondary encapsulation for unstable factor to attain a controlled-release effect in a gastrointestinal environment. In this work, assembled nanoparticles engineered by nobiletin (NOB), zein, and tannin acid (TA) were first reported for drug delivery systems. The TA added was capable of obtaining further assembly to stabilize nobiletin in comparison with NOB-loaded zein NPs only. Sunflower pollens (SPGs) were selected as carriers for further oral delivery, while zein was chosen as a coating material for capping SPGs absolutely. As a result, the NOB/zein/TA NPs (NZT NPs) obtained had a stable size of 100 nm after 48 h. Besides, they could improve the chemical stability of NOB for at least 120 days at 4 °C compared with zein NPs (ZT NPs). Owing to the secondary capping by SPGs, the final system was able to release selectively via an oral route, that is, achieving no release in a gastric environment and slow release in an intestine environment. Generally, our research proposed a secondary protection model to prevent drug-loaded NPs from resolving after oral administration, which provided a new perspective for nutraceutical or drug encapsulation and controlled-release delivery.

Bee pollen (BP) collected from different floras possesses various potential bioactivities, but the mechanism-related research on anti-inflammatory effects is limited. Here, three types of BP originating from Camellia sinensis L. (BP-Cs), Nelumbo nucifera Gaertn. (BP-Nn), and Brassica campestris L. (BP-Bc) were assessed using molecular and metabolomics methods to determine their anti-inflammatory effects. The differences in polyphenolic abundance of three types of BP extracts were determined by HPLC-DAD/Q-TOF-MS. In vitro anti-inflammatory effects of three BP extracts were evaluated in a lipopolysaccharide (LPS)-induced RAW 264.7 cells model. BP-Cs extract with the most abundant polyphenols was found to be the most effective in reducing inflammation by downregulating inflammatory-related genes expression and blocking the activation of MAPK and NF-κB signaling pathways. Polyphenol-rich BP-Cs was further evaluated for their in vivo anti-inflammatory effect in a LPS-induced acute lung injury mouse model. An UPLC-Q-TOF/MS-based metabolomics approach was applied to analyze metabolite changes in mouse serum. Weshowed that the pretreated BP-Cs extract alleviated inflammation and regulated glycerophospholipid metabolism significantly. Our findings provide a foundation for developing and justifying BP as a potential anti-inflammatory ingredient in functional foods or nutraceutical formulations.

The aim of the bioassay-guided fractionation was the selection of the most potent group of compounds responsible for the protection of sunflower bee pollen grains. Synthesis of prospective antifungal polyamides of hydroxycinnamic acids was based on previous structural elucidation of ethanol soluble fraction by 1 H, 1 H-PFG-COSY, 1 H, 13 C-HSQC, FT-IR, FT-Raman, and LC-MS experiments. The main compounds found were tri- p-coumaroylspermidines accompanied by other HCAA of spermidine and putrescine. Several model HCAA derivatives were prepared to test their antifungal activity against widespread spoilage fungi ( A. niger 42 CCM 8189, F. culmorum DMF 0103, and P. verrucosum DMF 0023). A. niger CCM 8189 and F. culmorum DMF 0103 exhibited higher resistance to the antifungal effects of hydroxycinnamic acid amides, whereas P. verrucosum DMF 0023 was the most sensitive strain. It has been discovered the effect of HCAA polarity on the role of secondary metabolites in the microbial protection of pollen grains. The combination of bioassay-guided fractionation, structural elucidation, selection of prospective compounds, and their synthesis to determine their antifungal properties could be considered as an original approach.

Flavonoids are characteristic in maize and have diverse biological functions. C-Glycosylflavones are neuroprotective against β-amyloid-induced tau hyperphosphorylation and neurotoxicity in SH-SY5Y cells, which is relevant to Alzheimer's disease prevention and treatment. The content of the flavonoids eriodictyol, luteolin, isoorientin, and maysin varied in pollens, silks, tassels, and seeds among five maize varieties. Eriodictyol content was high (51-322 ng/g dw) in pollens, while luteolin content was low (0.2-106 ng/g dw) in all four tissues. The isoorientin content was approximately 3- to 10-fold greater than eriodictyol in pollens and tassels, particularly in the hybrid M1 and sweet corn M5 varieties. Maysin content was high in most silks and tassels. The differential expression of five genes involved in the maysin biosynthesis correlated well with the profiles of the four flavonoids among tissues and varieties. The present study offers valuable data for maize breeding and the use of maize flavonoids as functional food components.

CJP-4 is an allergen found in pollen of the Japanese cedar Cryptomeria japonica. The protein is a two-domain family GH19 (class IV) Chitinase consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. Here, we produced recombinant CJP-4 and CBM18-truncated CJP-4 (CJP-4-Cat) proteins. In addition to solving the crystal structure of CJP-4-Cat by X-ray crystallography, we analyzed the ability of both proteins to hydrolyze chitin oligosaccharides, (GlcNAc) n , polysaccharide substrates, glycol chitin, and β-chitin nanofiber and examined their inhibitory activity toward fungal growth. Truncation of the CBM18 domain did not significantly affect the mode of (GlcNAc) n hydrolysis. However, significant effects were observed when we used the polysaccharide substrates. The activity of CJP-4 toward the soluble substrate, glycol chitin, was lower than that of CJP-4-Cat. In contrast, CJP-4 exhibited higher activity toward β-chitin nanofiber, an insoluble substrate, than did CJP-4-Cat. Fungal growth was strongly inhibited by CJP-4 but not by CJP-4-Cat. These results indicate that the CBM18 domain assists the hydrolysis of insoluble substrate and the antifungal action of CJP-4-Cat by binding to chitin. CJP-4-Cat was found to have only two loops (loops I and III), as reported for ChiA, an allergenic class IV Chitinase from maize.

Digestion and fermentation in vitro of polysaccharides from bee collected pollen of Chinese wolfberry (WBPPS) were investigated in the present study. It was found that WBPPS mainly consisted of mannose, ribose, rhamnose, galacturonic acid, glucose, galactose, xylose, and arabinose in a molar ratio of 0.38:0.09:0.17:0.64:0.22:0.67:0.08:1.03, respectively. WBPPS was not affected by human saliva. The fraction A (molecular weight 1340 kDa) of WBPPS was not broken down in simulated gastric and small intestinal juices, while the small fraction B (molecular weight 523 kDa) of WBPPS was degraded. Moreover, fermentation in vitro revealed that WBPPS could significantly enhance the production of short-chain fatty acids and modulate gut microbiota composition via increasing the relative abundances of genera Prevotella, Dialister, Megamonas, Faecalibacterium, and Alloprevotella and decreasing the numbers of genera Bacteroides, Clostridium XlVa, Parabacteroides, Escherichia/Shigella, Phascolarctobacterium, Parasutterella, Clostridium sensu stricto, and Fusobacterium.

Bee pollen (BP) is collected by honeybees from flower pollen mixed with nectar and its secretions with extensive nutritional and therapeutic properties. Lipids are known to be critical contributors for the therapeutic effects of BP and vary depending on different plant sources; however, lipid profiles of BP are not available. Here, an UPLC-Q-Exactive Orbitrap/MS method was established for comprehensive lipidomics analysis of BP derived from three major nectar plants (Brassica campestris L., Camellia sinensis L., and Nelumbo nucifera Gaertn.). A total of nine lipid classes, including phosphatidylcholine (41 species), phosphatidylethanolamine (43 species), phosphatidylglycerol (9 species), phosphatidylserine (10 species), lysophosphatidylcholine (12 species), ceramide (8 species), diglyceride (27 species), triglyceride (137 species), and fatty acids (47 species), were first identified and quantified in the three BPs. In vitro anti-inflammatory activity was also discovered in the lipid extracts of three BPs, which has potential relevance to the abundance of phospholipids and unsaturated fatty acids in BP. Our comprehensive lipidomics profiling and in vitro anti-inflammatory properties of BP provide evidence for its future application.

Pyrrolizidine alkaloids (PAs) in honey can be a potential human health risk. So far, it has remained unclear whether PAs in honey originate from pollen or floral nectar. We obtained honey, nectar, and plant pollen from two observation sites where Echium vulgare L. was naturally abundant. The PA concentration of honey was determined by targeted analysis using a high pressure liquid chromatography-mass spectrometry system (HPLC-MS/MS), allowing the quantification of six different PAs and PA-N-oxides present in E. vulgare. Echium-type PAs were detected up to 0.153 μg/g in honey. Nectar and plant pollen were analyzed by nontargeted analysis using ultrahigh pressure liquid chromatography-high resolution-mass spectrometry (UHPLC-HR-MS), allowing the detection of 10 alkaloids in small size samples. Echium-type PAs were detected between 0.3-95.1 μg/g in nectar and 500-35000 μg/g in plant pollen. The PA composition in nectar and plant pollen was compared to the composition in honey. Echimidine (+N-oxide) was the main alkaloid detected in honey and nectar samples, while echivulgarine (+N-oxide) was the main PA found in plant pollen. These results suggest that nectar contributes more significantly to PA contamination in honey than plant pollen.

1,2-Dehydropyrrolizidine alkaloids are common toxic metabolites isolated from plants within the Boraginaceae, in particular from the genera Heliotropium and Echium. Previous studies have deduced the structures of these often potent bioactives based upon mass spectrometric evidence, but these identifications have not established conclusive connectivity and configurational data. Herein, we describe the isolation and full structural characterization of echivulgarine, occurring in the pollen of Echium vulgare and correct the structure previously ascribed to the compound, using a comprehensive combination of both experimental and calculated nuclear magnetic resonance and electronic circular dichroism spectroscopic data.

To determine the botanical origin of Coffea honey, a new method using proton nuclear magnetic resonance ((1)H NMR) is proposed. Integration of the aromatic region of the NMR spectrum of Coffea honey diluted in deuterated water allowed us to simultaneously quantify caffeine, theobromine, and trigonelline, as well as other compounds. The amounts of the three markers listed are significantly higher than those previously reported for Citrus spp. honey: caffeine ranged from 15 to 98 mg/kg, theobromine from 25 to 160 mg/kg, and trigonelline from 23 to 86 mg/kg. The concurrent presence of these three substances is proposed as an indicator of the botanical origin of Coffea honey. Excellent correlation was found between these markers and the relative amounts of Coffea pollen measured in the same samples.

In this study, a simple and convenient method for the determination of trans-resveratrol (TRA) in peanut oils based on pollen grain solid-phase extraction (SPE) was developed. Pollen grains were used as normal-phase SPE sorbent to separate TRA from peanut oils for the first time. As a naturally occurring material, pollen grains exhibited an excellent adsorption capacity for polyphenolic compounds due to their particular functional structures such as hydroxyl groups, saturated and unsaturated aliphatic chains with aromatics. Their stable compositions as well as adequate particle size (30-40 μm) also make them suitable for SPE. Several parameters influencing extraction performance were investigated. Coupled with high-performance liquid chromatography-ultraviolet detection (HPLC-UV), a green purification method for fast determination of TRA in peanut oils using pollen grain cartridges as sorbents was established. The linearity range of the proposed method was 10-2500 ng · g(-1) with a satisfactory correlation coefficient (r(2)) of 0.9999. The limit of detection (LOD) for TRA in peanut oils was 2.7 ng · g(-1), and the recoveries in spiked oil samples were from 70.2% to 98.4% with the relative standard deviations (RSDs) less than 4.9% (intraday) and 5.2% (interday). This method was successfully applied to the analysis of TRA in several peanut oils with different brands from local market as well as other kinds of vegetable oils.

Beehive products could be powerful monitors of pesticide residues originating in agroecosystems during production cycles. Their ready availability provides enough samples to perform analytical determinations, but their chemical complexity makes residue analysis a real challenge. Taking advantage of the plasticity of QuEChERS coupled to LC-MS/MS, validated methodologies were developed for bees, honey, beeswax, and pollen and applied to real samples for the simultaneous determination of 19 of the most employed pesticides in intensive cropping fields. Beehives placed in Uruguayan agroecosystems accumulated the pesticides thiacloprid, imidacloprid, methomyl, carbaryl, hexythiazox, azoxystrobin, pyraclostrobin, tebuconazole, and haloxyfop-methyl at 0.0001-0.01 mg/kg levels. The oscillations on the amount and occurrence of residue findings for specific apiaries were correlated statistically with the sampling season and the agroecosystem where the beehives were located, showing the potential of bees and bee products to record relevant information to survey the chemicals applied in their surroundings.

Bee pollen is used as a dietary supplement, being promoted as a health food. Echium plantagineum L. bee pollen fractions enriched in flavonols (fraction I) or anthocyanins (fraction II) and the whole extract were characterized by HPLC-DAD. Both in the whole extract and in fraction II seven flavonols and five anthocyanins were identified, while fraction I contained six flavonols (in higher levels than fraction II) and small amounts of petunidin-3-O-rutinoside. Antioxidant capacity was evaluated in Caco-2 cells under oxidative stress induced by tert-butyl hydroperoxide (t-BHP). Fraction I pre-exposure imparted a tendency to protect cells, while fraction II and the whole extract aggravated t-BHP toxicity at some concentrations. The protective effects seem to be correlated with the levels of total glutathione, while no correlation between cellular viability and reactive species was seen. The extracts displayed no significant effect on antioxidant enzymes activity. Overall, anthocyanins seem to abrogate the antioxidant potential of flavonoid-rich extracts.

The study of plant secondary chemistry has been essential in understanding plant consumption by herbivores. There is growing evidence that secondary compounds also occur in floral rewards, including nectar and pollen. Many pollinators are generalist nectar and pollen foragers and thus are exposed to an array of secondary compounds in their diet. This review documents secondary compounds in the nectar or pollen of poisonous rangeland plants of the western United States and the effects of these compounds on the behavior, performance, and survival of pollinators. Furthermore, the biochemical, physiological, and behavioral mechanisms by which pollinators cope with secondary compound consumption are discussed, drawing parallels between pollinators and herbivores. Finally, three avenues of future research on floral reward chemistry are proposed. Given that the majority of flowering plants require animals for pollination, understanding how floral reward chemistry affects pollinators has implications for plant reproduction in agricultural and rangeland habitats.

Total and individual carotenoids, fatty acid composition of total lipids, and main lipid classes of 16 fresh bee-collected pollen samples from Romania were determined by high-performance liquid chromatography with photodiode array detection and capillary gas chromatography with mass detection. Analyzed samples were found rich in lutein, whereas β-criptoxanthin and β-carotene were present in a wide range of amounts correlated with predominant botanical origin of the samples. High amounts of lutein were correlated with the presence of Callendula officinalis, Taraxacum officinale and Anthylis sp. The highest amount of total lipids was found in samples where pollen from Brassica sp. was predominant. Lipid classes were dominated by polyunsaturated fatty acids. Saturated fatty acids were determined in variable amounts. Lipid and carotenoid contents present great variability, explained by the various botanical species present in the samples.

This study quantitatively measured neonicotinoids in various foods that are common to human consumption. All fruit and vegetable samples (except nectarine and tomato) and 90% of honey samples were detected positive for at least one neonicotinoid; 72% of fruits, 45% of vegetables, and 50% of honey samples contained at least two different neonicotinoids in one sample, with imidacloprid having the highest detection rate among all samples. All pollen samples from New Zealand contained multiple neonicotinoids, and five of seven pollens from Massachusetts detected positive for imidacloprid. These results show the prevalence of low-level neonicotinoid residues in fruits, vegetables, and honey that are readily available in the market for human consumption and in the environment where honeybees forage. In light of new reports of toxicological effects in mammals, the results strengthen the importance of assessing dietary neonicotinoid intakes and the potential human health effects.

LC-UV-MS analyses of inflorescence extracts of Sambucus nigra L. (elder, Adoxaceae) revealed the presence of numerous acyl spermidines, with isomers of N,N-diferuloylspermidine and N-acetyl-N,N-diferuloylspermidine being most abundant. Pollen was the main source of the acyl spermidines in the inflorescence. Three of the major acyl spermidines were isolated and their structures determined by NMR spectroscopy as N⁵,N¹⁰-di-(E,E)-feruloylspermidine and the new compounds N¹-acetyl-N⁵,N¹⁰-di-(Z,E)-feruloylspermidine and N¹-acetyl-N⁵,N¹⁰-di-(E,E)-feruloylspermidine. An isomer of N,N,N-triferuloylspermidine was also obtained and identified as N¹,N⁵,N¹⁰-tri-(E,E,E)-feruloylspermidine. In addition to stereoisomers of the isolated acyl spermidines, other acyl spermidines detected by the positive ion LC-UV-MS were isomers of N-caffeoyl-N,N-diferuloylspermidine, N-coumaroyl-N,N-diferuloylspermidine, N-caffeoyl-N-feruloylspermidine, N-coumaroyl-N-feruloylspermidine, N-acetyl-N-caffeoyl-N-feruloylspermidine, and N-acetyl-N-coumaroyl-N-feruloylspermidine. Analysis of commercial elderflower drinks showed that acyl spermidines were persistent in these processed elderflower products. Examination of inflorescence extracts from Sambucus canadensis L. (American elder) revealed the presence of acyl spermidines that were different from those of S. nigra.

Bee pollen, promoted as a natural food supplement, is consumed increasingly by people to maintain a healthy diet. Depending on environmental conditions, pollen can also be an optimum medium for growth of molds such as Fusarium and Penicillium . A quick, easy, cheap, rapid, and safe (QuEChERS) extraction procedure followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) determination of eight selected Fusarium toxins in bee pollen was developed and optimized. Recovery studies at 20, 80, and 1000 μg/kg showed values between 73 and 95% with relative standard deviations (RSDs) of <15% for all studied mycotoxins. Limits of quantitation (LOQ) ranged from 1 to 4 μg/kg. The proposed method was applied to the analysis of 15 commercial samples. Two of 15 samples showed quantifiable values for neosolaniol and nivalenol.

Bee pollen has been praised for its good nutrition and therapeutic values. China is the largest producer in the world. Twelve common varieties of monofloral bee pollen collected from China's main producing regions were selected for nutritional composition analysis, including proximate contents, dietary fibers, amino acid distribution, fatty acid composition, and mineral elements. The proximate compositions mostly met the specifications regulating pollen load quality of China. Proline and glutamic acids were found to be the predominant amino acids in the form of both total amino and free amino acids. Lysine was the relative limiting amino acid. The percentage of total essential amino acids (TEAA) to total amino acids (TAA) reached the nutrition recommendation of the Food and Agricultural Organization (FAO). The major fatty acids, presented as mean values, were C18:3 (25.1%), C16:0 (19.6%), C18:1 (17.3%), C18:2 (8.78%), C22:0 (4.07%), and C18:0 (2.96%) acids. The proportions of C18:3 were generally higher than those of C18:2, and the ratio of total unsaturated fatty acids (TUS) to total saturated fatty acids (TS) was >1.0, except for Nelumbo nucifera Gaertn. pollen for the characteristic absence of C18:3 acids. High levels of beneficial elements such as K, Ca, Mg, Zn, Fe, Mn. and Cu were observed in pollen samples. The contents of detrimental trace elements of Cd, Pb, and Hg were primarily lower or not detected. However, more attention should be paid to a large amount of Al, with a concentration of >100 mg/kg DW in most samples. There were some significant differences between samples. On the whole, the Chinese bee pollen was evaluated as a good complement to diet.

Cornflower (Centaurea cyanus) honey can be characterized by a greenish yellow color and an intense flavor with a bitter aftertaste. Because cornflower honey contains only a limited amount of pollen for the verification of its floral origin, one objective was the characterization of its polyphenol and norisoprenoid contents to assign floral markers. Here, lumichrome (18.8-43.5 mg/kg), 7-carboxylumichrome, (Z/E)-3-oxo-retro-α-ionol, and 3-oxo-α-ionol appeared to be quite suitable for distinguishing cornflower honey from other unifloral honeys. Additionally, due to its comparably high hydrogen peroxide content (0.5-0.9 mM/h) and the associated antibacterial activity, cornflower honey was used as an alternative treatment of digital dermatitis on an organic dairy farm. Cows affected by this hoof disease often show severe lameness and a subsequent decline in milk yield and loss of body condition. The cows' hooves treated with cornflower honey showed significantly faster healing than the control group without any treatment.

Neonicotinoids are systemic insecticides widely used on many pollinated agricultural crops, and increasing evidence indicates that they move to some extent into pollen and nectar. This study measured levels of neonicotinoid residues in pollen and nectar from a pumpkin crop treated with formulated products containing imidacloprid, dinotefuran, and thiamethoxam using different timings and application methods. Environmental conditions have a significant effect on overall residue levels; nectar residues were 73.5-88.8% less than pollen residues, and metabolites accounted for 15.5-27.2% of the total residue amounts. Foliar-applied treatments and chemigated insecticides applied through drip irrigation during flowering resulted in the highest residues of parent insecticide and metabolites, which may reach average levels up to 122 ng/g in pollen and 17.6 ng/g in nectar. The lowest levels of residues were detected in treatment regimens involving applications of insecticides at planting, as either seed dressing, bedding tray drench, or transplant water treatment.

Seven bee pollen samples (C1-C7) with different palynological sources were harvested from Pindamonhangaba municipality (Southeast Brazil). Methanol extracts of untreated samples (control), samples frozen at -18 °C and samples frozen and then dried were analyzed by HPLC/PAD/ESI/MS/MS. Flavonoid diglycosides of quercetin, kaempferol, isorhamnetin and patuletin were detected, together with hydroxycinnamic acid amide derivatives, such as N',N'',N'''-tris-p-feruloylspermidine and N',N'',N'''-tris-p-coumaroylspermidine. Distinct phenolic profiles characterized the analyzed samples, but no differences were noted as resulting from different treatments. Total phenolic contents determined with the Folin-Ciocalteau reagent ranged from 1.7 to 2.2%. Antioxidant activities above 75%, based on the DPPH method, were observed for all extracts, not correlated with total phenolic contents. Among samples from the same origin, those frozen were more active than samples untreated (control), and the samples frozen and then dried were the most active.

A set of experiments was carried out to validate a method for inorganic contaminants in honeybee-collected pollen, consisting of digestion of the samples in a closed microwave-assisted system and quantification of 10 inorganic contaminants by ICP OES. Forty-three samples of Brazilian bee pollen, collected in southeastern Brazil during one year, were analyzed. Determination of these analytes is important both as bioindicators of pollution and to verify the safety of consuming the pollen itself. The method had satisfactory performance, with good accuracy and precision. The ranges of the mean levels were 10.4-268.0 mg/kg for Al, <0.01-1.38 mg/kg for As, 2.78-17.63 mg/kg for Ba, 0.003-0.233 mg/kg for Cd, <0.01-1.11 mg/kg for Co, <0.01-2.32 mg/kg for Cr, <0.10-1.13 mg/kg for Ni, <0.01-0.44 mg/kg for Pb, <0.035-1.33 mg/kg for Sb, and <0.0004-0.0068 mg/kg for Hg. Contamination seemed to occur in the following decreasing order: Sao Paulo > Minas Gerais > Espirito Santo. Generally higher levels of all studied contaminants were observed in samples produced in an urban site, compared to those of a rural site. Al, Cd, Co, and Pb tended to have higher levels during the dry months (July-October). Ingestion estimates showed that Al and As would have the highest contributions to the adult diet, reaching 27 and 8%, respectively, of the provisional tolerable weekly intake (PTWI) values, considering a daily portion of 25 g.

An analytical method was refined for the extraction and determination of neonicotinoid pesticide residues and their metabolites in honey bees and bee products. Samples were extracted with 2% triethylamine (TEA) in acetonitrile (ACN) followed by salting out, solid phase extraction (SPE) cleanup, and detection using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated in triplicate at three fortification concentrations in each matrix. Good recoveries were observed for most analytes and ranged between 70 and 120% with relative standard deviations between replicates of <20% in most cases. The method limits of detection were 0.2 ng/g for the parent neonicotinoid pesticides and ranged between 0.2 and 15 ng/g for the neonicotinoid metabolites. This refined method provides lower detection limits and improved recovery of neonicotinoids and their metabolites, which will help researchers evaluate subchronic effects of these pesticides, address data gaps related to colony collapse disorder (CCD), and determine the role of pesticides in pollinator decline.

The soluble aggregates of Cry j 1, a major allergen of Japanese cedar pollen, were formed without any coagulates during heat treatment at acidic pH 5, as shown in HPLC and SDS-PAGE patterns. A remarkable change in the CD spectrum was observed between native and heat-denatured Cry j 1 at a linear rate of 1 degrees C/min from 40 to 90 degrees C. The negative peak of native Cry j 1 at 222 nm was moved to 218 nm, suggesting the transition of an alpha-helix to beta-structure during heat denaturation. The increase in beta-structure was also observed during heat denaturation by monitoring the fluorescence with Thioflavin T. These results suggest that Cry j 1 forms intermolecular cross-beta-structure between denatured proteins during heating at 90 degrees C. The antigenicity of Cry j 1 detected by dot-blotting was greatly diminished during heating at a linear rate of 1 degrees C/min from 40 to 90 degrees C without any coagulates. These results suggest that IgE epitopes exposed on the molecular surface of Cry j 1 was buried inside soluble aggregates through intermolecular beta-structure formed by heating.

This work on the botanical origin of various types of honey produced in Slovenia and based on the mineral content analyses by the total reflection X-ray spectrometry (TXRF) is a continuation of this group's preliminary work (Golob, T.; Doberšek, U.; Kump, P.; Nečemer, M. Food Chem. 2005, 91, 593-600), which introduced the analytical methodology and employed only a simple statistical evaluation and which examined the possibility to determine the botanical origin of honey samples via elemental content. A much more comprehensive study on a total of 264 major types of honey samples harvested in 2004, 2005, and 2006 and interpreting the results with up to date chemometric methods was performed in this work. Slovenia is a small country by surface area, but it is pedologically and climatically diverse, therefore offering interesting possibilities for studying the influence of these diversities on the elemental content of natural products. By employing principal component analysis (PCA) and regularized discriminant analysis (RDA) it was established that from all of the measured elements only the four characteristic key elements Cl, K, Mn, and Rb could be used to best discriminate the types of honey. It was established that the employed combination of a simple, fast, and inexpensive multielement TXRF analytical approach and the evaluation of data by chemometric methods has the potential to discriminate the botanical origins of various types of honey.

The importance of geographical origin determination is an increasing and pressing requirement for all foods. Honey is one of the largest studied foods due to its nutritional and medicinal properties in a correct diet. In this paper, a total of 41 honey samples (polyfloral and acacia) from different countries have been analyzed in terms of (1)H NMR spectroscopy coupled with multivariate statistical methods. Unsupervised principal component analysis resulted as an efficient tool in distinguishing (1)H NMR spectra of polyfloral and acacia honey samples and for geographical characterization of the latter ones. Hierarchical projection to latent structures discriminant analysis was successfully applied for the discrimination among polyfloral honey samples of different geographical origins. (13)C NMR spectroscopy was applied to honey samples with the aim to investigate possible sugar isoforms differentiation. Our preliminary data indicated a different isoforms ratio between betaFP and betaFF only for polyfloral Argentinean samples, while Hungarian samples showed resonance shifts for some carbons of alphaFF, betaFP, betaFF, and alphaGP isoforms for both varieties. These data confirmed the potentiality of (13)C spectroscopy in food characterization, especially in sugar-based foods.

The protein complement of tea pollen collecting from tea tree (Camellia sinensis) was compared under different storage conditions. Protein was partially identified using a combination of the 2D-PAGE, MALDI-TOF/MS, MASCOT, and Xproteo search engine. Two hundred and sixty-nine and 396 proteins were detected in pollen stored at room temperature (RT) and -20 degrees C, respectively. Forty-three of the identified proteins were assigned to defense-related functions, energy metabolism, cytoskeleton, nucleic acid metabolism, membrane transport, amino acid metabolism, stress response, protein metabolism, transcription, fat metabolism, others, and function unknown proteins. The abundance analysis showed that more proteins related to stress response, nucleic acid metabolism, fat metabolism, and membrane transport are lost at RT than at -20 degrees C, while proteins related to defense and energy metabolism showed a reverse relation. For the others, no differences were found between the two storage conditions. During the determination of the peptides mass fingerprinting (PMF) of each spot, 35 proteins were identified in tea pollen for the first time. Thus, our data present an initial molecular picture of bee collected tea pollen, and our results suggest that freezing is the best way to maintain the quality of tea pollen.

Using HPLC-ESI-MS, several saturated and 1,2-dehydropyrrolizidine alkaloids were detected, mainly as their N-oxides, in fresh pollen collected from flowers of the pyrrolizidine alkaloid-producing plants Echium vulgare, E. plantagineum, Senecio jacobaea, S. ovatus, and Eupatorium cannabinum, and/or pollen loads from bees (bee pollen) that foraged on those plants. A major alkaloidal metabolite in S. ovatus was tentatively identified, using its mass spectrometric data and biogenic considerations, as the previously unreported, saturated alkaloid, 2-hydroxysarracine. Heating had very little effect on the 1,2-dehydropyrrolizidine alkaloids and their N-oxides from a variety of sources. Considered in conjunction with international concerns about the adverse effects of these alkaloids, the results strongly indicate a need for monitoring pollen supplies intended for human consumption, at least until conditions for processing and/or selection are clearly defined such as to significantly reduce the hepatotoxic (and potentially carcinogenic and genotoxic) pyrrolizidine alkaloid content of bee pollen.

Honeybee-collected pollen is promoted as a health food with a wide range of nutritional and therapeutic properties. A high-performance capillary electrophoresis with amperometric detection method has been developed for the simultaneous determination of bioactive ingredients in 10 samples of honeybee-collected pollen in this work. Under the optimum conditions, 13 phenolic components can be well-separated or nearly baseline-separated (apigenin and vanillic acid peaks) within 29 min at the separation voltage of 14 kV in a 50 mM borax running buffer (pH 9.0), and adequate extraction was obtained with ethanol for the determination of the above 13 compounds. Recovery (94.1-104.0%), repeatability of the peak current (<5.4%), and detection limits (6.9 x 10(-7)-6.4 x 10(-9) g mL(-1)) for the method were evaluated. This procedure was successfully used for the analysis and comparison of the phenolic content of honeybee-collected pollen samples originating from different floral origins based on their electropherograms or "phenolic profiles".

Thyme honey is the most appreciated unifloral Greek honey in Greece as well as around the world. In an effort to investigate the headspace composition of this type of honey, 28 samples were analyzed by means of solid-phase microextraction coupled to a gas chromatography-mass spectrometry system. The botanical origin of the samples was ascertained by pollen analysis, and samples displayed relative frequencies of thyme pollen between 18 and 41%. A total of 62 compounds were isolated, and phenylacetaldehyde was the most abundant (32.9% of the total peak area). Possible botanical markers are 1-phenyl-2,3-butanedione (13.4%), 3-hydroxy-4-phenyl-2-butanone, 3-hydroxy-1-phenyl-2-butanone (14.7%), phenylacetonitrile (4.8%), and carvacrol (0.9%), since these compounds are found only in thyme honey. Additionally, high proportions of phenylacetaldehyde are also characteristic ( F = 12.282, p < 0.001). The average concentrations of seven compounds were significantly different ( p < 0.05), namely phenylacetaldehyde, acetophenone, octanoic acid, carvacrol, phenylethyl alcohol, nonanal, and hexadecane. Applying principal component analysis to the data, six components were extracted, explaining 85.4% of the total variance. The first component explained 46.2% of the total variance and was positively correlated to phenylacetaldehyde, nonanoic acid, acetophenone, decanoic acid, benzaldehyde, phenylacetonitrile, isophorone, and nonanal. The extracted components were used as variables to the discriminant analysis, which showed good discrimination, especially for samples from Crete. A leave-one-out classification showed 85.7% of cross-validated grouped cases correctly classified. These results are promising to establish a discrimination model for these geographical regions. This is crucial for local beekeeper corporations on their effort to produce honey with geographical origin label.

Fipronil is a pesticide suspected of having harmful effects on honey bees at microgram per kilogram levels. Considering the lack of methodology, it thus appeared to be necessary to develop a method for the determination of the lowest amounts of fipronil and its metabolites in pollen. This paper describes a new analytical method with a limit of quantification (LOQ) of 0.1 microg/kg for a representative sample weight of 5 g. In the case of a field study, this tool was used for checking the possible existence of fipronil and/or metabolites in pollen samples, but none of them contained residues higher than the LOQ. This three-step rapid method uses liquid-solid solvent extraction with mechanical grinding, followed by liquid-liquid partitioning and Florisil solid-phase extraction for the two cleanup steps. The quantification is made by liquid chromatography with electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Indeed, combined with an adequate sample treatment, this technique offers good sensitivity and selectivity in such a complex matrix. The method has given good recoveries of 74-104% with relative standard deviations of 5.6-18.2%.

With the objective of evaluating the utility of the amino acid profile in the characterization of honey samples, 39 honey samples of two different harvests from a particular production zone in Córdoba, Argentina, were analyzed. Multivariate statistical techniques, such as principal component analysis (PCA), cluster analysis (CA), and multiple correspondence analysis (MCA), were applied to verify the correlation among the amino acid profiles, pollen percentages, and different harvests. PCA, CA, and MCA demonstrate the presence of differences of amino acid profiles between samples of the two harvests, such differences being mainly due to differences in pollen availability. Variation of the flora surrounding the apiary due to agricultural practices makes the analysis of amino acid profile typical for those cases with stabilized flora.

Fast protein liquid chromatography on a Superdex 75 HR column has been applied to analyze the proteins of 29 honeys, 12 of floral origin and 17 from honeydew. The molecular masses were comprised between 13100 and 94000 Da. Seven peaks have been separated; four of them were present in all of the honeys, and three were only present in some honeys. Direct observation of the chromatograms of the floral and honeydew honeys did not reveal any information about their botanical origins. However, both types of honeys can be distinguished with the percentages of the areas of four of the seven chromatographic peaks obtained.

The systemic imidacloprid is one of the most used insecticides in the world for field and horticultural crops. This neurotoxicant is often used as seed-dressing, especially for maize, sunflower, and rape. Using a LC/MS/MS technique (LOQ = 1 microg/kg and LOD = 0.1 microg/kg), the presence of imidacloprid has been measured in maize from field samples at the time of pollen shed, from less than 0.1 microg/kg up to 33.6 microg/kg. Numerous random samples were collected throughout France from 2000 to 2003. The average levels of imidacloprid measured are 4.1 microg/kg in stems and leaves, 6.6 microg/kg in male flowers (panicles), and 2.1 microg/kg in pollen. These values are similar to those found previously in sunflower and rape. These results permit evaluation of the risk to honeybees by using the PEC/PNEC ratios (probable exposition concentrations/predicted no effect concentration). PEC/PNEC risk ratios were determined and ranged between 500 and 600 for honeybees foraging on maize treated with imidacloprid by seed dressing. Such a high risk factor can be related to one of the main causes of honeybee colony losses.

The pyrrolizidine alkaloids previously identified in floral honey attributed to Echium vulgare (Boraginaceae) have been detected (8000-14 000 ppm) in pure pollen collected from the anthers of Echium vulgare. Pyrrolizidine alkaloids and/or their N-oxides were isolated from the aqueous acid extracts of pollen by use of strong cation-exchange, solid-phase extraction and identified by liquid chromatographic/mass spectrometric (LCMS) analysis. The pyrrolizidine alkaloids in the pollen are present mainly as the N-oxides. In addition to seven previously described pyrrolizidine alkaloids and/or their N-oxides (echimidine, acetylechimidine, uplandicine, 9-O-angelylretronecine, echiuplatine, leptanthine, and echimiplatine), one unidentified (echivulgarine), but previously found in honey, and two previously undescribed (vulgarine and 7-O-acetylvulgarine) pyrrolizidine alkaloids and/or their N-oxides were identified in the pollen. Tentative structures for these unidentified pyrrolizidine alkaloids are proposed on the basis of the mass spectrometric data and biogenetic considerations. The implications of these results for identifying the source and subsequent concentrations of pyrrolizidine alkaloids in honeys and commercial bee pollen are briefly discussed.

We report the development of a rapid, specific, and sensitive enzyme-linked immunoassay (ELISA) for the evaluation of sunflower pollen in honey as a method alternative to melissopalynology, which is considered the standard technique for the evaluation of floral origin of honey. Two 33-36 kDa proteins, identified as characteristic of sunflower pollen, were isolated and used as coating antigens in the competitive ELISA. We verified its analytical performance by evaluating reproducibility, specificity, and exactitude in relation to melissopalynology. The competitive ELISA developed during this work is able to quantify sunflower pollen in honey, with a detection limit of 10%, showing linear response between 10 and 90%. The method afforded low cross reactivity with honey from other floral origin, thus evidencing an adequate selectivity. We also observed a significant correlation (r = 0.975; p < 0.001) when the proposed ELISA was referenced to melissopalynology. Hence, we conclude that the competitive ELISA constitutes a valuable and feasible alternative for authentication of sunflower honey. This work opens the possibility to develop similar assays for other pollen types.

Bee pollen is a major substrate for mycotoxins growth when no prompt and adequate drying is performed by the beekeeper after collection by bees. Regulatory limits for aflatoxins and ochratoxin A are currently in force in the European Union for a rising list of foodstuffs, but not for this. An immunoaffinity column cleanup process has been applied prior to the analysis of aflatoxins B(1), B(2), G(1), and G(2) and ochratoxin A (OTA). Optimization of the HPLC conditions has involved both a gradient elution and a wavelength program for the separation and fluorimetric quantitation of all five mycotoxins at their maximum excitation and emission values of wavelength in a single run. The higher limit of detection (mug/kg) was 0.49 for OTA and 0.20 for aflatoxin B(1). Repeatability (RSDr) at the lower limit tested ranged from 9.85% for OTA to 6.23% for aflatoxin G(2), and recoveries also at the lower spiked level were 73% for OTA and 81% for aflatoxin B(1). None of the 20 samples assayed showed quantifiable values for the five mycotoxins.

Imidacloprid, a neonicotinic insecticide, has been used as a seed dressing (Gaucho) to protect crops against soil and aerial insects. However, French beekeepers observed abnormal behavior of bees foraging on sunflowers and suspected a link between the imidacloprid seed treatment and the observed bee syndrome. This work studies the distribution of [(14)C-imidazolidin]imidacloprid (1 mg/seed) in three stages of Gaucho-treated sunflowers grown in an outdoor lysimeter. Plants absorbed <10% of [(14)C]imidacloprid spiked on seeds, and 75% of that absorbed radioactivity was found in cotyledons. Concentrations in the upper leaves were 20 times lower than in the first leaves. From the extracted radioactivity, imidacloprid accounted for 50% and metabolites for the other 50%. Four major metabolites can be detected, in variable concentrations, among which the hydroxy- and olefin-imidacloprid have toxicities equivalent to that of imidacloprid. In pollen, concentrations of imidacloprid were 13 ng x g(-1). Thus, imidacloprid residues from Gaucho seed treatment contaminated sunflower pollen, involving the translocation of imidacloprid within the plant.

The high incidence of food allergies, including oral allergy syndrome, represent major considerations when introducing new crops and foods. A new structural database of allergenic proteins, SDAP-Food, http://fermi.utmb.edu/SDAP/, has been developed to aid in predicting the IgE-binding potential of novel food proteins and cross-reactivities among known allergens. The site is designed to facilitate the first steps of a decision tree approach to determine the allergenicity of a given protein, based on the sequence and structural similarity to known allergens and their IgE binding sites. Immunological tests can then be used to confirm the predictions. A hierarchical procedure for identifying potential allergens, using a physical property-based sequence similarity index, has been designed to identify regions that resemble known IgE binding sites. As an example, SDAP tools were used to find food allergen sequences similar to an IgE binding site of the Jun a 3 allergen from mountain cedar pollen. The SDAP sequence similarity search matched the Jun a 3 epitope to regions in several food allergens, including cherry (Pru av 2), apple (Mal d 2) and pepper (Cap a 1), which are, like Jun a 3, members of the plant pathogenesis-related (PR-5) protein family. Homology modeling, using our EXDIS/DIAMOD/FANTOM program suite, indicated a similar surface location and structure for the potential epitope region on all of these allergens. The quantitative approach presented here can be used as part of a screening process for potential allergenicity of recombinant food products.

Eight fatty acid esters of triterpene alcohols (1-8), four free triterpene alcohols (9, 12, 17, and 18), four diterpene acids (19-22), two tocopherol-related compounds (23 and 24), four estolides (25-28), three syn-alkane-4,6-diols (29-31), one 1,3-dioxoalkanoic acid (32), and one aliphatic ketone (33), along with the mixture of free fatty acids, were isolated from the diethyl ether extract of the pollen grains of sunflower (Helianthus annuus). Among these compounds, 14 (2-8, 12, 23, 25-28, and 33) were new naturally occurring compounds, and their structures were determined on the basis of spectroscopic methods. Twenty-four terpenoids and lipids (1-4, 6-9, 12, and 19-33) and six free triterpene triols (10, 11, and 13-16), derived from their fatty acid esters (2, 3, and 5-8) by alkaline hydrolysis, were evaluated with respect to their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), in Raji cells, which is known to be a primary screening test for antitumor promoters. Among the 30 compounds tested, 21 compounds possessing a di- or a polycyclic ring system in the molecule (1-4, 6-16, and 19-24) showed potent inhibitory effects on EBV-EA induction (91-100% inhibition at 1 x 10(3) mol ratio/TPA).

Headspace analyses of pollen, whole flowerheads, ligulate and tubular florets, flower buds, involucral bracts, and leaves have been performed on the food plant Chrysanthemum coronarium L. (Asteraceae). The analyses permitted differences in the pattern of volatiles emitted by the different floral parts to be observed and the site and phenological stage of emission of these chemicals to be verified. Camphor and cis-chrysanthenyl acetate were emitted mainly by ligulate and tubular florets; the production of myrcene and (Z)-ocimene was higher in the flower buds, whereas beta-caryophyllene, (E,E)-alpha-farnesene, and (E)-beta-farnesene seemed attributable mainly to the involucral bracts. The leaves showed a quite different volatile profile, with (Z)-ocimene as the main constituent. Pollen showed a completely different composition of its volatiles, with perilla aldehyde, cis-chrysanthenyl acetate, and camphor among the principal compounds; many carbonylic compounds and linear hydrocarbons have been detected exclusively in pollen. Furthermore, the essential oils obtained from flowerheads and leaves have been studied. These samples showed mainly quantitative differences. Camphor (22.1%) and cis-chrysanthenyl acetate (19.9%) were the main constituents of the oil from flowers, whereas the oil from the leaves contained mainly (Z)-ocimene (45.4%) and myrcene (28.2%).

Bee-collected pollen ("bee pollen") is promoted as a health food with a wide range of nutritional and therapeutic properties. The objective of the current study is to evaluate the contribution made through the free radical scavenging capability of bee-collected floval pollens by their flavonoid/phenolics constituents, and to determine whether this capability is affected by aging. The free radical scavenging effectiveness of a bee pollen (EC(50)) as measured by the DPPH method is shown to be determined by the nature and levels of the constituent floral pollens, which can be assayed via their phenolics profiles by HPLC. Each pure floral pollen has been found to possess a consistent EC(50) value, irrespective of its geographic origin or date of collection, and the EC(50) value is determined to a large extent (ca. 50%) by the nature and the levels of the pollen's flavonoids and phenolic acids. Non-phenolic antioxidants, possibly proteins, account for the balance of the activity. Pollen aging over 3 years is demonstrated to reduce the free radical scavenging activity by up to 50% in the most active floral pollens, which tend to contain the highest levels of flavonoids/phenolic acids. It is suggested that the freshness of a bee pollen may be determined from its free radical scavenging capacity relative to that of fresh bee pollen containing the same floral pollen mix.

The headspace analyses of pollen, whole living female and male flowers, and staminoids have been performed on Laurus nobilis L. (Lauraceae) from Italy to determine whether there are differences in the volatiles emitted in order to give a contribution to the roles of the different flower parts in the pollination ecology of dioecious plants. Also, the essential oils obtained from male and female plants have been studied to evaluate a possible correlation between the spontaneously emitted volatiles and the constituents stored in the glandular tissues. Furthermore, the headspace sampling technique has been improved, with respect to previously employed methods, by means of solid-phase microextraction (SPME).

We report on the development of a novel alternative method for the assessment of floral origin in honey samples based on the study of honey proteins using immunoblot assays. The main goal of our work was to evaluate the use of honey proteins as chemical markers of the floral origin of honey. Considering that honeybee proteins should be common to all types of honey, we decided to verify the usefulness of pollen proteins as floral origin markers in honey. We used polyclonal anti-pollen antibodies raised in rabbits by repeated immunization of Sunflower (Elianthus annuus) and Eucalyptus (Eucalyptus sp.) pollen extracts. The IgG fraction was purified by immunoaffinity. These antibodies were verified with nitrocellulose blotted pollen and unifloral honey protein extracts. The antibodies anti-Sunflower pollen, bound to the 36 and 33 kDa proteins of Sunflower unifloral honey and to honey containing Sunflower pollen; and the antibodies anti-Eucalyptus sp. pollen bound to the 38 kDa proteins of Eucalyptus sp. unifloral honey in immunoblot assays. Satisfactory results were obtained in differentiating between the types of pollen analyzed and between Sunflower honey and Eucalyptus honey with less cross reactivity with other types of honey from different origin and also with good sensitivity in the detection. This immunoblot method opens an interesting field for the development of new antibodies from different plants, which could serve as an alternative or complementary method to the usual melissopalynological analysis to assess honey floral origin.