Development of a competitive ELISA for the evaluation of sunflower pollen in honey samples

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.     

Flavonoids in monospecific eucalyptus honeys from Australia

The HPLC analyses of Australian unifloral Eucalyptus honeys have shown that the flavonoids myricetin (3,5,7,3',4', 5'-hexahydroxyflavone), tricetin (5,7,3',4',5'-pentahydroxyflavone), quercetin (3,5,7,3',4'-pentahydroxyflavone), luteolin (5,7,3', 4'-tetrahydroxyflavone), and kaempferol (3,5,7, 4'-tetrahydroxyflavone) are present in all samples. These compounds were previously suggested as floral markers of European Eucalyptus honeys. The present results confirm the use of flavonoid analysis as an objective method for the botanical origin determination of eucalyptus honey. Honeys from E. camaldulensis (river red gum honey) contain tricetin as the main flavonoid marker, whereas in honeys from E. pilligaensis (mallee honey), luteolin is the main flavonoid marker, suggesting that species-specific differences can be detected with this analysis. The main difference between the flavonoid profiles of Australian and European Eucalyptus honeys is that in the Australian honeys, the propolis-derived flavonoids (pinobanksin (3,5, 7-trihydroxyflavanone), pinocembrin (5,7-dihydroxyflavanone), and chrysin (5,7-dihydroxyflavone)) are seldom found and in much smaller amounts.     

Identification of flavonoid markers for the botanical origin of Eucalyptus honey

European Eucalyptus honeys showed a common and characteristic HPLC profile in which the flavonoids myricetin (3,5,7,3',4', 5'-hexahydroxyflavone), tricetin (5,7,3',4',5'-pentahydroxyflavone), quercetin (3,5,7,3',4'-pentahydroxyflavone), luteolin (5,7,3', 4'-tetrahydroxyflavone), and kaempferol (3,5,7, 4'-tetrahydroxyflavone) were identified. Their contents, and relative amounts, in the analyzed honey samples were quite constant and supported their floral origin. In addition, ellagic acid and the propolis-derived flavonoids pinobanksin, pinocembrin, and chrysin were detected in most samples. The contents of these nonfloral phenolics were much more variable as could be expected for their propolis origin. Myricetin, tricetin, and luteolin had not been identified as floral markers in any other honey sample previously analyzed in our laboratory (chestnut, citrus, rosemary, lavender, acacia, rapeseed, sunflower, heather, lime tree, etc.) or reported in the literature, suggesting that these could be useful markers. Only in some individual heather samples produced in Portugal has tricetin previously been detected in minor amounts. These samples, however, were contaminated with Eucalyptus as revealed by their pollen analysis and the lack of tricetin or their glycosides in heather floral nectar. It remains to be established if myricetin, tricetin, and luteolin originate from Eucalyptus floral nectar where the corresponding glycosides should be present.     

Quantitative high-performance liquid chromatography analyses of flavonoids in Australian Eucalyptus honeys

Flavonoids of nine Australian monofloral Eucalyptus honeys have been analyzed and related to their botanical origins. The mean content of total flavonoids varied from 1.90 mg/100 g of honey for stringybark (E. globoidia) honey to 8.15 mg/100 g of honey for narrow-leaved ironbark (E. crebra) honey, suggesting that species-specific differences occur quantitatively among these Eucalyptus honeys. All of the honey samples analyzed in this study have a common flavonoid profile comprising tricetin (5,7,3',4',5'-pentahydroxyflavone), quercetin (3,5,7,3',4'-pentahydroxyflavone), and luteolin (5,7,3',4'-tetrahydroxyflavone), which, together with myricetin (3,5,7,3',4',5'-hexahydroxyflavone) and kaempferol (3,5,7,4'-tetrahydroxyflavone), were previously suggested as floral markers for European Eucalyptus honeys. Thus, flavonoid analysis could be used as an objective method for the authentication of the botanical origin of Eucalyptus honeys. Moreover, species-specific differences can also be found in the composition of honey flavonoid profiles. Among these honeys, bloodwood (E. intermedia) honey contains myricetin and tricetin as the main flavonoid compounds, whereas there is no myricetin detected in yapunyah (E. ochrophloia), narrow-leaved ironbark (E. crebra), and black box (E. largiflorens) honeys. Instead, these types of Eucalyptus honeys may contain tricetin, quercetin, and/or luteolin as their main flavonoid compounds. Compared to honeys from other geographical origins, the absence or minor presence of propolis-derived flavonoids such as pinobanksin, pinocembrin, and chrysin in Australian honeys is significant. In conclusion, these results demonstrate that a common flavonoid profile exists for all of the Eucalyptus honeys, regardless of their geographical origins; the individual species-specific floral types of Eucalyptus honey so common in Australia could be possibly differentiated by their flavonoid profile differences, either qualitatively or quantitatively or both.     

Immunochemical approach to detection of adulteration in honey: physiologically active royal jelly protein stimulating TNF-alpha release is a regular component of honey

The presence of royal jelly (RJ) proteins in honey collected from nectars of different plants, origin, and regions and in honeybee's pollen was detected by Western-blot analysis using polyclonal antibodies raised against water-soluble RJ-proteins. The most abundant RJ-protein in honeybee products corresponded to a 55 kDa protein. The N-terminal amino acid sequence of 55 kDa protein was N-I-L-R-G-E. This sequence is identical to the apalbumin-1, the most abundant protein of RJ. Apalbumin-1 is a regular component of honeybee products and thus is a suitable marker tool for proving adulteration of honey by means of immunochemical detection. Its presence in all tested samples of honeys and honeybee pollen was confirmed also by Western-blot analysis using polyclonal antibodies raised against recombinant apalbumin-1. It has been found that major RJ-proteins, apalbumin-1, and apalbumin-2, stimulate mouse macrophages to release TNF-alpha, which demonstrates that physiologically active proteins of honey could be used for its biological valuation.     

Authentication of the botanical and geographical origin of honey by front-face fluorescence spectroscopy

Front-face fluorescence spectroscopy, directly applied on honey samples, was used for the authentication of 11 unifloral and polyfloral honey types (n = 371 samples) previously classified using traditional methods such as chemical, pollen, and sensory analysis. Excitation spectra (220-400 nm) were recorded with the emission measured at 420 nm. In addition, emission spectra were recorded between 290 and 500 nm (excitation at 270 nm) as well as between 330 and 550 nm (excitation at 310 nm). A total of four different spectral data sets were considered for data analysis. Chemometric evaluation of the spectra included principal component analysis and linear discriminant analysis; the error rates of the discriminant models were calculated by using Bayes' theorem. They ranged from <0.1% (polyfloral and chestnut honeys) to 9.9% (fir honeydew honey) by using single spectral data sets and from <0.1% (metcalfa honeydew, polyfloral, and chestnut honeys) to 7.5% (lime honey) by combining two data sets. This study indicates that front-face fluorescence spectroscopy is a promising technique for the authentication of the botanical origin of honey and may also be useful for the determination of the geographical origin within the same unifloral honey type.     

Lumichrome and phenyllactic acid as chemical markers of thistle (Galactites tomentosa Moench) honey

HPLC-DAD-MS/MS chromatograms of thistle (Galactites tomentosa Moench) unifloral honeys, previously selected by sensory evaluation and melissopalynological analysis, showed high levels of two compounds. One was characterized as phenyllactic acid, a common acid found in honeys, but the other compound was very unusual for honeys. This compound was extracted from honey with ethyl acetate and purified by SPE using C(18), SiOH, and NH(2) phases. Its structure was elucidated on the basis of extensive 1D and 2D NMR experiments as well as HPLC-MS/MS and Q-TOF analysis, and it was identified as lumichrome (7,8-dimethylalloxazine). Lumichrome is known to be the main product of degradation obtained in acid medium from riboflavin (vitamin B(2)), and this is the first report of the presence of lumichrome in honeys. Analysis of the G. tomentosa raw honey and flowers extracts confirmed the floral origin of this compound. The average amount of lumichrome in thistle honey was 29.4 ± 14.9 mg/kg, while phenyllactic acid was 418.6 ± 168.9 mg/kg. Lumichrome, along with the unusual high level of phenyllactic acid, could be used as a marker for the botanical classification of unifloral thistle (G. tomentosa) honey.     

Authentication of the botanical origin of honey by near-infrared spectroscopy

Fourier transform near-infrared spectroscopy (FT-NIR) was evaluated for the authentication of eight unifloral and polyfloral honey types (n = 364 samples) previously classified using traditional methods such as chemical, pollen, and sensory analysis. Chemometric evaluation of the spectra was carried out by applying principal component analysis and linear discriminant analysis. The corresponding error rates were calculated according to Bayes' theorem. NIR spectroscopy enabled a reliable discrimination of acacia, chestnut, and fir honeydew honey from the other unifloral and polyfloral honey types studied. The error rates ranged from <0.1 to 6.3% depending on the honey type. NIR proved also to be useful for the classification of blossom and honeydew honeys. The results demonstrate that near-infrared spectrometry is a valuable, rapid, and nondestructive tool for the authentication of the above-mentioned honeys, but not for all varieties studied.     

Authentication of the botanical and geographical origin of honey by mid-infrared spectroscopy

The potential of Fourier transform mid-infrared spectroscopy (FT-MIR) using an attenuated total reflectance (ATR) cell was evaluated for the authentication of 11 unifloral (acacia, alpine rose, chestnut, dandelion, heather, lime, rape, fir honeydew, metcalfa honeydew, oak honeydew) and polyfloral honey types (n = 411 samples) previously classified with traditional methods such as chemical, pollen, and sensory analysis. Chemometric evaluation of the spectra was carried out by applying principal component analysis and linear discriminant analysis, the error rates of the discriminant models being calculated by using Bayes' theorem. The error rates ranged from <0.1% (polyfloral and heather honeys as well as honeydew honeys from metcalfa, oak, and fir) to 8.3% (alpine rose honey) in both jackknife classification and validation, depending on the honey type considered. This study indicates that ATR-MIR spectroscopy is a valuable tool for the authentication of the botanical origin and quality control and may also be useful for the determination of the geographical origin of honey.     

Authentication of the botanical origin of honey by front-face fluorescence spectroscopy. A preliminary study

The potential of front-face fluorescence spectroscopy for the authentication of unifloral and polyfloral honey types (n = 57 samples) previously classified using traditional methods such as chemical, pollen, and sensory analysis was evaluated. Emission spectra were recorded between 280 and 480 nm (excit: 250 nm), 305 and 500 nm (excit: 290 nm), and 380 and 600 nm (excit: 373 nm) directly on honey samples. In addition, excitation spectra (290-440 nm) were recorded with the emission measured at 450 nm. A total of four different spectral data sets were considered for data analysis. After normalization of the spectra, chemometric evaluation of the spectral data was carried out using principal component analysis (PCA) and linear discriminant analysis (LDA). The rate of correct classification ranged from 36% to 100% by using single spectral data sets (250, 290, 373, 450 nm) and from 73% to 100% by combining these four data sets. For alpine polyfloral honey and the unifloral varieties investigated (acacia, alpine rose, honeydew, chestnut, and rape), correct classification ranged from 96% to 100%. This preliminary study indicates that front-face fluorescence spectroscopy is a promising technique for the authentication of the botanical origin of honey. It is nondestructive, rapid, easy to use, and inexpensive. The use of additional excitation wavelengths between 320 and 440 nm could increase the correct classification of the less characteristic fluorescent varieties.     

Nectar Flavonol rhamnosides are floral markers of acacia (Robinia pseudacacia) honey

With the objective of finding floral markers for the determination of the botanical origin of acacia (robinia) honey, the phytochemicals present in nectar collected from Robinia pseudacacia flowers were analyzed by high-performance liquid chromatography-tandem mass spectrometry. Eight flavonoid glycosides were detected and characterized as kaempferol combinations with rhamnose and hexose. Acacia honey produced in the same location where the nectar was collected contained nectar-derived kaempferol rhamnosides. This is the first time that flavonoid glycosides have been found as honey constituents. Differences in the stability of nectar flavonoids during honey elaboration and ripening in the hive were shown to be due to hydrolytic enzymatic activity and to oxidation probably related to hydrogen peroxide (glucose-oxidase) activity. Acacia honeys contained propolis-derived flavonoid aglycones (468-4348 microg/100 g) and hydroxycinnamic acid derivatives (281-3249 microg/100 g). In addition, nectar-derived kaempferol glycosides were detected in all of the acacia honey samples analyzed (100-800 microg/100 g). These flavonoids were not detected in any of the different honey samples analyzed previously from different floral origins other than acacia. Finding flavonoid glycosides in honey related to floral origin is particularly relevant as it considerably enlarges the number of possible suitable markers to be used for the determination of the floral origin of honeys.     

Comparison of the volatile composition in thyme honeys from several origins in Greece

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.     

Evaluation of the botanical origin of estonian uni- and polyfloral honeys by amino acid content

The free amino acid content of 61 honey samples from Estonia has been determined by HPLC-UV with precolumn derivatization with diethyl ethoxymethylenemalonate. Analyzed samples were seven types of unifloral honeys and polyfloral honeys. The main amino acids found in Estonian honeys were proline and phenylalanine. The resulting data have been analyzed by t test and principal component analysis (PCA). t Test revealed that some amino acids (alpha-alanine, beta-alanine, asparagine, gamma-aminobutyric acid, glutamine, glycine, histidine, ornithine, phenylalanine, proline, serine, and tryptophan) are more potent for assigning honey botanical origin than others. PCA enabled differentiation of some honey types by their botanical origin. In the space of the two first principal components, heather honeys form a cluster that is clearly separable from, for example, polyfloral honeys. It is concluded that analysis of the free amino acid profile may serve as a useful tool to assess the botanical origin of Estonian honeys.     

Usefulness of amino acid composition to discriminate between honeydew and floral honeys. Application to honeys from a small geographic area

With the aim of finding methods that could constitute a solid alternative to melissopalynological and physicochemical analyses to determine the botanical origin (floral or honeydew) of honeys, the free amino acid content of 46 honey samples has been determined. The honeys were collected in a small geographic area of approximately 2000 km(2) in central Spain. Twenty-seven honey samples were classified as floral and 19 as honeydew according to their palynological and physicochemical analyses. The resulting data have been subjected to different multivariant analysis techniques. One hundred percent of honey samples have been correctly classified into either the floral or the honeydew groups, according to their content in glutamic acid and tryptophan. It is concluded that free amino acids are good indicators of the botanical origin of honeys, saving time compared with more tedious analyses.     

Pyrrolizidine alkaloids of Echium vulgare honey found in pure pollen

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.     

Analysis of volatiles from Spanish honeys by solid-phase microextraction and gas chromatography-mass spectrometry

Headspace solid-phase microextraction (SPME), followed by gas chromatography (GC)-mass spectrometry (MS) determination, has been used for the analysis of honey volatiles. Two SPME fibers were employed to study the composition of volatiles from various types of Spanish honeys. The best results were obtained with the Carboxen/PDMS fiber, using a homogenization time of 1 h at 70 degrees C and a sampling period of 30 min. A total of 35 compounds were detected, most of them identified by GC-MS and quantified using external standards. Differences in the composition of honey volatiles were obtained, and these results allowed the differentiation of honeys. However, further studies are necessary to confirm the utility of this technique as an alternative tool for the characterization of the floral origin of honeys.     

Analysis of phenolic and other aromatic compounds in honeys by solid-phase microextraction followed by gas chromatography-mass spectrometry

The solid-phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS) was used for the analysis of phenolic and other aromatic compounds in honey samples from different floral origin. Different parameters affecting the efficiency of the extraction, such as the type of the stationary phase of the fiber, NaCl and acetic acid addition, and extraction time, were optimized for the detection of the maximum number of compounds in the shortest analysis time. A total of 31 compounds were detected, with most of them identified and quantified by GC-MS. The principal component analysis (PCA) was applied to the data matrix; the results allowed for the differentiation between honeydew and nectar honeys on the basis of the salicylic acid concentration. It was found that this acid has a high contribution in the honeydew group (71.2-705.9 microg/100 g of honey) compared to the nectar honey group (0-47.6 microg/100 g of honey). The comparison of data in each honey group enabled us to characterize the floral source of some honeys using some aromatic compounds as markers.     

Botanical origin causes changes in nutritional profile and antioxidant activity of fermented products obtained from honey

Honey as rich source of enzymatic and nonenzymatic antioxidants serves as health-promoting nutrient in the human body. Here, we present the first time a comparative study of nutritional profiles (e.g., acidities, sugar, organic acid profile, total polyphenolic, flavonoid content) for different unifloral, multifloral honeys and their fermented products, in correlation with their antioxidant activity. Additionally, an optimized method for HPLC separation of organic acids from honey was established. The total phenolic content of honey samples varied widely among the honey types compared to fermented products. High amounts of total flavonoids were quantified in heather honey, followed by raspberry, multifloral, black locust, and linden honey. A positive correlation between the content of polyphenols, flavonoids, and antioxidant activity was observed in honey samples. After fermentation, the flavonoid content of dark honey fermented products decreased significantly. Black locust and linden honeys are more suitable for fermentation because the decrease in antioxidant substances is less pronounced.     

Age-induced diminution of free radical scavenging capacity in bee pollens and the contribution of constituent flavonoids

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.     

A 50 kDa maize gamma-zein has marked cross-reactivity with the almond major protein

Cross-reactivity of antibodies against almond major protein (AMP, a legumin), the major almond allergen, with cereal proteins may cause problems in detecting almond contaminants in cereal products when antibody-based assays are used. Rabbit polyclonal IgG antiserum produced against AMP was used to test cross-reactivity with protein extracts from maize, a cereal commonly found in breakfast and snack foods. Gradient SDS-PAGE followed by Western blotting was performed, and two cross-reactive proteins were detected by chemiluminescence. A fraction of maize proteins purified by elution from an IgG anti-AMP affinity column followed by electrophoreseis and immunoblotting showed a high degree of cross-reactivity with a minor 50 kDa protein of maize, as well as low cross-reactivity with the 27 kDa gamma-zein. The 50 kDa cross-reactive protein was identified as the 50 kDa gamma-zein by immunoreaction with anti-50 kDa gamma-zein antiserum. Notably, the 50 kDa maize gamma-zein also reacted with IgE from pooled human sera from patients with self-reported severe almond allergies. The high immunoreactivity of the 50 kDa gamma-zein should be considered in maize quality improvement programs, and such notable cross-reactivity is of relevance in the design of antibody-based assays for almond allergen detection.