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.     

Identification of botanical biomarkers in Argentinean Diplotaxis honeys: flavonoids and glucosinolates

To select and establish floral biomarkers of the botanical origin of Diplotaxis tenuifolia honeys, the flavonoids and glucosinolates present in bee-deposited nectar collected from hive combs (unripe honey) and mature honey from the same hives fron which the unripe honey samples were collected were analyzed by LC-UV-PAD-ESI-MS(n). Glycosidic conjugates of the flavonols quercetin, kaempferol, and isorhamnetin were detected and characterized in unripe honey. D. tenuifolia mature honeys contained the aglycones kaempferol, quercetin, and isorhamnetin. The differences between the phenolic profiles of mature honey and freshly deposited honey could be due to hydrolytic enzymatic activities. Aliphatic and indole glucososinolates were analyzed in unripe and mature honeys, this being the first report of the detection and characterization of glucosinolates as honey constituents. Moreover, these honey samples contained different amounts of propolis-derived flavonoid aglycones (1765-3171 μg/100 g) and hydroxycinnamic acid derivatives (29-1514 μg/100 g). Propolis flavonoids were already present in the freshly deposited nectar, showing that the incorporation of these compounds to honey occurs at the early steps of honey production. The flavonoids quercetin, kaempferol, and isorhamnetin and the glucosinolates detected in the samples could be used as complementary biomarkers for the determination of the floral origin of Argentinean Diplotaxis honeys.     

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.     

Floral classification of honey using mid-infrared spectroscopy and surface acoustic wave based z-Nose Sensor

Fourier transform infrared spectroscopy (FTIR) and z-Nose were used as screening tools for the identification and classification of honey from different floral sources. Honey samples were scanned using microattenuated total reflectance spectroscopy in the region of 600-4000 cm(-1). Spectral data were analyzed by principal component analysis, canonical variate analysis, and artificial neural network for classification of the different honey samples from a range of floral sources. Classification accuracy near 100% was achieved for clover (South Dakota), buckwheat (Missouri), basswood (New York), wildflower (Pennsylvania), orange blossom (California), carrot (Louisiana), and alfalfa (California) honey. The same honey samples were also analyzed using a surface acoustic wave based z-Nose technology via a chromatogram and a spectral approach, corrected for time shift and baseline shifts. On the basis of the volatile components of honey, the seven different floral honeys previously mentioned were successfully discriminated using the z-Nose approach. Classification models for FTIR and z-Nose were successfully validated (near 100% correct classification) using 20 samples of unknown honey from various floral sources. The developed FTIR and z-Nose methods were able to detect the floral origin of the seven different honey samples within 2-3 min based on the developed calibrations.     

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.     

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.     

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.     

Solid-phase extraction and LC-MS analysis of pyrrolizidine alkaloids in honeys

Strong-cation-exchange, solid-phase extraction of pyrrolizidine alkaloids and their N-oxides from honey samples was followed by reduction of the N-oxides and subsequent analysis of total pyrrolizidine alkaloids using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. A limited survey of 63 preprocessing samples of honey, purposefully biased toward honeys attributed to floral sources known to produce pyrrolizidine alkaloids, demonstrated levels of pyrrolizidine alkaloids up to approximately 2000 parts per billion (ppb) in a sample attributed to Echium plantagineum. Up to 800 ppb pyrrolizidine alkaloids was detected in some honeys not attributed by the collector to any pyrrolizidine alkaloid-producing floral source. No pyrrolizidine alkaloids were detected in approximately 30% of the samples in this limited study, while some honeys showed the copresence of pyrrolizidine alkaloids from multiple floral sources such as E. plantagineum and Heliotropium europaeum. In addition, retail samples of blended honeys (with no labeling to suggest that pyrrolizidine alkaloid-producing floral sources were used in the blends) have been shown to contain up to approximately 250 ppb pyrrolizidine alkaloids.     

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.     

Evolution of invertase activity in honey over two years

Invertase activity is a good parameter for evaluating honey freshness. Invertase activity evolution was determined on 57 fresh, unheated, commercially purchased Galician (northwestern Spain) floral honey samples. All honeys were stored in darkness at room temperature for up 24 months and analyzed each 6 months so as to determine the invertase activity evolution tendency for the first time. Invertase activity analysis was carried out according to Siegenthaler's method and in a simple assay, the latter showing a good precision (coefficient of variation between 0.35 and 0.66%). Initial invertase activity mean value was 163.9 (48.4-251.0) micromol of 4-nitrophenyl-alpha-D-glucopyranoside hydrolyzed/kg of honey/min. After application of the SPSS statistical package, the values of invertase activity showed five types of temporal behavior: exponential (56% of samples), linear (25% of samples), logarithmic (11% of samples), inverse (5% of samples), and quadratic (3% of samples). Linear regression equations were used to predict the invertase activity at 6, 12, 18, and 24 months from the initial Galician honeys' invertase activities; no statistical differences were found between experimental data and the activities calculated from the linear regression equations.     

Homogentisic acid: a phenolic acid as a marker of strawberry-tree (Arbutus unedo) honey.

Analysis of organic acids in strawberry-tree (Arbutus unedo) honey showed the presence of an unknown acid as the main constituent. This compound was isolated and identified as homogentisic acid (2, 5-dihydroxyphenylacetic acid) by MS and NMR techniques. Its average content in honey was 378 +/- 92 mg/kg. Analysis of nectar confirmed the floral origin of the compound found in honey. Since this acid was not detected in any of the different monofloral honeys, it could be used as a marker of strawberry-tree (A. unedo) honey.     

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.     

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.     

The use of carbohydrate profiles and chemometrics in the characterization of natural honeys of identical geographical origin

A study of the real possibilities of carbohydrate profiles and chemometrics to characterize the botanical origin of honey from a single geographical area, the Province of Soria (Spain), is presented. To this end, 14 carbohydrates were quantified using high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD) in 77 natural honeys, the botanical origins of which were ling, spike lavender, French lavender, thyme, forest, and multifloral. Principal component analysis has been employed as a first approach to characterize the honey samples analyzed, showing similarities between spike lavender and multifloral honeys. The best discrimination among groups is obtained when four canonical discriminant analyses were carried out sequentially, origin by origin, achieving an overall percentage of success of 90% following cross-validation.     

Determination of volatile organic compound patterns characteristic of five unifloral honey by solid-phase microextraction-gas chromatography-mass spectrometry coupled to chemometrics

We report the evaluation of the floral origin of honey by analysis of its volatile organic compounds (VOCs) profile, joined with the use of combined pattern recognition techniques. Honey samples, from five floral origins, were analyzed by headspace solid-phase microextraction-gas chromatography-mass spectrometry, selecting 35 VOCs out of the entire profiles, which were analyzed by hierarchical cluster analysis (HCA), stepwise discriminant analysis (SDA), and K-nearest-neighbor (KNN). Both HCA and SDA were used as exploratory tools to select a group of VOCs representing similitude and differences among studied origins. Thus, six out of 35 VOCs were selected, verifying their discriminating power by KNN, which afforded 93% correct classification. Therefore, we drastically reduced the amount of compounds under consideration but kept a good differentiation between floral origins. Selected compounds were identified as octanal, benzeneacetaldehyde, 1-octanol, 2-methoxyphenol, nonanal, and 2-H-1-benzopyran-2-one. The analysis of VOC profiles, coupled to HCA, SDA, and KNN, provides a feasible alternative to evaluate the botanical source of honey.     

Improved method for extraction and LC-MS analysis of pyrrolizidine alkaloids and their N-oxides in honey: application to Echium vulgare honeys

A method for analyzing honey samples was developed that enabled the simultaneous detection and identification of pyrrolizidine alkaloids and their N-oxides. Honey samples were treated with methanol or dilute sulfuric acid and then centrifuged to remove insoluble material. Subsequent strong cation exchange, solid-phase extraction of the supernatant provided a fraction that was analyzed for the presence of pyrrolizidine alkaloids and their N-oxides using high-pressure liquid chromatography coupled to electrospray ionization mass spectrometry. The procedure was validated using extracts of Echium plantagineum and authenticated standards of pyrrolizidine alkaloids and their N-oxides from other plant sources. Of several variations of the solid-phase extraction method assessed in this study, the best combination for generic use involved the dilution of honey with 0.05 M sulfuric acid and the subsequent application of the centrifuged solution to solid-phase extraction columns at the rate of a maximum of 10 g of honey per solid-phase extraction column. The method was applied to the analysis of nine floral honeys, five of which were attributed by the apiarist to Echium vulgare. Seven of the honey samples were positive for pyrrolizidine alkaloids and N-oxides characteristic of E. vulgare.     

Changes in the free amino acid contents of honeys during storage at ambient temperature

This study was carried out to establish the changes in the free amino acid contents of floral honeys, honeydew honeys, and blend honeys during storage at room temperature and to test the capacity of the amino acids to distinguish the origin of the honeys after storage. For this purpose, 54 artisanal honeys (39 floral, 5 honeydew, and 10 blend) were studied. Samples were taken from recently collected honeys and at 3, 6, 9, 12, 16, 20, and 24 months after harvesting. The contents of most of the free amino acids were found to decrease with storage time, with the greatest reduction observed in the first 9 months. The contents of the amino acids aspartic acid, beta-alanine, and proline increased in the first few months after storage, reaching maximum values at 6 months, suggesting the possible existence of enzymatic activities. The application of stepwise discriminant analysis to the free amino acid content data demonstrated that the contents of the amino acids valine, beta-alanine, gamma-aminobutyric acid, serine, isoleucine, alpha-alanine, ornithine, and glutamine correctly assigned 87% of honeys to their group of origin: floral, honeydew, or blend.     

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.     

Assessment of the floral origin of honey by SDS-page immunoblot techniques

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.