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.     

Amino acid composition and antioxidant capacity of Spanish honeys

The amino acid composition of 53 honey samples from Spain, consisting of 39 floral, 5 honeydew, and 9 blend honeys, has been determined. Physicochemical characteristics, polyphenolic content, amino acid composition, and estimation of the radical scavenging capacity against the stable free radical DPPH of the honey samples were analyzed. The resulting data have been statistically evaluated. The results showed that pH, acidity, net absorbance, electrical conductivity, and total polyphenolic contents of the honeys showed a strong correlation with the radical scavenging capacity. The correlation between the radical scavenging capacity of honey and amino acid contents was high with 18 of the 20 amino acids detected, with correlation values higher than those obtained for polyphenolic content. These results suggest that the amino acid composition of honey is an indicator of the sample's scavenging capacity.     

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.     

Protein analysis of honeys by fast protein liquid chromatography: application to differentiate floral and honeydew honeys

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.     

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.     

Classification of Italian honeys by 2D HR-NMR

The importance of honey has been recently increased because of its nutrient and therapeutic effects, but the adulteration of honey in terms of botanical origin has increased, too. The floral origin of honeys is usually determined using melisso-palynological analysis and organoleptic characteristics, but the application of these techniques requires some expertise. A number of papers have confirmed the possibility of characterizing honey samples by selected chemical parameters. In this study high-resolution nuclear magnetic resonance (HR-NMR) and multivariate statistical analysis methods were used to identify and classify honeys of five different floral sources. The 71 honey samples (robinia, chestnut, citrus, eucalyptus, polyfloral) were analyzed by HR-NMR using both 1H NMR and heteronuclear multiple bond correlation spectroscopy (HMBC). Spectral data were analyzed by application of unsupervised and supervised pattern recognition and multivariate statistical techniques such as principal component analysis (PCA) and general discriminant analysis (GDA). The use of 1H-(13)C HMBC coupled with appropriate statistical analysis seems to be an efficient technique for the classification of honeys.     

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.     

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.     

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.     

Comparison of free amino acids profile in honey from three Argentinian regions

The physical chemistry characteristics of honey are directly related to floral origin and, as a result, to the production region. There are some available methods that can determine the botanical or geographical origin of honey such as the free amino acids profile analysis. This paper reports data on the free amino acid composition, determined by reversed-phase high-performance liquid chromatography UV detection on 56 honey samples from three different Argentine regions, with characteristic apiarian flora. To evaluate if the quantified amino acid could be used to verify the geographical or botanical origin of honey, statistical analyses were performed. The cluster analysis showed that samples were grouped in clusters related to sampling regions and more strictly to apiarian flora around apiaries. Each cluster appears associated, in accordance with the principal component analysis, to high or low concentrations of different amino acids.     

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.     

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.     

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.     

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.     

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.     

Classification and characterization of manuka honeys based on phenolic compounds and methylglyoxal

Manuka honey from New Zealand is often considered to be a medicinal product of special value due to its high level of antimicrobial activity. Therefore, the distinct authentication of its botanical origin is of great importance. Aside from the common pollen analysis, it is in this respect particularly the analysis of the phenolic acids, flavonoids, and norisoprenoids that is described as useful. In the present study, numerous manuka honeys were analyzed by UPLC-PDA-MS/MS after solid-phase extraction and compared to other kinds of honey to define marker substances characteristic for manuka honeys. The PDA profiles obtained differed markedly from each other so that the individual honey samples could be assigned to three groups. For the honeys of group 1 the comparably high concentrations of 4-hydroxybenzoic acid, dehydrovomifoliol, and benzoic acid proved to be typical, whereas the profiles of group 2 showed high kojic acid and 2-methoxybenzoic acid intensities. The manuka honeys of group 3, on the other hand, yielded high amounts of syringic acid, 4-methoxyphenyllactic acid, and methyl syringate. Furthermore, the comprehensive comparison of manuka honeys to other unifloral honeys revealed that especially kojic acid, 5-methyl-3-furancarboxylic acid, leptosin, unedone, 2-methoxybenzoic acid, 4-methoxyphenyllactic acid, 3-hydroxy-1-(2-methoxyphenyl)penta-1,4-dione, and methyl syringate were useful for distinguishing manuka honeys from the other kinds of investigated honeys. Moreover, kojic acid, unedone, 5-methyl-3-furancarboxylic acid, 3-hydroxy-1-(2-methoxyphenyl)penta-1,4-dione, and lumichrome were identified in manuka honey for the first time.     

Multivariate correlation between color and mineral composition of honeys and by their botanical origin

The mineral content and color characteristics of 77 honey samples were analyzed. Eighteen minerals were quantified for each honey. Multiple linear regression (MLR) was used to establish equations relating the colorimetric CIELAB coordinates to the mineral data. The results obtained shown that lightness (L) was significantly correlated with S, Ca, Fe, As, Pb, and Cd for the dark honey types (avocado, heather, chestnut, and honeydew). For the light and brown honey types (citrus, rosemary, lavender, eucalyptus, and thyme), C(ab) and b showed the lower correlation with the mineral content of the honeys; their regression functions involve a few independent variables (Mg and Al for b and only Al for C(ab)). Furthermore, by means of application of linear discriminant analysis to the mineral content, it was possible to obtain a model that classifies the honeys by their lightness. The prediction ability of the built model, determined with the test set method, was 85%.     

Identification and quantification of antioxidant components of honeys from various floral sources

Little is known about the individual components of honey that are responsible for its antioxidant activity. The present study was carried out to characterize the phenolics and other antioxidants present in honeys from seven floral sources. Chromatograms of the phenolic nonpolar fraction of the honeys indicated that most honeys have similar but quantitatively different phenolic profiles. Many of the flavonoids and phenolic acids identified have been previously described as potent antioxidants. A linear correlation between phenolic content and ORAC activity was demonstrated (R(2) = 0.963, p < 0.0001). Honeys were separated by solid-phase extraction into four fractions for sugar removal and separation based on solubility to identify the relative contribution of each fraction to the antioxidant activity of honey. Antioxidant analysis of the different honey fractions suggested that the water-soluble fraction contained most of the antioxidant components. Specific water-soluble antioxidant components were quantified, including protein; gluconic acid; ascorbic acid; hydroxymethylfuraldehyde; and the combined activities of the enzymes glucose oxidase, catalase and peroxidase. Of these components, a significant correlation could be established only between protein content and ORAC activity (R(2) = 0.674, p = 0.024). In general, the antioxidant capacity of honey appeared to be a result of the combined activity of a wide range of compounds including phenolics, peptides, organic acids, enzymes, Maillard reaction products, and possibly other minor components. The phenolic compounds contributed significantly to the antioxidant capacity of honey but were not solely responsible for it.     

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.     

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.