Rapid Analysis of Glucose,Fructose, Sucrose,and Maltose in Honeys from Different Geographic Regions using Fourier Transform Infrared Spectroscopy and Multivariate Analysis

ABSTRACT: Quantitative analysis of glucose, fructose, sucrose, and maltose in different geographic origin honey samples in the world using the Fourier transform infrared (FTIR) spectroscopy and chemometrics such as partial least squares (PLS) and principal component regression was studied. The calibration series consisted of 45 standard mixtures, which were made up of glucose, fructose, sucrose, and maltose. There were distinct peak variations of all sugar mixtures in the spectral “fingerprint” region between 1500 and 800 cm⁻¹. The calibration model was successfully validated using 7 synthetic blend sets of sugars. The PLS 2nd-derivative model showed the highest degree of prediction accuracy with a highest R² value of 0.999. Along with the canonical variate analysis, the calibration model further validated by high-performance liquid chromatography measurements for commercial honey samples demonstrates that FTIR can qualitatively and quantitatively determine the presence of glucose, fructose, sucrose, and maltose in multiple regional honey samples.     

Simultaneous determination of nine acaricides and two metabolites in comb honey by LC/MS/MS

ABSTRACT

We developed a method for the simultaneous determination of acaricides in comb honey using LC/MS/MS. Because methods for honey analysis had not previously been applied to comb honey, we modified three techniques for sample preparation and LC/MS/MS conditions. First, we used a modified QuEChERS method that changed the extraction solution from ethyl acetate to acetonitrile. Second, we replaced the InertSep® MA-1 (30 mg, 1 ml) clean-up cartridge with an Oasis® HLB (60 mg, 3 ml). Third, we changed the ionisation mode from ESI to atmospheric pressure chemical ionisation (APCI). With these modifications, sample matrices had no effect on the identification and quantification of analytes, using an external solvent calibration curve. We verified this new method with nine acaricides and two metabolites on comb honey and honey samples from three different honey origins. The trueness ranged from 74.0 to 99.4%. The relative standard deviation of repeatability (RSD_r) ranged from 0.8 to 14.8% and that of within-laboratory reproducibility (RSD_WR) ranged from 1.3 to 14.8%. All criteria met Japanese validation guidelines. The LOQ was 1.0 μg kg^–1 for all analytes. We applied this method to 10 comb honey and 31 honey samples commercially available in Tokyo. From the results of the analysis of 41 samples, we observed that amitraz remained as N-(2,4-dimethylphenyl)-N-methylformamidine (DMPF) in 9 comb honey and 23 honey samples and that their residual concentrations were less than 20 μg kg^–1. Using this new method, we improved recovery and precision, which enabled precise quantitative determination. Furthermore, the residual amitraz value in honey determined by both this new and the previous method were in good agreement.     

Determination and surveillance of nine acaricides and one metabolite in honey by liquid chromatography-tandem mass spectrometry

A simple and accurate analytical method for the determination of acaricides in honey was developed and validated in accordance with Japanese validation guidelines. Analytes – amitraz, N-2,4-dimethylphenyl-N-methylformamidine (DMPF), etoxazole, fenpyroximate, fipronil, hexythiazox, propargite, pyridaben and spirodiclofen – were extracted with ethyl acetate under basic conditions and subsequently cleaned up using an InertSep^® MA-1 polymer-based anion-exchange column. The method was validated by fortified recovery tests at three different concentrations (1, 5 and 10 µg kg^?1) performed with three samples daily on five different days. The method exhibited recoveries of 77–116% and precision (relative standard deviations – RSDs) of repeatability and within-laboratory reproducibility ranged from 2% to 22% and from 3% to 23%, respectively. The sample solution was successfully cleaned up to enable quantification using external solvent calibration curves. The limits of quantification (LOQs) were estimated to be 1 µg kg^?1 for all analytes. The method was applied to honey samples commercially available in Tokyo, Japan. Analysis of 250 honey samples indicated that amitraz was present in 127 samples, and that its residual concentration was less than 20 µg kg^?1. Propargite was detected in 23 samples at concentrations less than 1 µg kg^?1.     

Quantitative analysis of physical and chemical measurands in honey by mid-infrared spectrometry

Fourier transform infrared spectroscopy (FT-IR) was used to determine 20 different measurands in honey. The reference values for 144 honey samples of different botanical origin were determined by classical physical and chemical methods. Partial least squares regression was used to develop the calibration models for the measurands studied. They were validated using independent samples and proved satisfying accuracies for the determination of water (R ²=0.99), glucose (0.94), fructose (0.84), sucrose (0.91), melezitose (0.98) and monosaccharide content (0.82) as well as fructose/glucose ratio (0.98), glucose/water ratio (0.94), electrical conductivity (0.98), pH-value (0.87) and free acidity (0.96). The prediction accuracy for hydroxymethylfurfural, proline and the minor sugars maltose, turanose, erlose, trehalose, isomaltose and kojibiose was rather poor. The results demonstrate that mid-infrared spectrometry is a valuable, rapid and non-destructive tool for the quantitative analysis of the most important measurands in honey.     

Determination of the botanical origin of honey by Fourier-transformed infrared spectroscopy: an approach for routine analysis

Fourier-transformed infrared spectroscopy has been established for analysing most of the physical–chemical parameters of honey. Additionally this technique can be used for determination of the botanical origin of a honey sample by comparison of the mid-infrared spectra. In this study calibrations for authentification of the main regional honey types should be developed to be able to measure simultaneously physical–chemical properties and the botanical origin at a minimum of time and at low costs. Honey samples from local beekeepers were collected and characterized by standard methods. The most common honey types were used for calibration. We recorded the mid infrared spectrum from each honey sample. Classification models were achieved by PCA-calibration and validated with samples from various botanical origins. Honey of Brassica spp., Robinia pseudoacacia, Calluna vulgaris, Trifolium spp., Tilia spp., Helianthus annuus, Centaurea cyanus and honeydew honey was used for calibration. Afterwards the calibration models were improved during routine analysis. Most of the honey samples from rape, false acacia, heather and honeydew can be classified correctly by the FTIR in consideration of the physical–chemical and sensorial properties.     

Detection of inverted beet sugar adulteration of honey by FTIR spectroscopy

A combination of Fourier transform infrared (FTIR) spectroscopy and multivariate statistics as a screening tool for the determination of beet medium invert sugar adulteration in three different varieties of honey is discussed. Honey samples with different concentrations of beet invert sugar were scanned using the attenuated total reflectance (ATR) accessory of the Bio‐Rad FTS‐6000 Fourier transform spectrometer. The spectral wavenumber region between 950 and 1500 cm^?1 was selected for partial least squares (PLS) regression to develop calibration models for beet invert sugar determination in honey samples. Results from the PLS (first derivative) models were slightly better than those obtained with other calibration models. Predictive models were also developed to classify beet sugar invert in three different varieties of honey samples using discriminant analysis. Spectral data were compressed using the principal component method, and linear discriminant and canonical variate analyses were used to detect the level of beet invert sugar in honey samples. The best predictive model for adulterated honey samples was achieved with canonical variate analysis, which successfully classified 88–94 per cent of the validation set. The present study demonstrated that Fourier transform infrared spectroscopy could be used for rapid detection of beet invert sugar adulteration in different varieties of honey. © 2001 Society of Chemical Industry     

Pyrrolizidine alkaloids in food: downstream contamination in the food chain caused by honey and pollen

In recent years, there has been a steadily growing number of published data on pyrrolizidine alkaloids (PAs) in honey and pollen. This raises the question whether honey and/or pollen used as ingredients in food processing might provoke a downstream contamination in the food chain. Here we addressed two different facets in connection with PAs in honey and pollen. First, we analysed the PA content of several food types such as mead (n?=?19), candy (n?=?10), fennel honey (n?=?9), soft drinks (n?=?5), power bars and cereals (n?=?7), jelly babies (n?=?3), baby food (n?=?3), supplements (n?=?3) and fruit sauce (n?=?1) that contained honey as an ingredient in the range of 5% to approximately 37%. Eight out of 60 retail samples were tested as being PA-positive, corresponding to 13%. Positive samples were found in mead, candy and fennel honey, and the average PA content was calculated to be 0.10?µg?g^–1 retronecine equivalents (ranging from 0.010 to 0.484?µg?g^–1). Furthermore, we investigated the question whether and how PAs from PA pollen are transferred from pollen into honey. We conducted model experiments with floral pollen of Senecio vernalis and PA free honey and tested the influence of the quantity of PA pollen, contact time and a simulated honey filtration on the final PA content of honey. It could clearly be demonstrated that the PA content of honey was directly proportional to the amount of PA pollen in honey and that the transfer of PAs from pollen to honey was a rather quick process. Consequently, PA pollen represents a major source for the observed PA content in honey. On the other hand, a good portion remains in the pollen. This fraction is not detected by the common analytical methods, but will be ingested, and it represents an unknown amount of ‘hidden’ PAs. In addition, the results showed that a technically and legally possible honey filtration (including the removal of all pollen) would not be an option to reduce the PA level of the final product significantly.     

Exploring stingless bee honey from selected regions of Peninsular Malaysia through gas chromatography–mass spectrometry–based untargeted metabolomics

Volatile organic compounds in honey are known for their considerable impact on the organoleptic properties of honey, such as aroma, flavor, taste, and texture. The type and composition of volatile organic compounds are influenced by entomological, geographical, and botanical origins; thus, these compounds have the potential to be chemical markers. Sixty-two volatile compounds were identified using gas chromatography–mass spectrometry from 30 Heterotrigona itama (H. itama) honey samples from 3 different geographical origins. Hydrocarbons and benzene derivatives were the dominant classes of volatile organic compounds in the samples. Both clustering and discriminant analyses demonstrated a clear separation between samples from distant origins (Kedah and Perak), and the volcano plot supported it. The reliability and predictability of the partial least squares–discriminant analysis model from the discriminant analysis were validated using cross-validation (R²: 0.93; Q²: 0.83; accuracy: 0.97) and the permutation test (p < 0.001), and the output depicted that the model is legitimate. In combination with the variable importance of projection (VIP > 1.0) and the Kruskal–Wallis test (p < 0.01), 19 volatile organic compounds (encompassed aldehydes, benzene derivatives, esters, hydrocarbons, and terpenoids) were sorted and named potent chemical markers in classifying honey samples from three geographical origins. In brief, this study illustrated that volatile organic compounds of stingless honey originated from the same bee species, but different geographical origins could be applied as chemical markers.     

Rheological behaviour and physicochemical properties of kefir with honey

The effect of the flower and pine honey on the rheological behavior of kefir was investigated. Major aim was to produce a new functional fermented milk beverages with honey. Soluble solid was changed from 6.37 to 25.28; refractive index values were between 1.342 and 1.374; pH was changed from 4.04 to 4.65 and color values, L of samples were determined as 63.10–73.18. The rheological properties of the samples were determined using a controlled stress rheometer. It was observed that an empirical power-law model was suitable to describe the rheological behavior of kefir with honey with correlation coefficients (R ²) between 0.991 and 0.96. The apparent viscosity of the kefir with honey decreased with increasing shear rate. Flow behavior index (n) and consistency index (K) values for the kefir with honey ranged from 0.3711 to 0.4691, 130 to 204 mPa s, respectively. The model kefir with honey was found to exhibit non-Newtonian behavior.     

Removing τ‐fluvalinate residues from press‐extracted honey

Honey is obtained from the hives of European honeybees. Honeybees can be infested by Varroa parasitism, which affects honey production. The acaricide τ‐fluvalinate used in beehives to control Varroa mites, can leave its residues in honey, pollen and wax. A monitoring study was undertaken to determine the τ‐fluvalinate residues in honey samples taken from treated hives. The τ‐fluvalinate residues were determined by gas chromatography using a short, non‐polar packed column with electron capture detection. Analysis of 50 samples showed an average residue level of 15 µg kg⁻¹. The same samples were filtered through Whatman filter paper to separate the honey from residual suspended particles. The filtered honeys showed no acaricide residues above the detection limit of 3 µg kg ⁻¹, while the average residue level determined in the sediments was 392 µg kg ⁻¹. Thus the residues of τ‐fluvalinate in honey can be eliminated by separating the honey from residual suspended particles that contain acaricide residues. Copyright © 2003 Society of Chemical Industry     

Comparative study of the physicochemical and palynological characteristics of honey from Melipona subnitida and Apis mellifera

Twenty‐four samples of Apis mellifera honey and twenty‐four samples of Melipona subnitida (Jandaira) honey were collected in the northeast of Brazil. Moisture, hydroxymethylfurfural, free acidity, insoluble solids in water, diastase activity, ashes, electrical conductivity, proteins, lipids, total carbohydrates, energy and sugars were the parameters analysed. The efficiency of the qualitative tests (Fiehe's test, Lugol's reaction, Lund's reaction) was tested. Pollen types and the corresponding plant species were identified in all samples (3 in Apis and 1 in Melipona). Apis mellifera honey samples demonstrated parameters in accordance with the Brazilian Legislation, while the Melipona subnitida honey samples displayed moisture (24.80%) and diastase activity (null) in discordance with the established by the regulation for Apis mellifera honeys. Apis honey samples presented higher values of electric conductivity (284.00 μS cm^?1) than the obtained from the Jandaira honey samples (102.77 μS cm^?1) as well as a darker colour (26.67 mmPfund) when compared with Jandaira honey (7.00 mmPfund). The concentration of the glucose, fructose and sucrose was higher in the Apis honeys than in the Jandaira honey. The characteristics of the two types of honey were very different, highlighting the need of developing specific legislation for stingless bees' honey.     

Invertase activity in fresh and processed honeys

The enzymatic activities of 147 samples of commercially produced Spanish polyfloral and monofloral honeys are reported. Important variations in invertase activity and peroxide accumulation were determined to obtain objective information related to honey quality. Variations from 4.04 (Robinia pseudoacacia) to 25.61 g sucrose hydrolysed per 100 g h⁻¹ (SN) (Castanea sativa) and from 11.31 (Erica vagans) to 45.25 µg H₂O₂ g⁻¹ h⁻¹ (Hedysarum coronarium) were detected, with a range from 10.57 to 46.2 SN in polyfloral honeys. Honeys from Erica spp had different invertase activities—high for E cinerea (18.3 SN) and low for E vagans (8.36 SN). The different heating processes of honey have been evaluated to determine the best treatment for preserving invertase activity. The hydroxymethylfurfural content is not a sufficiently good indicator of the degree of deterioration of honey. Another objective was to assess the use of invertase activity in honey quality legislation. © 2000 Society of Chemical Industry     

Investigating the impact of botanical origin and harvesting period on carbon stable isotope ratio values (13C/12C) and different parameter analysis of Greek unifloral honeys: A chemometric approach for correct botanical discrimination

The aim of this study was to investigate the impact of botanical origin and harvesting period on carbon stable isotope ratio (¹³C/¹²C), colour intensity (CI), radical scavenging activity (%RSA), P and Sn content of Greek unifloral honeys. Thus, twenty‐four honey samples were collected during harvesting periods 2011–2012 and 2012–2013, from four different regions in Greece. ¹³C/¹²C ratios and minerals were determined using isotope ratio mass spectrometry (IRMS) and inductively coupled plasma optical–emission spectroscopy (ICP‐OES), respectively. CI and %RSA were measured using spectrophotometric assays. Results showed that only ¹³C/¹²C values and %RSA were affected by both botanical origin and harvesting period (P < 0.05). Applying then chemometric analyses to the collected data set, honeys were correctly classified according to honey type (correct classification rate 87.5% and 79.2% using the original and cross‐validation method, respectively). The use of different origin parameters has the potential to aid in honey authentication.     

APPLICATION OF WLF AND ARRHENIUS KINETICS TO RHEOLOGY OF SELECTED DARK-COLORED HONEY

The rheological properties of Common Black Horehound, Globe Thistle, and Squill types of dark‐colored Jordanian honey were examined. The types of honey used were identified via assessing the source of nectar using pollen analysis (Melissopalynology). The apparent viscosity, η, was measured as a function of the shear rate, γ. In addition, the apparent viscosity was measured, at constant shear rate (6.12 s^?1), as a function of shearing time. Newton's law of viscosity (i.e., τ=ηγ) was found to adequately (R²～ 0.99) describe the flow behavior of honey samples. The apparent viscosity was found to decrease with temperature, and the temperature dependence of viscosity was contrasted versus both Arrhenius model (η=η_oe^Ea/RT) and WLF model (η/η_G= 10 (C₁(T–T)/C₂+(T–T_G))). Although Arrhenius kinetics may fit the viscosity versus temperature data for the examined types of honey, nevertheless, it gives a relatively high value of activation energy that is quite comparable with, if not even larger than, that of a typical chemical reaction. On the other hand, WLF‐model was found to adequately describe the data while at the same time it gives quite reasonable values of both T_G and η_G, which are in agreement with those cited in literature.     

Characterisation of the phenolic and flavonoid fractions and antioxidant power of Italian honeys of different botanical origin

BACKGROUND: Twenty‐seven Italian honey samples of different floral origin were analysed for total phenolic and flavonoid contents by a spectrophotometric method and for antioxidant power and radical‐scavenging activity by the ferric‐reducing/antioxidant power (FRAP) and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) assays respectively. In addition, the phenolic and flavonoid profiles were analysed using high‐performance liquid chromatography with UV detection (HPLC‐UV). RESULTS: The results of this study showed that honey contains copious amounts of phenolics and flavonoids. HPLC‐UV analysis showed a similar qualitative polyphenolic profile for all honey samples analysed. The main difference among samples was in the contribution of individual analytes, which was affected by floral origin. Total phenolic and flavonoid contents varied from 60.50 to 276.04 mg gallic acid equivalent kg^?1 and from 41.88 to 211.68 mg quercetin equivalent kg^?1 respectively. The antioxidant capacity was high and differed widely among samples. The FRAP value varied from 1.265 to 4.396 mmol Fe²⁺ kg^?1, while the radical‐scavenging activity expressed as DPPH‐IC₅₀ varied from 7.08 to 64.09 mg mL^?1. Correlations between the parameters analysed were found to be statistically significant (P < 0.05). CONCLUSION: The present study shows that honey contains high levels of phenolics and flavonoids and that the distribution of these compounds is influenced by the honey's floral origin. Copyright © 2009 Society of Chemical Industry     

Compositional analysis and antimicrobial activity of various honey types of Pakistan

Antibacterial activities of various honey samples were assessed by using clinical isolates like S. aureus (Gram‐positive), E. coli, P. aeruginosa and K. pneumonia (Gram‐negative). It was observed that acacia and citrus honey has inhibited growth of all bacterial strains as compared to standard antibiotics (Gentamicine). Inhibition zones for S. aureus (27.4 ± 0.5 mm), E. coli (26.5 ± 0.7 mm), K. pneumonia (24.4 ± 0.5 mm) and P. aeruginosa (22.4 ± 0.2 were observed. Minimum inhibitory concentration of S. aureu (0.068 mg mL^?1), E. coli (0.072 mg mL^?1), P. aeruginosa (0.084 mg mL^?1) and K. pneumonia (0.085 mg mL^?1) was obtained for various types of honey samples. Pronounced antibacterial activities as well as standard values of various quality parameters of honey samples are scientifically proven for its use in traditional medicine since ancient time throughout the world.     

Honey major protein characterization and its application to adulteration detection

The major proteins in honey have different molecular weights depending upon the honeybee species. To confirm the origin of major honey proteins, honey protein produced by Apis cerana or Apis mellifera were purified and analyzed by MALDI-TOF. Two major proteins were identified as a major royal jelly protein 1. Although two major proteins shared primary structure, they showed different molecular weights of 56 and 59 kDa, respectively. To discriminate the honeybee species producing honey using SDS–PAGE, artificial marker proteins, 56 and 59 kDa, were produced from Escherichia coli. Two artificial marker proteins were co-electrophoresed with honey samples and the difference in molecular weight was readily distinguished by SDS–PAGE. Therefore, the measurement of major proteins in honey is a useful method to discriminate the honey that produced from different honeybee species.     

SPECIFIC HEAT CAPACITY OF AUSTRALIAN HONEYS FROM 35 TO 165C AS A FUNCTION OF COMPOSITION USING DIFFERENTIAL SCANNING CALORIMETRY

Modulated temperature differential scanning calorimetry was used to investigate the specific heat capacity (C _p ) of 10 Australian honeys within the processing and handling temperatures. The values obtained were found to be different from the literature values at certain temperatures, and are not predictable by the additive model. The C _p of each honey exhibited a cubic relationship (P < 0.001) with the temperature (T, C). In addition, the moisture (M, %), fructose (F, %) and glucose (G, %) contents of the honeys influenced their C_p. The following equation (r² = 0.92) was proposed for estimating C_p of honey, and is recommended for use in the honey industry and in research:      

Investigation of aromatic compounds in Alicante and Jijona <Emphasis Type="Italic">turrón</Emphasis>

Alicante and Jijona turrón are honey- and almond-based confectionery products very famous in Spanish countries, especially during Christmas. In this study, the role of different types of honey (rosemary, orange blossom, and control—a mixture of sugars resembling the sugars composition of real honey) in the aroma and flavor of these confections was studied. Volatile compounds in turrón samples were extracted using simultaneous distillation–extraction and finally isolated, quantified and identified using GC-MS. Two different types of panels were used, trained and consumer, for studying the aroma and flavor intensities of turrón samples. Aldehydes, aromatic hydrocarbons and furans were the predominant chemical groups in turrón samples. The first conclusion was that terpenes and aromatic hydrocarbons decreased with the heating of the boixet step while pyrazines, furans, and pyrroles increased. Chemical feelings, such as cooling and pungency, are the best tool to discriminate among products containing and non-containing honey. Products containing honey were more aromatic and obtained higher satisfaction values than those prepared from a mixture of sugars.     

Determination of Rheological Behavior,Emulsion Stability,Color, and Sensory of Sesame Pastes (Tahin) Blended with Pine Honey

In this research, rheological properties of blends of pine honey (3%, 6%, and 9%) with sesame pastes (tahin) produced from hulled roasted sesame seeds, called simply tahin, and from unhulled roasted sesame seeds, so-called Bozkir tahin, were determined at temperatures ranging from 10°C to 60°C and at speeds ranging from 0.5 to 100 rpm. Tahin and Bozkir tahin blends with pine honey were found to exhibit non-Newtonian, pseudoplastic behavior at all temperatures. Apparent viscosities versus speed data were successfully fitted to the power law model. The flow behavior index, n, varied in the range of 0.4226–0.6228 for the tahin–pine honey blends, and in the range of 0.4661 to −0.7266 for the Bozkir tahin–pine honey blends. The consistency index, K, was in the range of 9.34–36.42 Pa·sⁿ for tahin–honey blends, and in the range of 9.92–37.53 Pa·sⁿ for Bozkir tahin–honey blends. The consistency index (K) increased with increasing honey levels in both tahin types. According to statistical analysis, the exponential model was a better model to describe the effect of the soluble solids on the viscosity of tahin samples represented by the pine honey percentage. The emulsion stability of both tahin types improved with the addition of pine honey. It was also correlated with activation energy (E _a), Arrhenius constant, some sensory properties such as spreadibility, firmness and overall acceptance, and color parameters such as the C and h. Temperature sensitivity of the consistency index was assessed by applying an Arrhenius type equation, and E _a value appeared in the range of 7.61–10.05 kJ/mol for tahin–honey blends and in the range of 9.02–10.50 kJ/mol for Bozkir tahin–honey blends.