Novel quality control methods in conjunction with chemometrics (multivariate analysis) for detecting honey authenticity

The importance of honey has been recently upgraded because of its nutrient and therapeutic effect. The adulteration of honey increased exponentially in terms of both geographic and/or botanical origin. Therefore, the need has arisen for more effective quality control methods aiming at detecting adulteration. Various novel, fast, and accurate methods like AAS, HPLC, GC-MS, ES-MS, TLC, HPAED-PAD, NMR, FT-Raman, and NIR have enriched the arsenal of analytical chemist in this direction. However, apart from these novel methods, the application of multivariate analysis and, in particular, PCA, CLA, and CA, proved to be extremely useful for grouping and detecting honey of various origins. Mineral and trace element analysis were repeatedly shown to be a very effective means for the classification purposes of honey of various origins (geographical and botanical).     

Classification of Honey According to Geographical and Botanical Origins and Detection of Its Adulteration Using Voltammetric Electronic Tongue

Main possible honey fraud is the addition of various sugar syrups. But, there are also other types of fraud, such as deception on the geographical and/or botanical origin product. Providing a product of the hive with full authenticity is therefore crucial for the preservation of beekeeping. In this pursuit, voltammetric electronic tongue (VE-tongue) was employed to classify honey samples from different geographical and botanical origins. Furthermore, VE-tongue was used to detect adulterants such as glucose syrup (GS) and saccharose syrup (SS) in honey. The data obtained were analyzed by three-pattern recognition techniques: principal component analysis (PCA), support vector machines (SVMs), and hierarchical cluster analysis (HCA). These methods enabled the classification of 18 honeys of different geographical origins and 7 honeys of different botanical origins. Excellent results were obtained also in the detection of adulterated honey. Therefore, this simple method based on VE-tongue could be useful in the honey packaging and commercialization industry.     

A review of the analytical methods to determine the geographical and botanical origin of honey

This review is concerned with analytical methods to prove the authenticity of honey. A special emphasis is put on suitable methods for the detection of the geographical and botanical origin of honey. Whereas the determination of some single parameters, such as 5-hydroxymethylfurfural (HMF), moisture, enzyme activity, nitrogen, mono- and disaccharides, and residues from medicinal treatment or pesticides in honey does not lead to any information about the botanical and geographical origin, there are some suitable methods based on the analysis of specific components or on multi-component analysis. Mostly, such methods give indications of the botanical origin, investigating flavonoids patterns, distribution of pollen, aroma compounds and special marker compounds. There are some other profiles of components which could probably be used for the detection of the geographical origin (e.g. oligosaccharides, amino acids, trace elements). In particular, the combination of methods could be a promising approach to prove authenticity, especially when modern statistical data evaluation techniques will be applied.     

Characterization of Spanish honeys with protected designation of origin "Miel de Granada" according to their mineral content

Honey attributes such as geographical origin or specified botanical sources often command a premium price due to their organoleptic or pharmacoactive properties. "Miel de Granada" is a highly quality product with protected designation of origin (PDO) which includes six monofloral honeys and two multifloral honeys. Our objective was the characterization of "Miel de Granada" according to their metal content. Metal content was specific enough and allowed discrimination from honeys of different botanical and geographical origins and confirmed the authenticity of PDO labelling as Granada product with the determination of only five elements (K, Na, Ca, Mg and Zn). Chemometric techniques as cluster analysis and ANOVA were used to classify honeys according to their botanical and geographical origin in the metal data. Metal content marks the differences in honey samples and can be used as a tool to assess the quality of honeys. ANOVA showed significant differences among rosemary honeys from different geographical areas despite the botanical factor weight. Our research contributes to the groundwork studies to determine the geographical origin of Spanish honeys.     

Honey—its characteristics,sensory aspects,and applications

Abstract

Honey is a natural, sweet, syrupy fluid collected by bees from nectar of flowers. The pleasant aroma and taste of this viscous liquid ranging in color from pale yellow to dark amber varies according to geographical and seasonal conditions. Its use as a sweetener is well known in different parts of the world. In early history, honey occupied a very important place on religious occasions. The high content of sugars, small amounts of amino acids, lipids, along with some vitamins and minerals imparts its high nutritional value. Honey has good medicinal and antimicrobial properties and is used in different cuisines. Proper processing is essential for a product of good sensory qualities. ISI/Agmark specifications for honey, its adulteration and detection of adulteration are well recorded. The application potential in bakery, confectionery, snack foods, fruit and vegetable products and beverages is ever increasing. A bird's‐eye view of these aspects along with possible lines of future research are discussed.     

Phenolic acids and abscisic acid in Australian Eucalyptus honeys and their potential for floral authentication

Seven phenolic acids related to the botanical origins of nine monofloral Eucalyptus honeys from Australia, along with two abscisic isomers, have been analyzed. The mean content of total phenolic acids ranges from 2.14 mg/100 g honey of black box (Eucalyptus largiflorens) honey to 10.3 mg/100 g honey of bloodwood (Eucalyptus intermedia) honey, confirming an early finding that species-specific differences of phytochemical compositions occur quantitatively among these Eucalyptus honeys. A common profile of phenolic acids, comprising gallic, chlorogenic, coumaric and caffeic acids, can be found in all the Eucalyptus honeys, which could be floral markers for Australian Eucalyptus honeys. Thus, the analysis of phenolic acids could also be used as an objective method for the authentication of botanical origin of Eucalyptus honeys. Moreover, all the honey samples analyzed in this study contain gallic acid as the main phenolic acid, except for stringybox (Eucalyptus globoidia) honey which has ellagic acid as the main phenolic acid. This result indicates that the species-specific differences can also be found in the honey profiles of phenolic acids. Furthermore, the analysis of abscisic acid in honey shows that the content of abscisic acid varies from 0.55 mg/100 g honey of black box honey to 4.68 mg/100 g honey of bloodwood honey, corresponding to the contents of phenolic acids measured in these honeys. These results have further revealed that the HPLC analysis of honey phytochemical constituents could be used individually and/or jointly for the authentication of the botanical origins of Australian Eucalyptus honeys.     

Flavonoids in Meliponinae honeys from Venezuela related to their botanical, geographical and entomological origin to assess their putative anticataract activity

 Honey was collected from 24 stinglessbee nests in Venezuela. The flavonoid compounds in the phenolic extracts were analysed and related to the botanical, geographical and entomological origin of the honey. Honeys produced in savannas were richer in flavonoids compared with honeys from the forests. It was found that phenolic extracts of honey of the same geographical origin had similar flavonoid profiles, whereas the same could not be said of honeys of the same entomological origin, although the botanical origin of the samples was variable. It is proposed that analysis of stinglessbee honey eyedrops in terms of their flavonoid content can be used as a basis of authenticating and controlling for their geographical origin. Received: 28 July 1997     

Identification of botanical biomarkers found in Corsican honey

Honeys from specified botanical sources often command a premium price due to their organoleptic or pharmacoactive properties. To prevent the fraudulent marketing of honey, analytical techniques are required to confirm its origin.     

Riboflavin and lumichrome in Dalmatian sage honey and other unifloral honeys determined by LC-DAD technique

Riboflavin (vitamin B(2)) and its metabolite lumichrome were quantified in 117 samples from 11 unifloral honeys types (Arbutus unedo L., Asphodelus microcarpus Salzm. et Viv., Citrus spp., Eucalyptus spp., Hedysarum coronarium L., Castanea sativa L. honeydew, Mentha spp., Paliurus spina-christi., Salix spp., Salvia officinalis L., Satureja spp.). The quantification of these two compounds was performed by LC-DAD method which does not require sample purification. The proposed method in our study has low limits of detection and quantification, very good linearity in a large concentration range and very good precision. It allows simultaneous determination of 5-hydroxymethylfurfural (HMF) and known chemical biomarkers of unifloral honeys such as abscisic acid diastereomers, homogentisic acid, methyl syringate and kynurenic acid. No statistical correlation was observed between riboflavin and lumichrome content. Although, the concentration of vitamin B(2) in honey may be too low (<6.1mg/kg) to generate interest in the field of nutrition, the presence of its main metabolite lumichrome may be useful to determine the botanical origin of certain unifloral honeys. In fact, the analysis of 11 unifloral honey types showed that Dalmatian sage (S. officinalis L.) honey is characterised by unusual high levels of lumichrome (20.2±2.6mg/kg). The botanical origin of lumichrome from sage flower was assessed by analysing bee-stomach extracts. Other analytical parameters, such as total phenols, antioxidant and antiradical activities, HMF and diastase activity were studied in Dalmatian sage honey.     

Characterization of Croatian Honeys by Right-Angle Fluorescence Spectroscopy and Chemometrics

The potential of right-angle fluorescence spectroscopy and selected chemical parameters for discrimination of botanical origin of Croatian honey types (n = 55) previously classified by physicochemical and melissopalynological analyses was evaluated. Systematic step-by-step fluorescence analysis included the measurement of complete excitation-emission matrix (EEM) in the range of excitation wavelengths from 260 to 400 nm, and emission wavelengths from 300 to 600 nm, followed by fluorescence intensity measurement at detected peaks of different excitation/emission wavelengths, and emission spectra recordings at selected specific excitation wavelengths of honey solutions in 50 mmol L^?1 phosphate buffer pH 7.0 and methanol. A total of five different sets of emission spectral data for buffer and two for methanolic honey solutions were considered for chemometric analysis of original and normalized emission spectra including principal component analysis and linear discriminant analysis. Additionally, chemical analysis of buffer and methanolic honey solutions included determination of protein, total polyphenol and reactive amino group content. Results showed that right-angle fluorescence spectroscopy of honey solutions has great potential for honey botanical origin discrimination, either by visual comparison of excitation-emission spectra landscapes, or even better, by normalized emission fluorescence spectra recordings at 260 and 280 nm of excitation. Moreover, increased honey discrimination was achieved in conjunction with chemometrics of fluorescence spectras. In addition, two rapid markers/indicators of honey authentication were found. Chestnut honey could be clearly discriminated from the other honey types by simple measurement of fluorescence intensity at 390/470 nm, while sage honeys by measurement of reactive amino group content.     

Different Aspects of the Elemental Analysis of Honey by Flame Atomic Absorption and Emission Spectrometry: A Review

The elemental composition of honey is correlated with the botanical provenience of nectar, pollen, and honeydew that are collected and ripened by bees. In addition to this, the geographical origin related to the locality of an apiary, the soil composition, and climatic conditions may contribute to the origin of elements in honey. The environmental pollution or other anthropogenic processes and activities also have an effect on the quality and the safety of honey since they may be accompanying sources that lead to its contamination and the presence of various trace elements (Cd, Cr, Cu, Fe, Ni, Pb, and Zn). This review article covers the literature devoted to the analysis of honey carried out by the most popular and commonly utilized flame atomic absorption and emission spectrometry, which was published from 1999 to 2011. Various aspects of such analysis are treated in detail, including ways of the sample preparation, the calibration, and the quality assurance of results. In addition, methods and results related to the fractionation analysis of elements in honey by means of chromatographic and non-chromatographic approaches are described.     

Characterization of honeys by their botanical and geographical origins based on physico-chemical properties and chemo-metrics analysis

Honey, because of its nutritional and medicinal values, is in high demand and has become one of the important commodities. However, the issue of its quality and authenticity remain as important factors in consumption and marketing of honey. To assess the possibility of discriminating honeys by their geographical and botanical origins; 30 fresh honey samples of different botanical and geographical origins were collected and their major physico-chemical properties such as: total dissolved sugar (TDS), total ash, sugar profile, acidity, metallic ions and electric conductivity (EC) were investigated. The data was subjected to different chemo-metric (Hierarchical Cluster, Principal components and stepwise discriminant) analysis. Among the 23 characters used in the analysis; only 11 (TDS, EC, acidity, total ash, colour, and some specific metallic ions) characters have showed significant variations among different origin honeys. According to the stepwise discriminant analysis; 11 variables confirmed the grouping of the honey samples into four cluster groups based on their botanical and geographical origins. The clustering of the honeys associated with dominant plant source & climatic conditions of their origins. The study generally revealed the successful discrimination of honeys into their botanical and geographical provenances using fewer physico-chemical characters supported with melissopalynological data through applying suitable chemo-metric analysis.     

油菜蜂蜜中挥发性成分指纹图谱的建立

分析油菜单花蜜中的挥发性成分,建立油菜单花蜜挥发性成分的指纹图谱。采用超声波辅助萃取法提取49种不同产地的油菜单花蜜中的挥发性物质,并利用气相-氢火焰化离子检测(gas chromatography-flame ionization detector,GC-FID)和气相色谱-质谱联用(gas chromatography-mass spectrometry,GC-MS)技术分析,对气相分析结果进行聚类分析和相似度分析。结果表明：49种油菜蜜分为5类,能明显区分未经加工的原料油菜蜜和市售油菜蜜;原料油菜蜜和市售油菜蜜的挥发性成分存在明显差异;推断苯甲酸-4-羟基-3,5-二甲氧基-酰肼可能作为油菜蜜植物源标记物。     

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.     

稳定同位素技术在蜂蜜真实性溯源中的研究进展

蜂蜜作为一种纯天然的食品,因丰富的营养价值以及良好的保健功效而深受消费者喜爱。然而,随着近年来蜂蜜消费量的不断增多,蜂蜜掺假、以次充好等现象日益严重。因此,蜂蜜的真实性溯源研究也变得更为迫切。稳定同位素技术是食品真实性溯源的有效的技术之一,在蜂蜜的真实性溯源研究方面也有较多成果。本文综述了稳定同位素技术在蜂蜜掺假、产地鉴别、品种识别方面的研究进展并分析该技术的优点与不足,为进一步推进蜂蜜的真实性溯源研究提供参考。     

Factors contributing to the variation in the volatile composition of chocolate: Botanical and geographical origins of the cocoa beans,and brand-related formulation and processing

The intrinsic characteristics of chocolate and the complex technological process complicate the assessment of the typical features of this product and the verification of its authenticity. In this study, the influence of the botanical and geographical origin of the cocoa beans, as well as the impact of brand-related processing on the volatile organic compound (VOC) composition of the resulting chocolates was examined. A total of ninety dark chocolates available on the Dutch market were analysed using Proton-Transfer-Reaction-Mass Spectrometry (PTR-MS). The VOC profiles generated by PTR-MS (136 masses per sample) were used as fingerprints and investigated using chemometric tools to elucidate information on production factors of cocoa and subsequent processing, in the finished product. Principal component analysis (PCA) showed some clustering of the chocolates according to the botanical and geographical origins of beans as well as according to the brand. Partial least square discriminant analysis (PLS-DA) further discriminated the samples according to the three classes (botanical origin, geographical origin, brand) and the models with the best classification results were used to investigate the relevant masses for each class. PCA clustering and PLS-DA classification highlighted that chocolate profiles are strongly affected by the processing applied by the different brands. However, reflection of the botanical and geographical origins of the beans was also mirrored in the VOC composition of the chocolates. PTR-Time of Flight-MS (PTR-ToF-MS) was used to tentatively identify the VOCs of the chocolates. These measurements allowed the identification of 36 spectrometric peaks which relate to the main classes of chocolate odorant compounds, in particular, aldehydes and pyrazines, products of Maillard reactions. Several compounds already present in unroasted beans were tentatively identified in the chocolates as well, such as, acetic acid, methylpropanoic acid, 2- and 3-methylbutanoic acid, 2-phenylethanol, and tetramethylpyrazine. The results of this study emphasize the impact of the brand-related formulation and processing on VOC profiles of dark chocolates. However, using chemometrics, VOC reflection of the botanical origin and geographical origin of the beans in the chocolates was revealed, which may be useful for a future cocoa/chocolate traceability.     

Botanical discrimination and classification of honey samples applying gas chromatography/mass spectrometry fingerprinting of headspace volatile compounds

A validated method for the discrimination and classification of honey samples performing GC/MS fingerprinting of headspace volatile compounds was developed. Combined mass spectra of honey samples originated from different plants and geographical regions of Greece were subjected to orthogonal partial least squares-discriminant analysis™ (OPLS™-DA), soft independent modelling of class analogy (SIMCA), and OPLS™-hierarchical cluster analysis (OPLS™-HCA). Analyses revealed an excellent separation between honey samples according to their botanical origin with the percentage of misclassification to be as low as 1.3% applying OPLS™-HCA. Fragments (m/z) responsible for the observed separation were assigned to phenolic, terpenoid, and aliphatic compounds present in the headspace of unifloral honeys. On the other hand, a variable classification for citrus and thyme honeys according to their geographical origin could be achieved. Results suggested that the developed methodology is robust and reliable for the botanical classification of honey samples, and the study of differences in their chemical composition.     

Fungal Honeydew Elements as Potential Indicators of the Botanical and Geographical Origin of Honeys

The present study applied indicator value analysis as a novel approach to estimate honey authenticity: microscopic indicators of honey origin were identified based on the distinct fungal content of honeys from different sources. The abundance and the IndVal index of 34 selected fungal spore types were quantified in 82 melissopalynological honey samples originating from various honeydew (Pinus, Abies) or nectar sources using multivariate statistical approaches. A dissimilarity matrix of honey samples was obtained by computing Bray-Curtis coefficients, and the distances were visualized using non-metric multidimensional scaling. K-means clustering was applied to sample coordinates to create a classification based on the frequency of selected fungal taxa. Strikingly, the resulting clusters were on a high level of agreement with the melissopalynological or geographical classification of samples. Various fungal taxa were shown to characterize groups of honey samples with a significant indicator value: floral honeys (Metschnikowia reukaufii), Pinus honeydew honeys (Capnobotrys sp., Antennatula sp.), Abies honeydew honeys from Greece (staurospore and scolecospore types) and honeydew honeys from Italy (Tripospermum spp. and Excipularia fusispora). Having revealed that the mere presence of distinct fungal taxa can indicate differences in the botanical and geographical source of honeys, the present findings encourage the confirmation of honey origin also by recording the occurrence of given honeydew elements during routine melissopalynological analysis.     

Combined use of HMF and furosine to assess fresh honey quality

BACKGROUND: The quality of honey can be affected by practices such as adulteration, inadequate storage or the application of severe heat treatments. Because hydroxymehylfurfural (HMF) is an indicator of honey freshness and furosine (ε‐2‐furoylmethyl lysine) has proved to be a useful chemical indicator of the progress of the Maillard reaction in foods, the aim of this work was to assess their usefulness as indicators of fresh honey quality. The effect of heat treatment, storage and adulteration with different types of syrups on HMF and furosine content has been studied. RESULTS: In fresh honey, HMF and furosine values ranged from 0.9 to 14.6 mg kg^?1 of product and from 3.06 to 12.06 g kg^?1 of protein, respectively. Heating of honey samples with different pH (3.76 and 5.14) produced slight increases in HMF content and negligible changes were detected in furosine values. The storage of fresh honey for 2 years caused a high increase in the HMF level, reaching values above EU limits. However, furosine showed a different behavior depending on the type of honey sample. Adulteration assays using different syrups produced an increase in HMF and a decrease of furosine values by dilution effect. HMF content of adulterated honey samples with syrup of known origin did not exceed EU limits. CONCLUSION: These results show the influence of long periods of storage or adulteration, using different percentages of corn or invert sugar syrups, on HMF and furosine content of fresh honey. This seems to indicate that the combination of HMF and furosine may be useful for evaluating the quality of fresh honey. Copyright © 2009 Society of Chemical Industry     

Trends in Chemometrics: Food Authentication,Microbiology, and Effects of Processing

In the last decade, the use of multivariate statistical techniques developed for analytical chemistry has been adopted widely in food science and technology. Usually, chemometrics is applied when there is a large and complex dataset, in terms of sample numbers, types, and responses. The results are used for authentication of geographical origin, farming systems, or even to trace adulteration of high value‐added commodities. In this article, we provide an extensive practical and pragmatic overview on the use of the main chemometrics tools in food science studies, focusing on the effects of process variables on chemical composition and on the authentication of foods based on chemical markers. Pattern recognition methods, such as principal component analysis and cluster analysis, have been used to associate the level of bioactive components with in vitro functional properties, although supervised multivariate statistical methods have been used for authentication purposes. Overall, chemometrics is a useful aid when extensive, multiple, and complex real‐life problems need to be addressed in a multifactorial and holistic context. Undoubtedly, chemometrics should be used by governmental bodies and industries that need to monitor the quality of foods, raw materials, and processes when high‐dimensional data are available. We have focused on practical examples and listed the pros and cons of the most used chemometric tools to help the user choose the most appropriate statistical approach for analysis of complex and multivariate data.