Automatic NMR-based protocol for assessment of honey authenticity.

1H NMR analysis of organic extracts of honey is a powerful technique to confirm its botanical origin, thanks to the presence of signals that are specific to each floral typology. Similarly, signals from bee metabolites provide an important tool to verify honey entomological origin. Here, we present a method for honey screening that does not require any detailed analysis of the NMR spectrum for the detection and quantification of such markers. Our approach is based on the measurement of two spectral parameters, named entomological factor (EF) and aromatic factor (AF), calculated by integration of well-defined regions of the NMR spectrum. The values of EF and AF can reveal direct or indirect dilution of honey with sugar syrups. This method was tested on honeys of different floral origins and could identify all adulterated samples previously recognized by official techniques. Notably, several samples found compliant by official methods were proven non-genuine by the proposed approach.

Sensory and physicochemical analysis of honeys of nine stingless bee species of Mexico and Guatemala.

Honeys produced by species other than Apis mellifera are little known. The objective of this study was to carry out sensory and physicochemical analyses of 62 samples to characterize honeys from nine species of stingless bees from Mexico and Guatemala. Analyses were performed at the sensory level and at the physicochemical level. Qualitative and quantitative data were studied using the multivariate non-metric multidimensional scaling (NMDS) statistical technique, with the Gower metric. According to the stress value (p > 0.2), the most significant parameters were at the sensory level, for visual evaluation, physical state, color, and appearance; for olfactory-taste evaluation, elemental sweet and acid tastes; for tactile evaluation, consistency, and crystallization, and for physicochemical evaluation, pH, electrical conductivity and reducing sugars. Data analysis showed that Melipona beecheii honeys formed a defined group, with similar sensory characteristics and physicochemical values, even taking into account their place of origin. Frieseomelitta nigra honeys also showed significant differences with the other honeys analyzed. Our work suggests that the entomological origin is the main factor that determines the characteristics of honey, and that the floral origin is the secondary factor, a hypothesis that should be verified in future works.

Impact of Different Storage Temperature on the Enzymatic Activity of Apis mellifera Royal Jelly.

Royal Jelly is a nutrient secretion of nurse bees and a high interest functional food in human nutrition. Very little information is available on its chemical composition integrity and enzymatic activity during shelf life and assessment of new freshness markers are desirable for its conservation. In this study, the activity of glucose oxidase, five proteases and two antioxidant enzymes in refrigerated and frozen Royal Jelly for different storage times was preliminary investigated. Refrigeration determined a significantly reduction in glucose oxidase and carboxypeptidase A-like activity in Royal Jelly after one year of storage while no differences were recorded in the activity of these enzymes in frozen samples. After one year of storage glucose oxidase and carboxypeptidase A-like activity resulted higher in frozen samples frozen than in refrigerate ones. Results obtained suggest that the activities of these enzymes may be good markers of Royal Jelly freshness within 1 year at refrigeration condition. Freezing could be a valid alternative storage method to ensure a higher preservation of glucose oxidase and carboxypeptidase A-like activities for at least 1 year. Further investigation to determine the timing of glucose oxidase inactivation/degradation under refrigerated conditions and the enzymatic activity trend under prolonged frozen conditions are desirable.

Floral origin modulates the content of a lipid marker in Apis mellifera honey.

The compound DAGE (DiAcyl Glyceryl Ether, 1-stearyl-2,3-dioleoyl glycerol), present in Apis mellifera honey, is a lipidic entomological marker secreted by the salivary glands of worker bees. Its content was determined by NMR, analyzing the organic extracts of a number of Italian honeys of different floral typology. We have found that the DAGE content is related to the botanical origin of honey. This dependence on floral typology was further confirmed by a linear correlation (R2 > 0.83) observed between the content of DAGE and the enzymatic activity of invertase and diastase in honey. Also these enzymes originate from bee salivary secretions and their concentrations in honey are known to depend on the floral source. DAGE content appears to be a sensitive parameter to some forms of honey manipulations, as indicated by the results of artificial bee-feeding experiments. This suggests its possible use as indicator of honey authenticity.

NMR carbohydrate profile in tracing acacia honey authenticity.

The sugar profile in honey can be used as a fingerprint to confirm the authenticity or reveal the adulteration of the product by sweetener addition. In this work, we have accurately determined the profile of 20 minor saccharides in a set of 46 European acacia honeys using a recently proposed NMR approach based on the CSSF-TOCSY experiment. Comparison of this reference profile with the sugar composition of several Chinese honey samples of the same declared botanical origin has revealed important differences. A detailed analysis of the saccharide profile of these Chinese honeys suggests product adulteration by overfeeding bee colonies with industrial sugars syrups during the main nectar flow period.

Investigation of water state during induced crystallization of honey.

This work studied water state of honey during crystallization, obtained statically and dynamically, by differential scanning calorimetry (DSC), water activity (aw) assessment and time domain nuclear magnetic resonance (TD-NMR). Crystallization was induced by adding 5% of crystallized honey to three honey samples with different fructose/glucose ratio, the key characteristic for honey crystallization. Samples were stored at 14 °C. Dynamic crystallization was obtained by using an impeller. DSC showed that the dynamic crystallization was faster than the static one, the latter characterized by two phases, showing different rates. The crystallization rate did not affect aw, that remained below 0.600. TD-NMR allowed to separately observe two kinds of protons, both pertaining to liquid sugars, one chemically exchanging with water and one not exchanging with it. The combination of techniques allowed speculating that the two crystallization methods led to crystals of different size and shape.

NMR assessment of European acacia honey origin and composition of EU-blend based on geographical floral markers.

A geographical discrimination of honey is an important prerequisite for quality and authenticity control. Here, we present a method based on an NMR-metabolomic analysis of organic extracts for a geographical discrimination of commercial European acacia honeys found on the Italian market. All 234 analysed samples show the characteristic 1H NMR profile of acacia (Robinia pseudoacacia) honey. However, a PLS2-DA model revealed variations among production zones allowing the successful geographical differentiation with a 100% of overall correct classification rate. Moreover, a PLS2 model is able to predict the proportions in binary blends of Italian and Eastern European acacia honeys. The geographical distinction and the possibility to characterize the content of blends derive from different markers content originating from minor nectar contributions of the acacia-accompanying flora.

Objective Definition of Monofloral and Polyfloral Honeys Based on NMR Metabolomic Profiling.

In this paper, a remarkably precise, simple, and objective definition of monofloral and polyfloral honey based on NMR metabolomics is proposed. The spectra of organic extracts of 983 samples of 16 botanical origins were used to derive one-versus-all OPLS-DA classification models. The predictive components of the statistical models reveal not only the principal but also the secondary floral origins present in a sample of honey, a novel feature with respect to the methods present in the literature that are able to confirm the authenticity of monofloral honeys but not to characterize a mixture of honey types. This result descends from the peculiar features of the chloroform spectra that show diagnostic resonances for almost each botanical origin, making these NMR spectra suitable fingerprints. The reliability of the method was tested with an additional 120 samples, and the class assignments were compared with those obtained by traditional analysis. The two approaches are in excellent agreement in identifying the floral species present in honeys and in the botanical classification. Therefore, this NMR method may prove to be a valid solution to the huge limitations of traditional classification, which is very demanding and complex.