Due to its multifunctional and complex role in the honey bee colony functioning and health (construction material allowing food storage, brood rearing, thermoregulation, mediation in chemical and mechanical communication, substrate for pathogens, toxins and waste), Apis mellifera beeswax has been widely studied over the last five decades. This is supported by a comprehensive set of scientific reports covering different aspects of beeswax research. In this article, we present an overview of the methods for studying chemical, biological, constructional, and quality aspects of beeswax. We provide a detailed description of the methods for investigating wax scales, comb construction and growth pattern, cell properties, chemical composition of beeswax using different analytical tools, as well as the analytical procedures for provenancing beeswax and beeswax-derived compounds based on the hydrogen isotope ratio (IRMS). Along with classical physico-chemical and sensory analysis, we describe more precise and accurate methods for detection of adulterants in beeswax (GC-MS and FTIR-ATR). Moreover, we present methods for studying the influence of beeswax (comb foundation) adulteration on comb construction. Analytical protocols for determining the pesticide residues using different chromatographic and spectroscopic techniques are also described. As beeswax is an agent of high risk for the transmission of bee diseases, we present methods for detection of pathogens in beeswax. To ensure the reproducibility of experiments and results, we present best practice approaches and detailed protocols for all methods described, as well as their advantages and disadvantages.

The present study was conducted to evaluate the quality and bio-functional properties of Portuguese honeys of different botanical and geographical origins. Quality parameter analyses included the determination of palynological (predominant, secondary, minor and isolated pollen percentage), physicochemical (°Brix, moisture content, pH, electrical conductivity, free acidity, total dissolved solids, salinity, vitamin C content and specific weight) including colour-metrics (CIELAB, Pfund and colour intensity determinations), along with volatile compounds identification using solid phase micro-extraction coupled to gas chromatography mass spectrometry. Bio-activity parameter analysis included the determination of in vitro antioxidant activity and total phenolic content using the 2,2-diphenyl-1-picryl-hydrazyl and Folin-Ciocalteu assays, respectively. Melissopalynological analysis showed that Portuguese honeys were classified as eucalyptus, chestnut and heather, recording significant variations (p < 0.05) among physicochemical, volatile and bio-activity parameter analyses according to both: botanical and geographical origin. Based on the multi-parameter analysis data Portuguese honeys could be characterized by a distinctive colour, a characteristic aroma, whereas conform to the European legislation relating to honey identity and quality. Specific attention should be given in the case of heather honey which showed the highest in vitro antioxidant activity and total phenolic content. Parameters that were also highly correlated using bivariate statistics.

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Botanical origin, physicochemical properties (ash, colour, diastase activity, electrical conductivity (EC), hydroxymethylfurfural (HMF), moisture, optical rotation (OP), pH, reducing sugars, total acidity, total soluble solids, and water activity), bioactive compounds (BC), and antioxidant activity obtained from strawberry tree honey from South Portugal were investigated. Results showed that the pollen analysis and physicochemical parameters were found within to meet international honey specifications. Significant differences (P<0.05) in results of ash content, EC, HMF, OP and colour when were compared with analogous famous Italian honey (Sardinia island). For BC, total phenolic and total flavonoid content were 94.47 mg gallic acid/100 g and 5.33 mg quercetin/100 g, respectively. Concerning Portuguese honey, it was also found that radical scavenging activity (DPPH assay) was 43.46% and antioxidant activity was 18.85 mg ascorbic acid equivalent/100 g and 9.92 mg quercetin equivalent/100 g. These results confirmed that Portuguese strawberry tree honey has the highest antioxidant activity, when compared with other kinds of honey. This complete report demonstrates advantages and can help to promote consumption and shown their benefical properties (e.g., antioxidant); which will may increase the commercial value.