Validation of analytical methods for the detection of beeswax adulteration with a focus on paraffin
Introduction
Beeswax has a complex role in the honey bee (Apis mellifera L.) colony where it serves as a construction material for the honeycomb (comb wax) that provides an infrastructure for brood rearing and food storage. It also has an important role in chemical communication (Breed et al., 1995a, Hepburn, 1998; Fröhlich et al., 2000; D'Ettorre et al., 2006), mechanical communication (Kirchner, 1993; Sandeman et al., 1996; Tautz, 1996; Tautz & Lindauer, 1997), and thermoregulation (Ellis et al., 2010; Humphrey & Dykes, 2008). It can thus be assumed that the use of good quality (genuine, authentic) beeswax in apiculture is important to preserve its natural multi-functionality for honey bees.
Beeswax production is a complex process comprising various biological and technological transformation steps. Beeswax is secreted by the honey bee workers in the form of wax scales and it is further being chewed by their mandibles which results in the transformation of texturally anisotropic scale wax into isotropic comb wax (Hepburn et al., 2014) supporting its multi-functionality in the honey bee colony. In apiculture, comb wax is commonly being recycled by melting the honeycomb and wax capping (after extraction of honey) and by removing foreign matter by melting it with boiling water which enables the production of crude beeswax - the final product being further commercialized. Beeswax recycling may also include industrial-scale decontamination methods for removing acaricides and heavy metals from beeswax (Navarro-Hortal et al., 2019), as well as discoloration, i.e. bleaching methods (Serra-Bonvehí & Orantes Bermejo, 2016). Nowadays, crude beeswax is used by various industry types, such as pharmaceuticals, cosmetics and food industry (e.g. food additive, packaging and coating) (Bogdanov, 2017a), along with its major (re-) use in apiculture in the form of comb foundations (Bogdanov, 2017b). Comb foundation represents a thin layer of beeswax intended for further use in apiculture (insertion in the beehives within wired frames) as a foundation for further comb wax construction by the bees (the cycle continues: wax scales - comb wax - crude beeswax - comb foundation). Although it is difficult to present exact figures on beeswax use, production and trade, it can be stated that the comb foundation production is probably the major use of beeswax (Bogdanov, 2017b; Crane, 1990; Krell, 1996).
There are only a few official statutory documents from the European Union and FAO/WHO providing a definition for beeswax, its description and quality (purity criteria). However, these documents only refer to its uses in the pharmaceutical sector as pharmaceutical grade beeswax defined by European Pharmacopoeia - Ph.Eur. 10th Edition, Supplement 10.2 (Council of Europe, 2020) and in the food industry as food additive E901 (Commission Regulation, 2012; Commission Directive, 2009; European Food Safety Authority (EFSA), 2007). Under those regulatory sectors, beeswax authentication relies on several classical physico-chemical analytical methods and corresponding physico-chemical criteria (range values) defining pure beeswax.
Most of the physico-chemical analytical range values characterizing pure beeswax in the existing legislations are generally accepted and widely used for beeswax research. The most commonly used methods are the determination of the melting point, acid value, saponification and ester value (Tulloch & Hoffman, 1972; Tulloch, 1973; Puleo & Rit, 1992; Bernal et al., 2005; Serra Bonvehı, 1990; Serra Bonvehí & Orantes Bermejo, 2012; Maia & Nunes, 2013, Svečnjak et al., 2015, 2019a), while some other methods have been used sporadically for the purpose of beeswax authentication, namely, determination of beeswax density (specific gravity), peroxide value (Bernal et al., 2005; Bogdanov, 2004), ash content and iodine value (Bernal et al., 2005; Puleo & Rit, 1992; Serra Bonvehı, 1990).
While the quality control of beeswax in pharmaceutical and food industry is currently covered by legislation, beeswax material used in apiculture (as comb foundations and crude beeswax used for their production) is not supported by any legislative framework. A proposal of purity criteria for beeswax used in apiculture based on physico-chemical parameters from existing legislation (Council of Europe, 2020) complemented with additional analytical testing (determination of refractive index, water content and ester/acid ratio) has been initiated by the world-wide network on honey and bee product science, the International Honey Commission (IHC), a decade ago (Bogdanov, 2009, 2017b). However, this initiative remained at a proposal level.
Currently, beeswax and its products used in the apiculture sector are classified as animal by-products (ABP) not intended for human consumption and sub-categorized as category 3 material (Commission Regulation, 2011; Regulation (EC), 2009). This category includes ABPs that do not present a potential risk for the food chain and, therefore, beeswax is not subject of any quality control before being marketed. This represents a vulnerable entry point to adulteration. Consequently, adulterated beeswax is frequently re-entering the apiculture sector via uncontrolled comb foundation trade. Comb foundations are the major target of adulteration which further leads to adulteration of crude beeswax. As reported in numerous studies, the most commonly used adulterant is paraffin wax (Tulloch, 1973; Bogdanov, 2004; Bernal et al., 2005; Serra Bonvehí and Orantes Bermejo, 2012; Maia et al., 2013; Svečnjak et al., 2015; Waś et al., 2016; Svečnjak et al., 2018; Tanner & Lichtenberg-Kraag, 2019; Špaldoňová et al., 2020), while stearin and/or stearic acid appears sporadically, especially in recent years (Reybroeck & Van Nevel, 2018, p. 115; Svečnjak et al., 2018; Tanner & Lichtenberg-Kraag, 2019). In chemistry, the term stearin (tristearin) corresponds to a tristearic acid triglyceride but commercially available stearin can also refers to stearic acid or a mixture of stearic acid and palmitic acid. These fatty acids are commonly used for beeswax adulteration.
In addition to the lack of legislation and defined purity criteria, another challenge in determining the beeswax purity is the reliability of the most commonly used physico-chemical methods (i.e. determination of the melting point, acid, saponification and ester value) for detecting adulterants in beeswax. As reported by Bernal et al. (2005), the minimum amount of the most common beeswax adulterants (paraffin, stearic acid, tallow and carnauba wax) that can be detected by physico-chemical methods is relatively high (between 2% and 50%, depending on the type of adulterant, as well as the method applied) and the determination of physico-chemical parameters does not guarantee beeswax purity (i.e. the absence of adulterants).
Furthermore, as indicated by Svečnjak et al. (2019a), one of the factors that may affect the analytical range values (primarily saponification value, and consequently, ester value and ester/acid ratio) is an exposure of beeswax to high heat treatment (Tulloch, 1973) applied as an integral part of the comb foundation production process (exposure to 125–130 °C) necessary to kill the spores of the heat-resistant Paenibacillus larvae, a bacteria responsible for the American foulbrood. A recent report by Špaldoňová et al. (2020) revealed small changes in the chemical composition of beeswax (i.e. simultaneous decrease in carboxylic acid groups and an increase in hydroxyl esters and palmitic acid methyl ester) caused by the heating treatment (i.e. exposure to 100 °C for 60 min). The results indicate that the heating process does not affect the quality of beeswax significantly but the effects of the most commonly used heating treatments during the comb foundation production process (125–130 °C) have yet to be investigated and correlated with the values of the physico-chemical parameters. Some deviations in analytical range values may also arise from a different geographical origin of the beeswax (Beverly et al., 1995), different A. mellifera subspecies producing the wax (Beverly et al., 1995; Fröhlich et al., 2000; Tulloch, 1980), comb age (Waś et al., 2014), and/or minor natural chemical variability of beeswax among colonies (Breed et al., 1995b; Svečnjak et al., 2015; 2019a). Still, the overall analytical deviations of the physico-chemical parameters are not yet fully explained. Sampling details are often not available in the scientific literature, so it can be assumed that anomalous analytical values may also be related to questionable sampling and/or to the origin of the beeswax samples.
Aiming to find robust and more reliable analytical methods for determining beeswax authenticity, advanced instrumental analytical methods have been developed and utilized in the last decade for the detection and quantification of adulterants in beeswax: gas chromatography (GC) coupled with different detectors (Jiménez et al., 2009; Serra Bonvehi & Orantes Bermejo 2012; Waś et al., 2015, 2016), and Fourier transform infrared spectroscopy (FTIR) coupled with Attenuated Total Reflectance (ATR) accessory (FTIR-ATR technique) (Maia et al., 2013; Svečnjak et al., 2015, Svečnjak et al., 2019a; Tanner & Lichtenberg-Kraag, 2019). Both analytical methods proved to be more reliable for beeswax adulteration detection compared to physico-chemical methods as they provide good detection limits (LOD<5%) for the most commonly used adulterants (i.e. paraffin, stearin/stearic acid). However, they have not yet been validated by inter-laboratory comparisons and, consequently, they are not yet incorporated in the current legislation on beeswax.
The aim of this study was to assess the effectiveness and reliability of the available analytical methods for the determination of beeswax purity with a focus on the detection of adulteration with paraffin as the most commonly used adulterant in apiculture. Based on the statistical validation of the analytical methods and the data available in the literature, a practical recommendation for the use of the analytical methods (and corresponding purity criteria) showing the best performance in detecting beeswax adulteration with paraffin was provided.
Section snippets
Materials and methods
The assessment of the analytical methods used for the detection of beeswax adulteration was performed on four levels (Fig. 1).
Inventory of analytical methods used for detection of paraffin and stearin/stearic acid in beeswax
The inventory of both classical routine and advanced analytical methods used for the detection of paraffin (Table 1) and stearin/stearic acid (Table 2) were summarized based on available literature data. These inventory tables were constructed based on the general method information (validation supported by legislation and scientific literature), indication of measurement uncertainty (limit of detection, reports on anomalous values for authentic beeswax), and crucial methods advantages and
Conclusions
In this study, an inventory of a comprehensive set of analytical methods (classical physico-chemical, and advanced instrumental techniques) and the corresponding purity criteria used for the detection of beeswax adulteration was established. This was achieved by retrieving the methods using an evidence-based data (covered by both legislation and scientific literature). The importance of selected methods was weighted in terms of their reliability and effectiveness in detecting targeted
Disclaimer
The views or positions expressed in this article do not necessarily represent in legal terms the official position of the European Food Safety Authority (EFSA). EFSA assumes no responsibility or liability for any errors or inaccuracies that may appear. This article does not disclose any confidential information or data. Mention of proprietary products is solely for the purpose of providing specific information and does not constitute an endorsement or a recommendation by EFSA for their use.
CRediT authorship contribution statement
Lidija Svečnjak: Conceptualization, Methodology, Investigation, Validation, Formal analysis, Resources, Writing - original draft, Visualization, Writing - review & editing. Fernando M. Nunes: Investigation, Methodology, Formal analysis, Resources, Writing - review & editing. Raquel Garcia Matas: Investigation, Data curation, Writing - review & editing. Jean-Pierre Cravedi: Investigation, Writing - review & editing. Anna Christodoulidou: Investigation, Writing - review & editing. Agnes Rortais:
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors wish to thank to Ewa Waś, PhD (Research Institute of Horticulture, Apiculture Department, Puławy, Poland) for participation in weight analysis by providing her expert opinion in weighting the importance of analytical methods for beeswax adulteration detection.
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