Characterization of E 472 food emulsifiers by high-performance thin-layer chromatography with fluorescence detection and mass spectrometry
Introduction
Fruit acid esters including acetic acid of mono- and diacylglycerols of fatty acids (MAG and DAG) are often used as food additives for the adjustment of the techno-functionality of foodstuffs. Based on their surface-active properties, they are named emulsifiers or more specifically emulsifiers of the type E 472. They are mainly used in food products like baked goods, infant formula, beverages, seasonings, dairy whipping cream, ice cream, and instant whipping cream to adjust for example emulsion stability, viscosity and foaming properties [1].
According to Regulation (EC) No 1333/2008 [2], emulsifiers of the type E 472 are approved food additives and categorized into six groups. These categories encompass acetic acid esters (E 472a, ACETEM), lactic acid esters (E 472b, LACTEM), citric acid esters (E 472c, CITREM), tartaric acid esters (E 472d, TATEM), mono-and diacetyl tartaric acid esters (E 472e, DATEM) and mixed acetic and tartaric acid esters (E 472f, MATEM) of MAG and DAG of fatty acids.
For emulsifiers of the type E 472, a direct esterification of glycerol with free fatty acids (FFA) or a transesterification of triacylglycerols (TAG), mainly obtained from hydrogenated edible oils, with glycerol are commercially performed syntheses. During these production processes, acetic acid, lactic acid, citric acid, tartaric acid or mixtures thereof are added, sometimes in the form of their activated derivatives [1]. Besides the rather complex mixtures of reactants, the reaction products show even more complexity leading to mixtures of various (fruit) acid esters of MAG and DAG including different (positional) isomers, by-products and the educts (Fig. S1, Supplementary information). The composition of the product is hardly controllable through reaction conditions like ratios of reactants or raw materials because of the variability and complexity of reactants and possible reactions [1]. Consequently, emulsifiers of the type E 472 are no defined single substances but very complex mixtures with strongly varying compositions as compared to the target compound and comprise many further components. The main constituents of E 472 are defined in Commission Regulation (EU) No 231/2012 [3], but for many of the present compounds no regulations exist.
It is known that the composition of food emulsifiers directly affects the food product structure, mainly properties like viscosity, emulsion stability and foaming stability [4], [5], [6], [7], [8]. In previous work [9] it was shown that different compositions of E 471 emulsifiers affect the techno-functional properties of instant whipping cream. These findings will very likely also hold true for E 472 emulsifiers due to their direct chemical relation to E 471 and their application for the similar product groups. However, apart from the work of Köhler [10], [11], [12], [13], who extensively studied the structure-function relationships of DATEM in baked food, until now, no data regarding composition and influence on the products is available for E 472 and their application in food is empirical. Therefore, the need of reliable and simple methods is urgent in order to evaluate and monitor the composition of E 472 emulsifiers.
Methods for the analysis of the complex E 472 emulsifiers are very scarce. Amara et al. [14] analyzed CITREM by high-performance thin-layer chromatography (HPTLC) in infant formula, when the main objective of the study was not the analysis of the citric acid esters but the investigation of their enzymatic digestion. Enzymatic hydrolysis was additionally evaluated by nuclear magnetic resonance and high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS). Apart from the work of Köhler [13], who synthesized DATEM and performed analysis by HPLC, no further methods are available for the analysis of E 472 emulsifiers.
Recently, own research led to a completely different strategy that was developed for the characterization of food emulsifiers [15]. A straightforward approach by HPTLC for the analysis of E 471 offered the characterization and comparison of many samples directly on the HPTLC plate through simple visual evaluation by a fingerprint. This fingerprint technique was the basis of the present study, the aim of which was the development of a sensitive and selective screening method for the characterization of E 472 by HPTLC. The great range of separation possibilities in HPTLC allows the development of a selective chromatographic system to separate emulsifier constituents, while derivatization with a fluorescence dye will guarantee sensitive and selective detection. The visual evaluation will directly offer an overview of the emulsifiers’ composition and their characterization and enable the comparison of numerous samples in a single run. Furthermore, coupling with MS will provide the simple and easy identification of single components of the complex emulsifier mixtures.
Section snippets
Chemicals and materials
1-Stearoyl-rac-glycerol (>99%), 1,2-distearoyl-rac-glycerol (>99%), 1,3-distearoylglycerol (>99%) and stearic acid (>99.5%, all analytical standard grade), glyceryl tristearate (>99%) and primuline (dye content 50%) were obtained from Sigma-Aldrich (Steinheim, Germany). Chloroform (≥99.8%, for pesticide residue analysis, Chromasolv), t‑butyl methyl ether (TBME, ≥99.8%, HPLC, Chromasolv), diethyl ether (≥99.5%, GC, puriss.), n-heptane (≥99%, HPLC, Chromasolv) and methanol (LC–MS, Chromasolv)
Approach
An HPTLC method for the characterization of E 472 was developed. Therefore, a suitable system was evaluated regarding both the separation of the polar fruit acid esters of MAG and DAG constituents and the separation of the common lipid classes MAG, DAG, TAG and FFA. Applying the developed visual characterization, evaluation and comparison of several E 472 emulsifiers was possible, and through the coupling with MS, identification of prominent constituents of interest was enabled.
Method development
Four categories
Conclusions
Visual comparison by HPTLC–FLD was shown as an innovative and sensitive technique for the characterization of E 472 emulsifiers. Simultaneous analysis of numerous samples, which were simply dissolved in TBME, was enabled and offered characteristic fingerprint patterns for the different E 472 categories. Visualization by means of primuline guaranteed high sensitivity through fluorescence detection. The easy characterization and direct visual comparison of the emulsifier pattern (fingerprint)
Funding
This research project was supported by the German Ministry of Economics and Technology, via AiF (Arbeitsgemeinschaft industrieller Forschungsvereinigungen, Cologne, Germany) and the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn, Germany), project AiF 19355 N.
CRediT authorship contribution statement
Claudia Oellig: Conceptualization, Methodology, Validation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration, Funding acquisition, Investigation. Katharina Link: Investigation, Resources. Wolfgang Schwack: Writing - review & editing, Supervision, Project administration, Funding acquisition.
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 express many thanks to Merck (Darmstadt, Germany) for support with plate material and to Oleochemicals (Hamburg, Germany), DuPont Danisco (Neu-Isenburg, Germany) and BASF (Illertissen, Germany) for providing E 472 emulsifiers. In addition, the authors thank Fresenius Kabi (Bad Homburg, Germany), Molkerei Meggle (Wasserburg/Inn, Germany) and Müller Service (Freising, Germany) for providing E 472 emulsifiers.
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