Determination of milk fat and cocoa butter equivalents in milk chocolate

An analytical approach for the determination of milk fat and cocoa butter equivalents in milk chocolate is presented. By performing a single triacylglycerol analysis using gas liquid chromatography several useful pieces of information can be determined, i.e., (i) the milk fat content of the chocolate sample, (ii) the contribution of triacylglycerols originating from milk fat, (iii) the presence/absence of cocoa butter equivalents, and (iv) the amount of vegetable fat present in milk chocolate. The elaborated approach was collaboratively tested, thus proving its validity to be used by control laboratories to assess correct labelling of milk chocolate according to Directive 2000/36/EC (1). milk fat (MF) interfered with the detection and quantification of CBEs in the chocolate. When milk chocolate is analysed it is necessary to correct the observed TAG pattern for the presence of MF TAGs, requiring knowledge of the amount of MF present in the product. Currently, the analysis of butyric acid in a mixed fat is a widely applied method and has for instance already been used to quantitate small amounts of MF in chocolate fats (8-10). However, with respect to the correct labelling of chocolate, this method can only provide an answer to one out of three questions, i.e., what is the content of MF in the chocolate fat. The method is not satisfactory for addressing the other two questions, i.e., (i) is there any other fat in addition to CB present and if yes (ii) how much. Therefore, an improved analytical approach, named CoCal-2, for the determination of MF together with CBEs in chocolate fats was developed. PRINCIPLE OF COCAL-2 CoCal-2 is based on a database consisting of the TAG profile of more than 300 genuine MF samples from 21 European countries and more than 900 mixtures thereof with other chocolate fats. The TAG database, obtained by gas liquid chromatography, was employed for the selection of a marker compound, i.e., 1-palmitoyl-2-stearoyl-3butyroyl-glycerol (PSB), to be used to calculate the MF content in chocolate fats (Table 1). PSB fulfilled the requirements (i) to be present in reasonable amounts allowing a reliable quantification of even low MF proportions in chocolate fats, (ii) to have low natural variability and (iii) to be present only in MF and no other fats (Figure 1). The advantage of the developed method is that for further applications, i.e., determination of CBEs in chocolate fats, a single TAG analysis is performed, whereas for the same purpose the butyric acid method requires two different analytical methods. The test sample, i.e., the chocolate fat obtained from milk chocolate using a rapid fat extraction procedure is separated by gas liquid chromatography into TAG fractions according to their molecular weight and degree of unsaturation. Individual TAG fractions (PSB, POP, POS, POO, SOS, SOO) are used (i) to calculate INTRODUCTION Since 2003, chocolate and cocoa based products are governed in the European Union by Directive 2000/36/EC (1), authorising the use of vegetable fats other than cocoa butter (CB) in chocolate as long as they do not account for more than 5 percent of the final product. Cocoa butter equivalents (CBEs) is the general term for vegetable fats used to replace CB in chocolate. Annex II of the Directive 2000/36/EC (1) specifies explicitly that CBEs must by definition be fats low in lauric acid, rich in symmetrical monounsaturated triacylglycerols (TAGs) of the type 1,3-dipalmitoyl-2-oleoyl-glycerol (POP), 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol (POS) and 1,3distearoyl-2-oleoyl-glycerol (SOS), miscible with CB, and obtained only by refining and fractionation. As specified in Annex II of the Directive the following vegetable fats, obtained from the plants listed below, may be used singly or in blends, i.e., illipé, Borneo tallow or Tengkawang (Shorea spp.), palm oil (Elaeis guineensis, Elaeis olifera), sal (Shorea robusta), shea (Butyrospermum parkii), kokum gurgi (Garcinia indica), and mango kernel (Mangifera indica). Since the Directive per se does not cover aspects regarding methods of analysis for law enforcement, a need has been recognized within official control laboratories for reliable analytical methods to prove label compliance to protect consumers from fraudulent malpractice. Chocolate products, which contain vegetable fats other than CB, may be marketed in all of the Member States, provided that their labelling is supplemented by a statement, i.e., "contains vegetable fats in addition to cocoa butter". Member States' laws, regulations and administrative provisions have to comply with Directive 2000/36/EC (1) since August 2003. A method for the detection and quantification of CBEs in dark chocolate using TAG profiling by gas liquid chromatography was developed (2) and subjected to validation by an international collaborative tr ial (3), offering an important measure to assess compliance with labelling provisions. This allowed the implementation and enforcement of Directive 2000/36/EC (1) at least for dark chocolate. To facilitate the usage of the approach an analytical toolbox named "CoCal-1 (=cocoa butter calculation toolbox)" has been established, consisting of a validated method for detection of CBEs in dark chocolate (4), a validated method for quantification of CBEs in dark chocolate (5), both of them standardized by the International Organization for Standardization (ISO) (6), a certified cocoa butter reference material (IRMM-801) to calibrate the analyst's instruments (7), and an electronic evaluation sheet for Microsoft Excel® to calculate the final result. Up to now, this standardized analytical approach for dark chocolate could not be used for milk chocolate since TAGs deriving from milk or Table 1. Descriptive statistics of individual TAGs tested as potential markers for MF content predictions (n=310) Manuela Buchgraber