Detection and quantification of cocoa butter equivalents in cocoa butter and plain chocolate by gas liquid chromatography of triacylglycerols

The development and in-house testing of a method for the detection and quantification of cocoa butter equivalents in cocoa butter and plain chocolate is described. A database consisting of the triacylglycerol profile of 74 genuine cocoa butter and 75 cocoa butter equivalent samples obtained by high-resolution capillary gas liquid chromatography was created, using a certified cocoa butter reference material (IRMM-801) for calibration purposes. Based on these data, a large number of cocoa butter/cocoa butter equivalent mixtures were arithmetically simulated. By subjecting the data set to various statistical tools, reliable models for both detection (univariate regression model) and quantification (multivariate model) were elaborated. Validation data sets consisting of a large number of samples (n = 4050 for detection, n = 1050 for quantification) were used to test the models. Excluding pure illipé fat samples from the data set, the detection limit was determined between 1 and 3% foreign fat in cocoa butter. Recalculated for a chocolate with a fat content of 30%, these figures are equal to 0.3-0.9% cocoa butter equivalent. For quantification, the average error for prediction was estimated to be 1.1% cocoa butter equivalent in cocoa butter, without prior knowledge of the materials used in the blend corresponding to 0.3% in chocolate (fat content 30%). The advantage of the approach is that by using IRMM-801 for calibration, the established mathematical decision rules can be transferred to every testing laboratory.     

Gas-liquid chromatographic determination of milk fat and cocoa butter equivalents in milk chocolate: interlaboratory study

A collaborative trial was conducted to validate an analytical approach comprising method procedures for determination of milk fat and the detection and quantification of cocoa butter equivalents (CBEs) in milk chocolate. The whole approach is based on (1) comprehensive databases covering the triacylglycerol composition of a wide range of authentic milk fat, cocoa butter, and CBE samples and 947 gravimetrically prepared mixtures thereof; (2) the availability of a certified cocoa butter reference material for calibration; (3) an evaluation algorithm, which allows reliable quantitation of the milk fat content in chocolate; (4) a subsequent correction to take account of the triacylglycerols derived from milk fat; (5) mathematical expressions to detect the presence of CBEs in milk chocolate; and (6) a multivariate statistical formula to quantitate the amount of CBEs in milk chocolate. Twelve laboratories participated in the validation study. CBE admixtures were detected down to a level of 0.5 g CBE/100 g milk chocolate, without false-positive or -negative results. The applied quantitation model performed well at the statutory limit of 5% CBE addition to milk chocolate, with a prediction error of 0.7%, and HorRat values ranging from 0.8 to 1.5. The relative standard deviation for reproducibility (RSDR) values for quantitation of CBEs in analyses of chocolate fat solutions ranged from 2.2 to 3.8% and for analyses of real chocolate samples, from 4.1 to 4.7%, demonstrating that the whole approach, based solely on chocolate fat blends, is applicable to real milk chocolate samples.     

Comparative study of matrix-assisted laser desorption/ionization and gas chromatography for quantitative determination of cocoa butter and cocoa butter equivalent triacylglycerol composition

The triacylglycerol (TAG) composition study of cocoa butter (CB) and cocoa butter equivalents (CBEs) has been performed by gas chromatography (GC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). These two techniques provided comparable results. The advantage of the MALDI technique was the detection of each compound comprising the triacylglycerol classes (Cn). Moreover, comparison of the data obtained by these two techniques indicated that TAG relative percentages could be obtained quantitatively with the MALDI technique. These techniques have been applied for the composition determination of CB + CBE mixtures. Encouraging results showed that it is possible to quantify an admixture containing as little as 4% of CBE.     

Development of a rapid method for the detection of cocoa butter equivalents in mixtures with cocoa butter

A simple and rapid gas chromatographic (GC) method was developed for the detection of cocoa butter equivalents (CBEs) in cocoa buffer (CB). It is based on the use of a 5 m nonpolar capillary column for the separation of the main triglycerides of CB according to their acyl/carbon numbers. The GC procedure was optimized to avoid thermal degradation of the triglycerides. By computing the ratio C54/C50 and (C54/C50) x C52 and by 2-dimensional plotting of these values, authentic CB samples were clearly distinguished from samples containing various CBEs. The detection of little as 1% CBE in CB (corresponding to about 0.3% CBE in chocolate) in a model system was shown to be possible. Under real conditions, for a wide range of CBs, about 2.5% CBEs in CB were detected. With this method, quantitation was possible at a concentration of 5% CBEs in CB mixtures, which corresponds to around 1% in chocolate; this value is far below the maximum level of 5% CBEs allowed to be added to chocolate.     

The Use Of Hplc To Separate Triglycerides In Confectionery Fats Using Ultra Violet Detection

Abstract

The triglycerides of confectionery fats have been separated by reverse phase HPLC using mixtures of either acetonitrile and tetrahydrofuran or acetonitrile and methyl tertiary butyl ether using UV detection at 237 nanometres. The method has been applied to samples of cocoa butter, cocoa butter equivalents, milk fat and other vegetable fats. The fat from a milk chocolate has been separated by the above system.     

Analysis of steryl esters in cocoa butter by on-line liquid chromatography-gas chromatography.

On-line liquid chromatography-gas chromatography (LC-GC) has been applied to the analysis of steryl esters in cocoa butter. Separation of the steryl esters was achieved after on-line transfer to capillary GC. HPLC removes the large amount of triglycerides and pre-separates the components of interest, thus avoiding time-consuming sample preparation prior to GC analysis. The identities of the compounds were confirmed by GC-MS investigation of the collected HPLC fraction and by comparison of the mass spectra (chemical ionization using ammonia as ionization gas) to those of synthesized reference compounds. Using cholesteryl laurate as internal standard, steryl esters were quantified in commercial cocoa butter samples, the detection limit being 3 mg/kg and the quantification limit 10 mg/kg, respectively. Only slight differences in percentage distributions of steryl esters depending on the geographical origin of the material were observed. The patterns were shown to remain unchanged after deodorization. The method described might be a valuable tool for authenticity assessment of cocoa butter.     

Crystallization behavior of blends of cocoa butter and milk fat or melk fat fractions

Mixtures of milk fat or milk fat fractions, produced by melt crystallization, and cocoa butter were studied using isothermal calorimetry. Crystallization of cocoa butter (at 15, 20 and 25C) was observed, and induction time for nucleation, peak time and amount of heat produced were recorded. Melting profiles and X-ray spectra were also obtained, yielding information about extent of crystallization and type of polymorph obtained. Induction time for nucleation generally increased with increasing temperature. Peak time increased at 15C, but decreased at 20C. Amount of crystallized fat decreased with increasing level of milk fat.This work was supported by the National Dairy Promotion and Research Board and the Wisconsin Milk Marketing Board. Special thanks are extended to Jennifer Wood for valuable participation in this research and to Dr. George Zografi and his research group for their assistance with the DSC studies.     

Structure elucidation of triglycerides by chromatographic techniques. Part 2: RP HPLC of triglycerides and brominated triglycerides

The structure determination of triglycerides by RP HPLC is discussed, and the importance of temperature and mobile phase composition demonstrated. The formation of brominated triglycerides followed by RP HPLC analysis allows rapid determination of cocoa butter equivalents (CBE ) in cocoa butter and chocolate products.     

Polymorphous transitions in cocoa butter</o:p>

Summary Large experimental evidence was collected on polymorphous transitions of triacyl glycerols (TAG) in cocoa butter by means of DSC investigations. The cooling treatment (in conditions close to those of the industrial practice) and the annealing temperature significantly affect the overall crystal fraction and the distribution of the various polymorphs. These data allowed a quantitative, although purely phenomenological, kinetic parameterization of polymorphous transitions of cocoa butter. The evaluation of the relevant kinetic constants and their dependence on the temperature allowed prediction of the yield in every polymorph after a given thermal history. Similar evidences were attained for cocoa liquor and dark chocolate where TAG are sided by other ingredients. These results can be the basis for an industrial exploitation.</o:p>     

Preparation of Low-Diacylglycerol Cocoa Butter Equivalents by Hexane Fractionation of Palm Stearin and Shea Butter

Herein, we prepared 1,3-dipalmitoyl-2-oleoyl glycerol (POP)-rich fats with reduced levels of diacylglycerols (DAGs), adversely affecting the tempering of chocolate, via two-step hexane fractionation of palm stearin. DAG content in the as-prepared fats was lower than that in POP-rich fats obtained by previously reported conventional two-step acetone fractionation. Cocoa butter equivalents (CBEs) were fabricated by blending the as-prepared fats with 1,3-distearoyl-2-oleoyl glycerol (SOS)-rich fats obtained by hexane fractionation of degummed shea butter. POP-rich fats achieved under the best conditions for the fractionation of palm stearin had a significantly lower DAG content (1.6 w/w%) than that in the counterpart (4.6 w/w%) prepared by the previously reported method. The CBEs fabricated by blending the POP- and SOS-rich fats in a weight ratio of 40:60 contained 63.7 w/w% total symmetric monounsaturated triacylglycerols, including 22.0 w/w% POP, 8.6 w/w% palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol, 33.1 w/w% SOS, and 1.3 w/w% DAGs, which was not substantially different from the DAG content in cocoa butter (1.1 w/w%). Based on the solid-fat content results, it was concluded that, when these CBEs were used for chocolate manufacture, they blended with cocoa butter at levels up to 40 w/w%, without distinctively altering the hardness and melting behavior of cocoa butter.     

Cocoa Shell as a Step Forward to Functional Chocolates—Bioactive Components in Chocolates with Different Composition

Chocolate is considered as both caloric and functional food. Its nutritional properties may be improved by addition of fiber; however, this may reduce polyphenols content. The aim of this research was to determine the influence of cocoa shell addition (as a source of fiber) and its combination with different ingredients (cocoa butter equivalents (CBE), emulsifiers, dairy ingredients) on polyphenols of dark and milk chocolates. Total polyphenol (TPC) and total flavonoid (TFC) contents were determined spectrophotometrically, identification and quantification of individual compounds by high pressure liquid chromatography and antioxidant capacity by ferric reducing antioxidant power (FRAP) assay. Results showed that even though addition of cocoa shell to chocolate results in reduced contents of TPC, TFC, and individual compounds, it is not significant compared to ones reported by other authors for commercial chocolates. Other ingredients influence determined values for all investigated parameters; however, additional research is needed to reveal exact mechanisms and implications.     

Synthesis and properties of minor crypto-active triglycerides of cocoa butter

Summary 1. The crypto-active triglycerides sn-PPO, sn-SSO, and sn-OSS have been synthesized.2. It has been shown that the melting points of mixtures of the main racemic triglyceride components of cocoa butter are affected by more unsaturated triglycerides. Cryptoactive triglycerides scarcely affect the melting points of the mixtures.M. V. Lomonosov Moscow Institute of Fine Chemical Technology. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 8–11, January–February, 1975.     

Determination of flavanol and procyanidin (by degree of polymerization 1-10) content of chocolate, cocoa liquors, powder(s), and cocoa flavanol extracts by normal phase high-performance liquid chromatography: collaborative study

An international collaborative study was conducted on an HPLC method with fluorescent detection (FLD) for the determination of flavanols and procyanidins in materials containing chocolate and cocoa. The sum of the oligomeric fractions with degree of polymerization 1-10 was the determined content value. Sample materials included dark and milk chocolates, cocoa powder, cocoa liquors, and cocoa extracts. The content ranged from approximately 2 to 500 mg/g (defatted basis). Thirteen laboratories representing commercial, industrial, and academic institutions in six countries participated in the study. Fourteen samples were sent as blind duplicates to the collaborators. Results from 12 laboratories yielded repeatability relative standard deviation (RSDr) values that were below 10% for all materials analyzed, ranging from 4.17 to 9.61%. The reproducibility relative standard deviation (RSD(R)) values ranged from 5.03 to 12.9% for samples containing 8.07 to 484.7 mg/g. In one sample containing a low content of flavanols and procyanidins (approximately 2 mg/g), the RSD(R) was 17.68%. Based on these results, the method is recommended for Official First Action for the determination of flavanols and procyanidins in chocolate, cocoa liquors, powder(s), and cocoa extracts.     

Thermal stability and flame retardancy of guanidinium and imidazolium borate finished cotton fabrics

Dark chocolate is a complex food product in which sugar crystals and cocoa particles are surrounded by cocoa butter. Thermogravimetry and derivative thermogravimetry are proposed as fast, cheap, and sensitive tools to determine the composition of dark chocolate and consequently to confirm the cocoa percent declared by the producer or to check the production cycle.     

Lipase-Catalyzed Synthesis of Cocoa Butter Equivalent from Palm Olein and Saturated Fatty Acid Distillate from Palm Oil Physical Refinery

Cocoa butter equivalent was prepared by enzymatic acidolysis reaction of substrate consisting of refined palm olein oil and palmitic?Cstearic fatty acid mixture. The reactions were performed in a batch reactor at a temperature of 60?°C in an orbital shaker operated at 160?RPM. Different mass ratios of substrates were explored and the compositions of the five major triacylglycerol (TAG) of the structured lipids were identified and quantified using cocoa butter-certified reference material IRMM-801. The reaction resulted in production of cococa butter equivent with TAG compostion (POP 26.6 %, POS 42.1, POO 7.5, SOS 18.0 %, and SOO 5.8 %) and melting temperature between 34.7 and 39.6?°C which is close to that of the cocoa butter. The result of this research demonstrated the potential use of saturated fatty acid distillate (palmitic and stearic fatty acids) obtained from palm oil physical refining process into a value-added product.     

Interlaboratory evaluation of injection techniques for triglyceride analysis of cocoa butter by capillary gas chromatography

As part of two international collaborative studies, in which 14 laboratories applied capillary GLC to determine the triglyceride (TG) profile of cocoa butter, the performance of different sample introduction techniques, i.e. cold on-column injection (OCI), split injection and programmed-temperature vapouriser (PTV) injection, was compared. In both studies, the participants did not apply a uniform GLC procedure. Synthetic mixtures of triglycerides were chosen to permit an accurate determination of detector response factors. No statistically significant difference was found between the mean values obtained by different injection modes. The OCI, generally recommended as best practice, did not give superior results than the PTV or the split injection techniques.     

Comparison between cocoa and chocolate: characterisation and fatty acid content

The connection between chocolate and cocoa is studied. As cocoa is produced in different countries, samples coming from the same geographical region are analysed. The analyses focus on the characterisation of the considered samples for different aspects of quality and to individuate the original locations of production, also connecting this with the nutritional aspects. For this purpose, qualitative and quantitative traditional analysis and determination of fatty acid content are performed. The obtained experimental data show interesting correlations. The percentages of fatty acids computed allow for distinctions to be made among cocoa and chocolate samples. Furthermore, a relation can be assumed among the data obtained for primary matters and final products corresponding to countries being positioned on the same latitude, even if they are situated in different continents.     

The use of multivariate modelling of near infra-red spectra to predict the butter fat content of spreads

In order to obtain a rapid method that can detect adulteration of butter fats with cheaper vegetable fats, the use of NIR spectroscopy and multivariate modelling was explored. For model building and validation, an extensive set of samples was collected, consisting of 152 butter samples, 42 oils and 200 blends thereof. Variations in butter fat composition are reflected in distinct NIR spectral regions. Principal components analysis and partial least square discriminant analysis was used to inspect the variation within the sample set. As reference values for training partial least squares models, butter fat levels as declared by suppliers were taken, as well as C4:0 fatty acid levels as measured directly by GC. All samples were used for training, except for 100 blends, which were used later for validation. Different pre-processing and PLS approaches were explored, resulting in models that had a RMSEPs for butter fat and C4:0 fatty acid level in the range of 4.3-8.2 and 0.33-0.38% (w/w), respectively. The performance of NIR in assessment of C4:0 fatty acid levels is lower as for GC, but this disadvantage is outweighed by shorter measurement times and the lower skill levels required. Furthermore NIR is able to assess overall levels of butter fat, in addition to the indirect indicator provided by the C4:0 fatty acid level.     

TG-pyrolysis and FTIR analysis of chocolate and biomass waste

Four waste streams from a chocolate factory were examined in view of their possible usage as a fuel: cocoa shell, jute bags, and two qualities of chocolate waste (milk, white). Thus, proximate and ultimate analyses and thermogravimetric analyses coupled with Fourier transform infrared (TG-FTIR) analyses were conducted. It was observed that milk and white chocolates have similar thermal properties; chocolate has a high calorific value (24.5 MJ kg^?1). Pyrolysis of chocolate proceeds in two stages: the first from 190 to 300 °C and the second from 300 to 518 °C. During the first stage, alkaloids, such as theobromine and caffeine, evolve, and sugar decomposes, releasing acids, CO₂, and water. During the second pyrolysis stage, cocoa butter and proteins decompose releasing volatile organics such as esters, acids, amides, phenols, CH₄, CO, etc. Polyphenols such as catechin, procyanidins, etc. decompose during both pyrolysis stages. Generally, chocolate waste yields less CO₂ and CO than cocoa shell and jute. In principle, it appears to be a promising source of energy and could be utilized by both co-firing and pyrolysis, producing fuels or chemicals.     

Software (MSPECTRA) for automatic interpretation of triacylglycerol molecular mass distribution spectra and collision induced dissociation product ion spectra obtained by ammonia negative ion chemical ionization mass spectrometry

Rapid analysis of molecular mass distributions of triacylglycerol (TAG) mixtures and regioisomeric structures of selected molecular mass species is possible using ammonia negative ion chemical ionization mass spectrometry utilizing sample introduction by direct exposure probe. However, interpretation of spectra and calculation of results is time consuming, thus lengthening the total analysis time. To facilitate result calculation a software package (MSPECTRA 1.3) was developed and applied to automatic processing of triacylglycerol molecular mass distribution spectra and collision induced dissociation (CID) product ion spectra. The program is capable of identifying triacylglycerol molecular mass species possessing different ACN:DB (acyl carbon number:number of double bonds) ratios on the basis of m/z values of [M - H](-) ions. In addition to such identification the program also corrects spectra for abundances of naturally occurring (13)C isotopes and calculates relative proportions of triacylglycerol molecular species in the analyzed samples. If several replicate spectra are processed simultaneously the program automatically calculates an average and standard deviation of relative proportions of molecular species. In the case of CID spectra the program identifies fatty acid fragment ions [RCO(2)](-) and the corresponding [M - H - RCO(2)H - 100](-) ions, and calculates the relative proportions of ions in both groups. These proportions are then used automatically to calculate the fatty acid combinations comprising the parent triacylglycerol molecule and the regiospecific positions of fatty acids. Processing of several replicate product ion spectra simultaneously produces averaged proportions of regioisomers comprising the parent triacylglycerol molecular species and the standard deviation of the analysis. The performance of the program was tested by analyzing triacylglycerol samples of human milk, human milk substitutes, human chylomicron and cocoa butter, and by comparing results obtained by automated processing of the data with manually calculated results.