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.     

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.     

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.     

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.     

Lipase-Catalyzed Acidolysis of Palm Mid Fraction Oil with Palmitic and Stearic Fatty Acid Mixture for Production of Cocoa Butter Equivalent

Cocoa butter equivalent (CBE) was prepared by enzymatic acidolysis reaction of substrate consisting of refined palm mid fraction oil and palmitic–stearic 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 composition of the five major triacylglycerols (TAGs) of the structured lipids was identified and quantified using cocoa butter certified reference material IRMM-801. The reaction resulted in production of cocoa butter equivalent with the TAGs' composition (1,3-dipalmitoyl-2-oleoyl-glycerol 30.7 %, 1-palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol 40.1%, 1-palmitoy-2,3- dioleoyl glycerol 9.0 %, 1,3-distearoyl-2-oleoyl-glycerol 14.5 %, and 1-stearoyl-2,3-dioleoyl glycerol 5.7 %) and with onset melting temperature of 31.6 °C and peak temperature of 40.4 °C which are close to those of cocoa butter. The proposed kinetics model for the acidolysis reaction presented the experimental data very well. The results of this research showed that palm mid fraction oil TAGs could be restructured to produce value added product such as CBE.     

Stereospecific analysis of the triacylglycerols of a cottonseed oil hydrogenate and its fractions

A cottonseed oil hydrogenate has been fractionated into three fractions: high-melting, medium-melting, and liquid. It has been established that the fat of the second fraction, because of its physicochemical indices, consistency, and the distribution of the saturated fatty acyl radicals in the extreme positions of the TAG molecules, can be regarded as a cocoa butter substitute.     

Method validation for detection and quantification of cocoa butter equivalents in cocoa butter and plain chocolate

A European interlaboratory study was conducted to validate an analytical procedure for the detection and quantification of cocoa butter equivalents in cocoa butter and plain chocolate. In principle, the fat obtained from plain chocolate according to the Soxhlet principle is separated by high-resolution capillary gas chromatography into triacylglycerol fractions according to their acyl-C-numbers, and within a given number, also according to unsaturation. The presence of cocoa butter equivalents is detected by linear regression analysis applied to the relative proportions of the 3 main triacylglycerol fractions of the fat analyzed. The amount of the cocoa butter equivalent admixture is estimated by partial least-squares regression analysis applied to the relative proportions of the 5 main triacylglycerols. Cocoa butter equivalent admixtures were detected down to a level of 2% related to the fat phase, corresponding to 0.6% in chocolate (assumed fat content of chocolate, 30%), without false-positive or -negative results. By using a quantification model based on partial least-squares regression analysis, the predicted cocoa butter equivalent amounts were in close agreement with the actual values. The applied model performed well at the level of the statutory limit of 5% cocoa butter equivalent addition to chocolate with a prediction error of 0.6%, assuming a chocolate fat content of 30%.     

Analysis of triacylglycerols on porous graphitic carbon by high temperature liquid chromatography

The retention behaviour of several triacylglycerols (TAGs) and fats on Hypercarb, a porous graphitic carbon column (PGC), was investigated in liquid chromatography (LC) under isocratic elution mode with an evaporative light scattering detector (ELSD). Mixtures of chloroform/isopropanol were selected as mobile phase for a suitable retention time to study the influence of temperature. The retention was different between PGC and non-aqueous reversed phase liquid chromatography (NARP-LC) on octadecyl phase. The retention of TAGs was investigated in the interval 30-70 degrees C. Retention was greatly affected by temperature: it decreases as the column temperature increases. Selectivity of TAGs was also slightly influenced by the temperature. Moreover, this chromatographic method is compatible with a mass spectrometer (MS) detector by using atmospheric pressure chemical ionisation (APCI): same fingerprints of cocoa butter and shea butter were obtained with LC-ELSD and LC-APCI-MS. These preliminary results showed that the PGC column could be suitable to separate quickly triacylglycerols in high temperature conditions coupled with ELSD or MS detector.     

Kinetics of Crystallization in n-Hexadecane and Cocoa Butter Oil-in-Water Emulsions Accounting for Droplet Collision-Mediated Nucleation

Droplet collision is shown to be important in the propagation of nucleation through supercooled oil-in-water emulsions by the use of a novel membrane technique. On the other hand, nucleation mediated by Tween 20 micelles is shown to be of relatively much less importance in both n-hexadecane and cocoa butter oil-in-water emulsions. The droplet collision phenomenon probably occurs via a surfactant bridge between the colliding droplets. When this process is taken into account we show that the Turnbull model for crystal nucleation kinetics explains very well nucleation in cocoa butter oil-in-water emulsions by seed crystals. On the basis of this model we characterized the seed crystals in cocoa butter via isothermal crystallization studies at 14.2, 15.0, 15.5, and 15.8 degrees C. We suggest that there are few seed crystals whose size exceeds 0.28 μm at 80 degrees C. In our cocoa butter samples there were between 10(16) and 10(17) seed crystals m(-3) whose average size we inferred to be less than 0.09 μm. A value of 0.133 mJ m(-2) is obtained for the Gibbs free energy of the nucleating surface in our West African cocoa butter samples. There is evidence that the alpha-polymorph of POS comprises the nucleating layer in the seed crystal. There is no evidence that surfactant influences the primary nucleation of oil crystals. However, the surfactant has a big effect on the kinetics of the secondary nucleation process, mediated by droplet collision. Copyright 2000 Academic Press.     

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.     

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.     

Investigating Isothermal DSC Method to Distinguish Between Cocoa Butter and Cocoa Butter Alternatives

In the article thermal behaviours of cocoa butter and representatives of the 3 classes of cocoa butter alternative fats were investigated using isothermal DSC-method. Besides traditional parameters, Avrami transformation and polar qualification system (adapted from NIR-technique) were used for data evaluations.Using a new parameter, t_max^*, the influence of the temperature change could be avoided. This parameter gave 100% success in classification of the investigated confectionery fats (p<0.05).In comparison with traditional Avrami parameters such as n and lgk measured at different temperatures, a new parameter (t^*) gave the best result in distinguishing confectionery fats (approx. 71% correctly classified). The classification improved using lgk and n together (79%).Better classification could be achieved using polar qualification system. The percentage of correctly classified cases was 87.5% using either the point or the surface method (p<0.05). In every case there was a clear borderline between cocoa butter-CBE fat and CBR-CBS fats.Comparing Avrami method and PQS, it can be concluded that the latter is a more successful method in classification of unknown fat samples (pure cocoa butter alternative fats only). However PQS does not give any information about thermal behaviour of the sample.This revised version was published online in November 2005 with corrections to the Cover Date.     

Stereospecific analysis of the triacylglycerols of a cottonseed oil hydrogenate and its fractions

A cottonseed oil hydrogenate has been fractionated into three fractions: high-melting, medium-melting, and liquid. It has been established that the fat of the second fraction, because of its physicochemical indices, consistency, and the distribution of the saturated fatty acyl radicals in the extreme positions of the TAG molecules, can be regarded as a cocoa butter substitute.Institute of the Chemistry of Plant Substances of the Uzbek SSR Academy of Sciences, Tashkent. Institute of Bioorganic Chemistry of the Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 509–513, July–August, 1987.     

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.     

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.     

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.     

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.     

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.     

Fast Melt Cocoa Butter Tablet: Effect of Waxes,Starch, and PEG 6000 on Physical Properties of the Preparation

A fast melt tablet (FMT) is well regarded as an alternative delivery system that might help resolve a patient’s non-compliance issue. The main objective of this study was to develop a cocoa butter-based FMT. Additives, namely 5–15% of PEG 6000, beeswax, paraffin wax, and corn starch, were incorporated into the cocoa butter-based FMT to study the effects of these additives with the physical characteristic of a cocoa butter FMT. An optimum-based formulation was chosen according to the desired hardness and disintegration time and the taste masking property achieved with the model drug—dapoxetine. The analysis demonstrated that incorporating beeswax (15%) and paraffin wax (15%) could prolong the disintegration time by at least two-fold. On the contrary, the presence of corn starch was found to cause an increase in the hardness and reduction of the disintegration time. The disintegration mechanism might be presumed due to the synergistic effect of starch swelling and cocoa butter melting. The hardness value and in vitro disintegration time of the optimum formulation were recorded at 2.93 ± 0.22 kg and 151.67 ± 6.98 s. In terms of dissolution, 80% of dapoxetine was released within 30 min and the dissolution profile was comparable to the innovator product. The formulation was palatable and stable for at least 1 year. The exposure of the FMT formulation at 30 °C for 12 months was reported to be stable. Along with the sound palatability profile and high drug load capacity, the current formulation possesses the desired characteristics to be scaled up and marketed.     

Determination of triacylglycerol regioisomers using electrospray ionization-quadrupole ion trap mass spectrometry with a kinetic method

The kinetic method was applied to differentiate and quantify mixtures of regioisomeric triacylglycerols (TAGs) by generating and mass selecting alkali ion bound metal dimeric clusters with a TAG chosen as reference (ref) and examining their competitive dissociations in a quadrupole ion trap mass spectrometer. This methodology readily distinguished pairs of regioisomers (AAB/ABA) such as LLO/LOL, OOP/OPO and SSP/SPS and consequently distinguished sn-1/sn-3, sn-2 substituents on the glycerol backbone. The dimeric complex ions [ref, Li, TAG_{(AAB and/or ABA)}]⁺ generated by electrospray ionization mass spectrometry were subjected to collision induced dissociation causing competitive loss of either the neutral TAG reference (ref) leading to [Li(AAB and/or ABA)]⁺ or the neutral TAG molecule (TAG_{(AAB and/or ABA)}) leading to [ref, Li]⁺. The ratio of the two competitive dissociation rates, defined by the product ion branching ratio (R_iso), was related via the kinetic method to the regioisomeric composition of the investigated TAG mixture. In this work, a linear correlation was established between composition of the mixture of each TAG regioisomer and the logarithm of the branching ratio for competitive fragmentation. Depending on the availability of at least one TAG regioisomer as standard, the kinetic method and the standard additions method led to the quantitative analysis of natural TAG mixtures.