Quantitative Measurement of the Chemical Composition of Fatty Acid of Cocoa Butter and the Isotopic Content of Glycerol Contained in Cocoa Butter by the NMR 13C from the INEPT Sequence and Characterization of the Geographical Origin of the Cocoa

The determination of the chemical composition of cocoa butter and the isotopic contents of the carbons (C-2, C-1 and C-3) of the glycerol contained in the cocoa butter were measured by the NMR-C from the INEPT sequence. This technique has already been applied to triglycerides of animal origin. Thus, cocoa butter is made up of nearly 66% saturated fatty acid composed mainly of stearic acid and palmitic acid. Monounsaturated fatty acids represented by oleic acid have an average content of 31% and polyunsaturated fatty acids represent less than 4%. Likewise, the isotopic contents of the glycerol of the triglycerides of cocoa butter in C of the carbons C-2, C-1 and C-3 were measured directly and values vary from −11‰ to −19‰ for the C-2 and from −39‰ to −41‰ for all C-1 and C-3 because of the symmetry of glycerol.


Introduction
Lipids are produced by plants and animals, and they constitute the fat of living How to cite this paper: Diomandé, D., Dro, T.T., Akpa, J.S., Virginie, S., Tea, I. and Remaud, G.S. (2022) Quantitative Measurement of the Chemical Composition of Fatty Acid of Cocoa Butter and the Isotopic Content of Glycerol Contained in Cocoa Butter by the NMR 13 C from the things [1]. Lipids are used in cosmetics, pharmaceuticals and human food. Lipids are constituents of cell membranes, inter and intracellular messengers and an energy reserve for the human body [2] [3]. The form of lipids present in the body is that of fatty acid triglycerides. Fatty acids can be divided into three classes: saturated, monounsaturated and polyunsaturated. Unsaturated fatty acids, in particular oleic acid and linoleic acid, play an essential nutritional role.
They have a preventive action against cardiovascular disease by lowering cholesterol levels, which is not the case for certain saturated fatty acids [4]. The chemical composition in fatty acids is linked to the botanical or animal origin or to the processes used to obtain it, but also to the geographical origin [5]. Today's society is demanding on the origin of the products consumed. Thus each actor, from the product to the consumer, wants the origin of the animal or vegetable fats used to be guaranteed, whether in a raw or processed product, in order to control its quality. It is therefore in this sense that this work is part of the determination of the fatty acid composition but also of the determination of the 13 C isotopic signature of the glycerol contained in cocoa butter by the 13 C-INEPT method. This technique has already made it possible to analyze triacylglycerols in olive oil and also to quantify several fatty acids, triglycerols in egg yolks [6].

Preparation of the Sample
We worked on 41 samples from 21 producing countries. These samples were provided by the International Agricultural Research Center for Development (CIRAD) and those from Côte d'Ivoire were obtained from farmers in different producing regions. Cocoa butter is extracted from the cocoa beans separated from the shells. 10 g of cotyledons are finely ground and the powder obtained is extracted hot (90˚) at reflux, for 2 hours, with (3 × 100 mL) of cyclohexane (Prolabo). Other solvents such as hexane, acetone can also be used for the extraction of cocoa butter. After cooling and filtration, the residue constitutes lean cocoa. The filtrate is evaporated and gives cocoa butter (45% to 50%). After separation on a vacuum pump to remove traces of cyclohexane, a mass of 403.2 mg of cocoa butter is dissolved in 630 mg of CDCl 3 (Eurisotop). The solution is well homogenized and then introduced into a 5 mm diameter NMR tube.

Isotopic 13 C NMR Acquisition Parameters
Carbon has two isotopes, 12 C, which has an isotopic abundance of about 98.9%, and 13 C, which is only 1.1%. In the 13 C NMR study, the spectrum obtained gives low intensities, which does not allow spectrum analysis. In order to obtain a spectrum favorable to the analysis, polarization transfers from hydrogen to car-  13 C and secondly to detect information on the 13 C nucleus. The start of this sequence is a spin echo that inverts the population of a subpopulation of protons resulting in carbon selectivity. This results in a gain in intensity of the 13 C signals. The linear combinations of these different spectra then make it possible to separate the CH, CH 2 and CH 3 sub-spectra. However, this assumes that all the groups have identical (or in any case very close) J C-H coupling constants, which is rarely verified.
The 13 C-INEPT method has already been discussed in the CEISAM "Chemistry and Interdisciplinarity, Synthesis, Analysis, Modeling" laboratory in the EBSI team under these fundamental and application aspects [7]. The application with the resulting cocoa butter was carried out in collaboration with the NMR specialists of the EBSI team. The 13 C NMR experiments were carried out on the 500 MHz Bruker Avance III spectrometer equipped with a 13 C/ 1 H dual cryo probe with 2 H lock. All the experiments were carried out at a temperature of 293 K and without rotating the tubes.
The 13 C NMR isotopic analysis of the glycerol of the triglycerides of cocoa butter was also carried out using a 13 C acquisition sequence, decoupled 1 H consisting of a recovery time followed by the application of an excitation pulse of which l The rocking angle is equal to 90˚ and decoupling 1 H during the collection of the FID only (zgig sequence). This sequence will serve as a reference to calibrate the results obtained with the 13 C-INEPT sequence. The parameters used for this measurement are as follows:  For the two sequences, 5 spectra were carried out. However, all the spectra carried out on the cocoa butter samples were obtained with the refocused INEPT-sequence, only on one of the samples of the series the zgig sequence was operated in order to ensure that we always obtained the same results. The acquired 13 C NMR spectra were subsequently processed with a decreasing exponential type apodization function using a line broadening factor (lb) of 1.5 Hz.
The baseline correction was made by part with a polynomial of degree 5. The area of the different lines present on the NMR-13 C spectra was determined using the Perch software (University of Kuopio, Finland). The different components of the lines corresponding to the carbon sites relating to glycerol and to the fatty acids of the triglycerides of cocoa butter were deconvoluted by Lorentzian lines whose chemical shifts, the width at mid-height and the amplitude of the peaks were adjusted. In the case of the measurements obtained with the zgig sequence our results are perfectly validated, in the case of the INEPT-refocused sequence, by the sequence itself the areas of the signals are not only proportional to the number of 13 C nuclei [9]. Thus, an average corrective factor established on the results of the 9 samples measured with the zgig sequence was applied to the areas obtained with the INEPT-refocused sequence.

Application to Glycerol and Fatty Acids of the Triglycerides of Cocoa Butter
There are two types of information that can be obtained from the 13 Table 1.

Statistical Treatment of the Data Obtained
Analysis of these results shows that the chemical fatty acid composition of cocoa   Statistical processing was performed using R software [11]. their biosynthesis in plants [12]. As a result, the varieties of cocoa trees can influence the discrimination observed on the different PCA. In fact, linoleic fatty acid is synthesized in abundance by gradual desaturation of oleic acid over a short period during the development of seeds or leaves [13].     about 29‰ [16]. If carbon uptake were limited only by the carboxylation process, fractionation would be 29‰ and C3 plants would have an isotopic deviation of −37‰. At the other extreme, if carbon uptake were limited only by diffusion processes, fractionation would be 4‰ and plants would have an isotopic signature of −12‰. In reality, the total fractionation is between the fractionation due to diffusion and that due to carboxylation and the C3 plants have a between −22‰ and −30‰ [17] [18].
The isotopic composition in C4 plants differs substantially from that of C3 plants because the first carboxylating enzyme is much less discriminating for the heavy isotope [16]. After entering the plant by the same diffusion processes as for C3 plants, the CO 2 is taken over by the PEP-carboxylase enzyme. This reaction is equivalent to a 5‰ -6‰ fractionation. The products of this reaction are then taken over by the enzyme RuBP, which slightly discriminates against the heavy isotope of carbon [16]. Thus, the sudden fractionations during the diffusion and the action of the enzymes PEP-carboxylase and RuBP determine the isotopic composition of the C4 plants, which have a δ 13 C between −10‰ and −14‰ [19].
Therefore, isotopic fractionation at the carbon level is linked to the nature of the plant.

Conclusion and Perspectives
This study made it possible to determine the chemical composition of cocoa butter in fatty acids. Thus cocoa butter is mainly composed of saturated fatty acids and then come the monounsaturated ones. We have around 63% to 68% saturated fatty acids composed mainly of stearic acid and palmitic acid, while monounsaturated fatty acids (oleic acid) represent 30% to 32%. Polyunsaturated acids represent less than 4%. We were also able to measure the δ 13 C isotopic signature of the glycerol contained in cocoa butter. The statistical treatment of these different results made it possible to highlight a discrimination within the samples. However, a study of the different varieties of cocoa could better make it possible to bring out the most discriminating parameters while eliminating the varietal factor. Also taking into account seasonal parameters (precipitation, sunshine, atmospheric conditions, etc.) could allow a more in-depth study with a view to characterizing the geographical origin of the cocoa bean.