Structural and Vibrational Investigations of Mixtures of Cocoa Butter (CB), Cocoa Butter Equivalent (CBE) and Anhydrous Milk Fat (AMF) to Understand Fat Bloom Process

: Some studies found that the proportions of cocoa butter (CB), cocoa butter equivalent (CBE) and milk fatty acid (AMF) tend to inﬂuence the blooming delay when mixing them. The goal of our research is to determine the effects of the proportion of CB, CBE and AMF on the structural organization of the ﬁnal mixtures. X-ray, DSC, MIR and Raman spectroscopy were used to analyze the structural features and the vibrational modes of four mixtures: CB + 0.5AMF, CB + AMF, CB + 0.5AMF + CBE and CB + AMF + CBE. At room temperature, the triglycerides are ingredients of CB, and CBE and AMF do not fully exhibit the known crystalline forms V or VI, unlike a recent CB sample. Part of these triglycerides is in the form IV instead. The presence of the latter seems to be a key parameter that favors the deceleration of the transformation to the form VI, which is responsible for the development of fat


Introduction
The fatty blooming of chocolate is characterized by the loss of the initial gloss of its surface, giving it a more or less white appearance.For milk chocolate, this phenomenon is directly linked to the physico-chemical structure of cocoa butter (CB), cocoa butter equivalent (CBE) and white milk fatty acids (anhydrous milk fat: AMF) [1][2][3][4][5][6].
Sonvaï and Rousseau [1] have shown that the proportion of triglycerides in cocoa butter (CB), cocoa butter equivalent (CBE) and anhydrous milk fats (AMF) of chocolate strongly influences the speed of appearance of blooming.These authors have shown that the time in weeks before the appearance of polymorphic crystals in form VI responsible for the blooming of chocolate depended on the proportion by mass of the product of cocoa butter (CB), cocoa butter equivalent (CBE) and milk fatty acids (AMF): for the mixtures CB + 0.5AMF and CB + AMF, the blooming of chocolate appears after two weeks, the mixture CB + 0.5AMF + CBE after 20 weeks while for the mixture CB + AMF + CBE it appears after 30 weeks.
We notice that products containing cocoa butter equivalent (CBE) take much longer to bloom than products not containing it."CB + AMF" mixture, not containing CBE, only takes two weeks before blooming, compared to thirty weeks (i.e., fifteen times more) for "CB + AMF + CBE" mixture or to twenty weeks for "CB + 0.5AMF + CBE" mixture.CBE, therefore, appears to delay very significantly the transition of cocoa butter crystals in V-form to VI-form.In addition, it can be noted that a suitable amount of milk fatty acids (AMF) in a product containing CBE further delays the appearance of crystals in form VI. Indeed, "CB + AMF + CBE" mixture containing twice as much milk fatty acids as "CB + 0.5AMF + CBE" mixture takes 10 more weeks to bloom, that is to say, an additional delay in the transition of 50%.The more milk "chocolate" contains milk powder, in the presence of equivalent cocoa butter, the longer it would take to bloom.Adding fatty acid to milk to delay blooming has also been shown for dark chocolate.Indeed, a few years earlier, it had already been demonstrated that the addition of 1 to 2% of milk fatty acid in dark chocolate delayed the blooming of the product [7].This could be explained by the presence of triglycerides in the fatty acids of milk but not in cocoa butter.Another, more recent, study, conducted by Bisvas et al. [8], also demonstrated that the presence of cocoa butter substitute (from palm oil, noted CBS) in the composition of a dark chocolate delayed the blooming of the product.Indeed, they showed that after two weeks of storage at 29 • C ± 1 • C, a dark chocolate containing no cocoa butter substitute (CBS) bloomed, unlike a dark chocolate containing 20% CBS.However, their experiments also showed that chocolate containing only 5% CBS bloomed just like chocolate not containing CBS.The presence of CBS in dark chocolate therefore makes it possible to delay blooming, provided that its proportion in the product is sufficient.Da Silva et al. [9] also conclude that when the chocolate was subjected to temperature cycling, the resistance of CBS and CBE to the formation of fat bloom became more evident.
ATR-FTIR and Raman spectroscopy are non-destructive vibrational spectroscopy techniques which give sensitive information about molecular structure in solid and liquid TG conformational dependence [6,[10][11][12].Some bands of Raman spectra are particularly interesting to investigate the polymorphic structure of AMF.The bands in the spectral region 3200-2700 cm −1 correspond to the ν(C-H) stretching modes.The ν(C=O) ester carbonyl stretching region appears at 1800-1700 cm −1 , and the ν(C=C) stretching region (olefinic band) near 1660 cm −1 .
After separately studying the cocoa butter, the equivalent cocoa butter and the fatty acids of milk [13][14][15], we present a structural and vibrational study of the four mixtures studied by Sonvaï and Rousseau by X-ray diffraction, DSC, MIR and Raman spectroscopies.The objective is to better understand the phenomenon of fatty blooming of chocolate observed by Sonvaï and Rousseau for the same four mixtures.For this, we looked for markers of differentiation between these samples by structural studies according to the temperature and by vibrational studies.

Mixtures Preparation
Cocoa butter (CB) came from the Ivory Coast.CB, CBE and AMF were both purchased from the industry Cadbury (Canada).From mass spectrometry experiments by two different techniques, ESI-HRMS and MALDI-HRMS [6], we can conclude that our samples of CB and CBE are identical with respect to the types of triglycerides in them, but regarding the three main triglycerides, the components POS, SOS and POP, the proportions are different: in CB, POS dominates in quantity compared to the other two (practically 50% of the total), while for CBE, POP plays this role (practically 46% of the total) with a slight increase of SOS.
Cocoa butter, cocoa butter equivalent and milk fatty acids are solid fats at room temperature.They therefore had to be melted to make the mixtures at T = 60 • C.Then, the samples were stored in a refrigerator at T = 4 • C until used for the experiments at room temperature.Using a weighing scale accurate to one hundredth of a gram, the required mass of each sample was weighed.The samples were then melted using a heating magnetic stirrer, then mixed using the magnetic stirrer to obtain a homogeneous liquid.The cooling then took place naturally in the open air.In Table 1, the mixtures are presented by mass.

SAXS-WAXS Experiments
X-ray diffraction patterns were acquired using a microfocus X-ray tube (IµS, Incoatec, Geesthacht, Germany), selecting the Cu Kα radiation.It was used with an intensity of 1000 µA and a voltage of 50 kV.The incident beam was focused at the detector with multilayer Montel optics and 2D Kratky block collimator.Small-angle (SAXS) and wideangle (WAXS) X-ray scattering analyses were performed simultaneously using two positionsensitive linear detectors (Vantec-1, Bruker, Billerica, MA, USA) set perpendicular to the incident beam direction, up to 7 • (2θ) and at 19 • to 28 • (2θ) from it, respectively.The direct beam was stopped with a W-filter.The scattered intensity was reported as a function of the scattering vector q = 4π sin θ/λ where θ is half the scattering angle and λ is the wavelength of the radiation.The repeat distances d, characteristic of the structural arrangements, were given by q (Å −1 ) = 2π/d (Å).Silver behenate and tristearin (β form) were used as standards to calibrate SAXS and WAXS detectors, respectively.
All samples (~10 mg) were introduced into thin-walled glass capillaries (GLAS, Müller, Berlin, Germany) of 1.5 mm external diameter which were then placed in a specially designed temperature-controlled sample holder (Microcalix, Setaram, Lyon, France).For static measurements at 20 • C, the acquisition time was 10 min.For measurements with temperature, samples were heated at 2 • C/min and acquisition time was 1 min, leading to frame recording every 2 • C.

Differential Scanning Calorimetry (DSC)
DSC experiments were carried out on a Netzsch DSC 204 F1 Phoenix ® heat flux differential calorimeter at a heating rate of 2 • C/min under a constant argon flow with 200 mL/min.Each sample was heated from room temperature to T = 60 • C. Samples were weighed in aluminum sample pans covered with a pierced lid.An empty aluminum sample pan with a pierced lid was used as a reference.Three temperatures could be measured: T onset , T max and T offset , which correspond respectively to the beginning, the top and the end of thermal events.

MIR Spectroscopy
The MIR measurements were carried out at the Walloon Agricultural Research Center CRA-W, in Belgium.The apparatus consists of an FT-MIR Vertex 70 spectrometer (Brukeroptics, Ettlingen, Germany) equipped with a Golden Gate ATR (Attenuated Total Reflectance).This ATR consists of a monolithic diamond crystal.The incident beam contains radiations of 4000 to 600 cm −1 , which correspond to the medium infrared.The incident light penetrates the sample to a depth of 7 µm maximum, after having passed through the diamond.The reflected beam emerges through the diamond before reaching the detector.The spectral resolution was set at 1 cm −1 and the number of co-added spectra was set to 128 scans.The measurements were carried out at room temperature.Spectra of the ambient air was used as background.The ATR-FTIR spectra had undergone special processing, in order to be able to compare the intensity ratios between the samples.Before normalizing the spectra on the peak at 1729 cm −1 , which corresponded to the most intense peak in the spectral region 2000-50 cm −1 , we subtracted the baseline for each spectrum.

Raman Spectroscopy
The measurements were carried out at the Walloon Agricultural Research Center (CRA-W), Belgium.
RAMAN spectra were acquired using a SENTERRA II Bruker RAMAN spectrometer.This fully automated instrument combines excellent sensitivity and high resolution of 1.5 cm −1 .The experiments were carried out with a laser of wavelength λ 0 = 532 nm, of maximum power, Pmax = 25 mW, an acquisition time of 100 s and an addition of two spectra.This instrument makes it possible to obtain spectra ranging from 50 to 3470 cm −1 .The Raman spectra had undergone special processing, in order to be able to compare the intensity ratios between the samples.Before normalizing the spectra on the peak at 2885 cm −1 , which corresponded to the most intense peak in the spectral region 3200-50 cm −1 , or on the peak at 1743 cm −1 , which corresponded to the most intense peak in the spectral region 1800-1600 cm −1 , we subtracted the baseline for each spectrum.

Statistical Analysis
Regarding the determination of the Raman and ATR spectra, we chose as a research protocol to make five different spectra for each sample in order to observe the reproducibility of the results.In micro Raman, we proceeded to the determination of five spectra on five different positions of the incident laser for each sample, while in ATR-FTIR we carried out the same experiment five times by taking the same sample five times.Whether with ATR or Raman spectra, we observed no difference between the spectra for the same sample, confirming the reproducibility of our results.
In order to refine our study, we can perform models by Lorentzian functions of MIR and Raman spectra to determine the values of the wavenumbers as well as the areas of the associated peaks.The values in frequencies of the modes are obtained by the modeling carried out by the software ORIGIN 5.0 professional (from OriginLab, Northampton, MA, USA).The modeling method was proposed by Bresson et al. [16].Statistical analysis of the data was performed by analysis of variance (ANOVA) by the software ORIGIN 5.0.The level of significance was defined as p ≤ 0.05.
We used as peak function the following Lorentz function: where x c represents the value of the fit mode wavenumber, A the area of the peak and w the width at mid-height.We took an iteration number equal to 100.The error was estimated to be ±0.5 cm −1 .

Polymorphic Discrimination at 20 • C
Figure 1 exhibits the intensity of SAXS (Small Angle X-ray Scattering) (Figure 1a) and WAXS (Wide Angle X-ray Scattering) (Figure 1b) configurations at room temperature for "pure" compounds, CB, CBE and AMF, and for mixtures CB + 0.5AMF, CB + AMF, CB + 0.5AMF + CBE and CB + AMF + CBE.Peaks at small angles are assigned to long d-spacings, reflecting the lamellar structure of TG; peaks at wide angles correspond to short d-spacing, defining distances between chains of TG.
WAXS (Wide Angle X-ray Scattering) (Figure 1b) configurations at room temperature for "pure" compounds, CB, CBE and AMF, and for mixtures CB + 0.5AMF, CB + AMF, CB + 0.5AMF + CBE and CB + AMF + CBE.Peaks at small angles are assigned to long d-spacings, reflecting the lamellar structure of TG; peaks at wide angles correspond to short dspacing, defining distances between chains of TG.
Based on studies on CB, CBE and AMF [7,13,14], the diffraction peaks can be assigned: for SAXS, the peak at 63.5 Å corresponds to the first order of the triple chain structure 3L001, the peak at 32.1 Å to the second order of the same structure (3L002) and
Based on studies on CB, CBE and AMF [7,13,14], the diffraction peaks can be assigned: for SAXS, the peak at 63.5 Å corresponds to the first order of the triple chain structure 3L 001 , the peak at 32.1 Å to the second order of the same structure (3L 002 ) and the peak of center at 43.0 Å to the first order of a double length chain structure 2L 001 .For WAXS, we can identify CB V(β 1 ) form due to the last six peaks (4.03, 3.90, 3.79, 3.70 and 3.59 Å).The presence of the peaks at 4.03 and 3.79 Å indicates that V form is involved, and not VI form, according to the literature.Indeed, during the preparation of the mixtures, the pure compounds were melted up to T = 60 • C: the samples' fusion erased their polymorphic history.It is therefore not surprising that some TGs in the mixture are still in V form, especially when the sample has been stored under optimal conditions (at a temperature of 4 • C) as Bresson et al. [13] showed with the obtaining of the cocoa butter polymorph isolated protocol.The peak at 4.27 Å was present in the diffractogram of CBE at room temperature and was assigned to the 2L-β' structure of POP triglycerides [14,17].
In addition, we notice a strong variation in the intensity of the peak at 43 Å compared to the other two peaks at 63 Å and 33 Å depending on the type of mixture, or compared to CB and CBE.This will lead us to consider a new parameter ρ, which takes into account the part of the intensity of the I 43 peak at 43 Å compared to I 63 peak in SAXS: ρ = I 43 I 63 = I(2L 001 ) I(3L 001 ) .From the study of this parameter, we will be able to propose an approximation of the proportion of TGs in β' form relative to the whole mixture.At 20 • C temperature, this parameter ρ becomes worthy for CB and CBE: ρ CB = 0.127 and ρ CBE = 0.729.These results seem to indicate that for CBE there are 83% = ρ CBE −ρ CB ρ CBE more TGs in IV or β' form than for CB.
Concerning AMF, SAXS shows the typical 2L long spacing around 41 Å (q = 0.154 Å −1 and third order at 0.453 Å −1 ); WAXS are noisy, certainly because the solid fraction is low at room temperature and it is not possible to see the weak b' signature described in the literature [15].
Regarding the SAXS, it is interesting to note that the 2L structure seems to be imposed by the CB as the position of its third order, which is at 0.431 Å −1 instead of 0.453 Å −1 for AMF.This is less discriminant for the first orders, because the peak positions are very close but the peak at 0.145 Å −1 is similar to that of CB alone (0.146 Å −1 ) and slightly different to AMF alone (0.154 Å −1 ).
At T = 20 • C, the parameter ρ becomes worthy for this mixture: ρ CB+0.5AMF = 0.938.Compared to CB and CBE, this parameter is more important: it increases by 87% between CB and CB + 0.5AMF, whereas by mass TGs in β' form have increased by 50%, and by 22% between CBE and CB + 0.5AMF.
After increasing the amount of AMF in the CB + AMF mixture it can be seen that the peak positions are practically the same as for CB + 0.5AMF mixture.This is also true for 2L structure which keep the "positions of CB" even if the proportion of AMF is more important.In addition, the intensity of 2L 001 peak compared to 3L 001 and 3L 002 peaks is much greater for "CB + AMF" mixture than for "CB + 0.5AMF" mixture.
At T = 20 • C, we measure a value for the parameter ρ for CB + AMF of 1.531 while for ρ CB + 0.5AMF (T = 20 • C) = 0.936, i.e., a 38% increase of this ratio.We have to compare this parameter with the proportion by mass of TGs in IV or β form: between CB + 0.5AMF and CB + AMF, these TGs have increased by 33%.It would seem once again that the parameter ρ linked to the results obtained in X-ray for the SXAS follows the same evolution as the parameter µ linked to the mass distribution of TGs according to their polymorphic forms.
We find the same peaks as the last two mixtures presented.The peak assignment is therefore the same: the peaks at 64.8 Å, 44.2 Å and 32.9 Å correspond respectively to the structures 3L 001 , 2L 001 and 3L 002 ; and the peaks observed in WAXS correspond to β 1 form and to β form.
At T = 20 • C, we measure a value for the parameter ρ for CB + 0.5AMF + CBE of 0.694 while ρ CBE (T = 20 • C) = 0.729, i.e., very similar values.Regarding the CB + AMF + CBE mixture, the parameter ρ reaches 1.233 while ρ CB + 0.5AMF + CBE (T = 20 • C) = 0.694, i.e., a 44% increase of this ratio.For this mixture, the proportion by mass of TGs from AMF has increased by 40% comparatively to CB + 0.5AMF + CBE.It would seem that the ρ parameter is directly related to the mass distribution of TGs in IV form at the temperature T = 20 • C.

Behavior with Temperature
Figures 2 and 3 deal with the thermal behavior of the mixtures.More precisely, DSC curves obtained during first heating from room temperature to 50 • C are plotted in Figure 2.For all samples, the curve is not flat at the start of the experiment, which indicates that the samples are not completely solid at room temperature.Indeed, the mixtures have a pasty structure and are quite flexible at room temperature, unlike the pure compounds which seemed solid to the touch.
and to  form.
At T = 20 °C, we measure a value for the parameter ρ for CB + 0.5AMF + CBE of 0.694 while ρCBE(T = 20 °C) = 0.729, i.e., very similar values.Regarding the CB + AMF + CBE mixture, the parameter ρ reaches 1.233 while ρCB + 0.5AMF + CBE(T = 20 °C) = 0.694, i.e., a 44% increase of this ratio.For this mixture, the proportion by mass of TGs from AMF has increased by 40% comparatively to CB + 0.5AMF + CBE.It would seem that the ρ parameter is directly related to the mass distribution of TGs in IV form at the temperature T = 20 °C.

Behavior with Temperature
Figures 2 and 3 deal with the thermal behavior of the mixtures.More precisely, DSC curves obtained during first heating from room temperature to 50 °C are plotted in Figure 2.For all samples, the curve is not flat at the start of the experiment, which indicates that the samples are not completely solid at room temperature.Indeed, the mixtures have a pasty structure and are quite flexible at room temperature, unlike the pure compounds which seemed solid to the touch.Several events are clearly visible on the DSC traces with a mean peak between 30.8 • C and 34 • C (position of the peak maximum) and shoulders before (as is the case for CB + 0.5AMF, CB + 0.5AMF + CBE and CB + AMF + CBE mixtures) or after (as in the case of CB + AMF mixture).This suggests the presence of a second endothermic event of lower intensity.We deduce the coexistence of two distinct crystal structures at room temperature: one relating to the 2Lβ' form and the other in the 3Lβ form.This is confirmed by the SAXS patterns with temperature.For all the mixtures, the 3L structure (peaks at 64 and 32 Å) finishes to melt first, followed by the 2L organizations (peak at 44 Å) due to AMF and/or CBE.Appl.Sci.2022, 12, 6594 9 of 16
Bresson et al. [6] showed that CBE exhibits the same vibrational behavior in MIR spectroscopy as CB.It is questionable whether the presence of CBE, which delays chocolate blooming, manifests on MIR spectra.
We can distinguish different regions on the spectra shown in Figure 4a.Here, we will focus on the two following regions: • the spectral region 3200-2700 cm −1 corresponding to the ν(C-H) stretching mode (Figure 4b) • the spectral region 1800-1700 cm −1 corresponding to the ν(C=O) ester carbonyl stretching region (Figure 4c).If CBE has the same vibrational behavior in MIR spectroscopy MIR spectra of "CB + 0.5AMF" and "CB + AMF + CBE" mixtures s deed, both of these two mixtures contain one third (1/3) of AMF a cocoa butter (only CB or a mixture of CB and CBE).
In the spectral region 1800-1700 cm −1 (Figure 4c), it actuall no notable difference between the spectra of "CB + 0.5AMF" an mixtures.The models of MIR spectra of the four mixtures in this s sented in Figure 4b.In Figure 4a, the spectra obtained for CB, C temperature are presented for comparison [6].We observe four whose values of σ remain almost stable from one mixture to anot cm −1 ; 1743 cm −1 and 1751 cm −1 .
Moreover, it should be noted that the pure compounds also components for the vibrations ν(C=O) (see Figure 5).Bresson et al. [6] showed that CBE exhibits the same vibrational behavior in MIR spectroscopy as CB.It is questionable whether the presence of CBE, which delays chocolate blooming, manifests on MIR spectra.
We can distinguish different regions on the spectra shown in Figure 4a.Here, we will focus on the two following regions: • the spectral region 3200-2700 cm −1 corresponding to the ν(C-H) stretching mode (Figure 4b) • the spectral region 1800-1700 cm −1 corresponding to the ν(C=O) ester carbonyl stretching region (Figure 4c).
If CBE has the same vibrational behavior in MIR spectroscopy as CB in the mixtures, MIR spectra of "CB + 0.5AMF" and "CB + AMF + CBE" mixtures should be identical.Indeed, both of these two mixtures contain one third (1/3) of AMF and two thirds (2/3) of cocoa butter (only CB or a mixture of CB and CBE).
In the spectral region 1800-1700 cm −1 (Figure 4c), it actually seems that there is no notable difference between the spectra of "CB + 0.5AMF" and "CB + AMF + CBE" mixtures.The models of MIR spectra of the four mixtures in this spectral region are presented in Figure 4b.In Figure 4a, the spectra obtained for CB, CBE and AMF at room temperature are presented for comparison [6].We observe four components in total, whose values of σ remain almost stable from one mixture to another: 1729 cm −1 ; 1735.5 cm −1 ; 1743 cm −1 and 1751 cm −1 .
Moreover, it should be noted that the pure compounds also presented these four components for the vibrations ν(C=O) (see Figure 5).The values of the peaks of the four mixtures as well as those of the compou alone are grouped together in Table 2.The values of the peaks in cm −1 of the mixt and of the pure compounds are almost identical, except for the component at 17 cm −1 for AMF which is located at 1739.8 cm −1 .In Table 2, we present the values o areas of modes with each component.It can be seen that "CB + 0.5AMF" areas "CB + AMF + CBE" mixtures are similar.In conclusion, it would seem that the p ence of CBE instead of CB is not visible in MIR spectroscopy in the 1800-1700 area.In Figure 5b, it can be seen that some components have larger areas in some mixt than in others.It is therefore interesting to comment on the evolution of the intensity the area of each peak from one mixture to another, using the values of the areas of component (Table 3).For example, the peak area at 1743 cm −1 is the highest for "C AMF" mixture, which contains the most AMF (50%) (area = 26.2au) and the lowes The values of the peaks of the four mixtures as well as those of the compounds alone are grouped together in Table 2.The values of the peaks in cm −1 of the mixtures and of the pure compounds are almost identical, except for the component at 1735.5 cm −1 for AMF which is located at 1739.8 cm −1 .In Table 2, we present the values of the areas of modes with each component.It can be seen that "CB + 0.5AMF" areas and "CB + AMF + CBE" mixtures are similar.In conclusion, it would seem that the presence of CBE instead of CB is not visible in MIR spectroscopy in the 1800-1700 cm −1 area.In Figure 5b, it can be seen that some components have larger areas in some mixtures than in others.It is therefore interesting to comment on the evolution of the intensity and the area of each peak from one mixture to another, using the values of the areas of each component (Table 3).For example, the peak area at 1743 cm −1 is the highest for "CB + AMF" mixture, which contains the most AMF (50%) (area = 26.2au) and the lowest for "CB + 0.5AMF + CBE" mixture, which contains the least (20% of AMF) (area = 6.1 au)."CB + 0.5AMF" and "CB + AMF + CBE" mixtures, which both contain 33% of AMF, have an area of intermediate and almost identical values (area = 13.5 au for CB + AMF + CBE and area = 14.8 for CB + 0.5AMF).It would therefore seem that the peak at 1743 cm −1 mainly reflects the vibrations ν(C=O) of TGs of AMF.In conclusion, CBE TGs have the same vibrational behavior in MIR spectroscopy as CB TGs in mixtures.This is not very surprising, since the study of CB alone and CBE alone did not reveal any vibrational differences in MIR [14].On the other hand, the detailed study of spectra in MIR spectroscopy in the spectral zone 1800-1700 cm −1 allows us to know the major contribution of one of the elements of the mixtures for three vibrational modes: the peaks at 1751 and cm −1 are predominantly sensitive to AMF while the peak at 1735.5 cm −1 is sensitive to CB and CBE.

Raman Investigations
Bresson et al. [14] showed that MIR spectroscopy study of CB and CBE failed to find vibrational modes that could differentiate CB from CBE, unlike Raman spectroscopy.In addition, A. Lambert et al. showed a vibrational behavior for AMF quite different from CB and CBE in Raman spectroscopy at room temperature [15].Raman spectra at room temperature for the four mixtures are represented in Figure 7a, 7b and 7c respectively in the spectral zones 3100-600, 3100-2700 and 1770-1710 cm −1 .

Raman Investigations
Bresson et al. [14] showed that MIR spectroscopy study of CB and CBE failed to find vibrational modes that could differentiate CB from CBE, unlike Raman spectroscopy.In addition, A. Lambert et al. showed a vibrational behavior for AMF quite different from CB and CBE in Raman spectroscopy at room temperature [15].Raman spectra at room temperature for the four mixtures are represented in Figure 7a, 7b and 7c respectively in the spectral zones 3100-600, 3100-2700 and 1770-1710 cm −1 .The spectral region 1800-1700 cm −1 corresponding to the vibration of elongation of the carbon-oxygen double bonds, noted ν(C=O), was found to be very rich for the three pure compounds [14,15].It is therefore interesting to study this zone for mixtures.For this, one carries out modeling by Lorentzian functions for the four mixtures (see Figure 8b).In order to analyze this spectral region more easily, Raman spectra of the pure compounds at room temperature are presented (see Figure 8a).Modeling makes it possible to determine the value in cm −1 of the center of the peaks (Table 4) as well as the areas of each peak (Table 5).The spectral region 1800-1700 cm −1 corresponding to the vibration of elongation of the carbon-oxygen double bonds, noted ν(C=O), was found to be very rich for the three pure compounds [14,15].It is therefore interesting to study this zone for mixtures.For this, one carries out modeling by Lorentzian functions for the four mixtures (see Figure 8b).In order to analyze this spectral region more easily, Raman spectra of the pure compounds at room temperature are presented (see Figure 8a).Modeling makes it possible to determine the value in cm −1 of the center of the peaks (Table 4) as well as the areas of each peak (Table 5).The component at 1735 cm −1 does not exist in AMF, whereas it exists in all other sam ples [13].It can thus be used as a witness of the contribution of AMF in mixtures.In Tab 1, it can be seen that between "CB + 0.5AMF" and "CB + AMF" mixtures, the variations i the area ratios are very significant.The modes at 1743 and 1730 cm −1 are common to a mixtures.Between these two mixtures, AMF's contribution is doubled.We notice that th

Figure 5 .
Figure 5.The MIR carbonyl stretching region (1770-1710 cm −1 ) fitted by Lorentzian curves of CB, CBE and AMF (a) and of the four mixtures (b): CB + 0.5AMF, CB + AMF, CB + 0.5AMF + CBE and CB + AMF + CBE, at room temperature.The solid lines represent the components fitted by Lorentzian functions.

Figure 6 .
Figure 6.The MIR C-H stretching region (3050-27500 cm −1 ) fitted by Lorentzian curves of CB + 0.5AMF + CBE at room temperature.The solid lines represent the components fitted by Lorentzian functions.

Figure 6 .
Figure 6.The MIR C-H stretching region (3050-27500 cm −1 ) fitted by Lorentzian curves of CB + 0.5AMF + CBE at room temperature.The solid lines represent the components fitted by Lorentzian functions.

1 )Figure 8 .
Figure 8.The Raman carbonyl stretching region (1770-1710 cm −1 ) fitted by Lorentzian curves of C CBE and AMF (a) and of the four mixtures (b): CB + 0.5AMF, CB + AMF, CB + 0.5AMF + CBE an CB + AMF + CBE, at room temperature.The solid lines represent the components fitted by L rentzian functions.

Figure 8 .
Figure 8.The Raman carbonyl stretching region (1770-1710 cm −1 ) fitted by Lorentzian curves of CB, CBE and AMF (a) and of the four mixtures (b): CB + 0.5AMF, CB + AMF, CB + 0.5AMF + CBE and CB + AMF + CBE, at room temperature.The solid lines represent the components fitted by Lorentzian functions.

Table 1 .
Composition of the mixtures of cocoa butter (CB), cocoa butter equivalent (CBE) and milk fatty acids (AMF).

Table 2 .
Areas of the components of MIR spectra for C=O carbonyl group for CB, CBE, A and mixtures.

Table 2 .
Areas of the components of MIR spectra for C=O carbonyl group for CB, CBE, AMF and mixtures.

Table 4 .
Values of the ν(C=O) (cm −1 ) of the peaks of Raman spectra of CB, CBE, AMF and mi tures at room temperature.* values obtained by Lorentzian modeling.

Table 5 .
Evolution of the area ratios of the components of Raman spectra for the carbonyl grou C=O for the four mixtures.

Table 4 .
Values of the ν(C=O) (cm −1 ) of the peaks of Raman spectra of CB, CBE, AMF and mixtures at room temperature.* values obtained by Lorentzian modeling.

Table 5 .
Evolution of the area ratios of the components of Raman spectra for the carbonyl group C=O for the four mixtures.