Crystallization and melting behavior of cocoa butter in lipid bodies of fresh cacao beans

The present study aims at observing the crystallization and polymorphic behavior of cocoa butter (CB) in fresh cacao beans with DSC and X-ray diffraction techniques. Underlying idea of this study was to relate the necessary conditions of germination of cacao beans to the crystallization of CB, which are present in oil-in-water emulsion droplets (lipid bodies) having 1~2 μm diameters. Different cooling and heating conditions, with rates of 15, 2, 0.5 and 0.1°C/min, were applied to


INTRODUCTION
Chocolate is a solid system formed by a cocoa butter (CB)-continuous matrix containing dispersed particles of cacao mass, sugar and other ingredients. Within this complex microstructure, CB crystals are determinant for the unique sensory characteristics of flavor release, mouthfeel and melting properties of chocolate, which is manufactured from the beans of Theobroma cacao. When cacao pods ripen, they are harvested by cutting them directly from the trunk of the tree and, when opened, several cacao beans, which are covered by a white mucilaginous pulp, are extracted. Immediately following harvesting, cacao beans are subjected to fermentation and drying processes, during which flavor precursors are developed.
Cacao beans are dicotyledonous seeds, as they consist of two cotyledons and a small embryo, which are protected by a shell called testa (see schematic illustration in Figure 1). Cotyledons may become the embryonic primary leafs when the seed germinates, but they also contain storage cells for the development of the seedling. The most abundant cell types are tiny lipid/protein storage cells, which contain a large number of lipid vacuoles embedded in 4 cytoplasm. Thus, most of the energy is stored in the form of native oil-in-water emulsion, that is to say, in lipid droplets or lipid bodies mainly constituted by CB, which represent almost the 50% (w/w) in fresh cacao beans, as shown in Table 1. 1,2 Lipid bodies are, therefore, dynamic cellular organelles that serve as important reservoirs of lipids, but also as substrates for multiple cellular processes. The lipids contained are surrounded by a phospholipid monolayer and associated proteins, and this surface composition becomes determinant for regulating lipid bodies size and their ability to interact with other lipid bodies or organelles. 3,4  Cocoa butter can crystallize in six different polymorphic forms (form I to VI) and, among them, form V is industrially promoted due to its unique texture, snap, gloss, melting, and mouthfeel characteristics. 5 Much research has been carried out on the crystallization and polymorphic 5 behavior of CB and its three main component triacylglycerols (TAGs), which are 1,3dipalmitoyl-2-oleoyl-glycerol (POP), 1,3-distearoyl-2-oleoyl-glycerol (SOS), and rac-palmitoylstearoyl-2-oleoyl-glycerol (POS). [6][7][8][9][10][11][12] However, to the best of our knowledge, most work related to the crystallization behavior of CB was carried out by using CB in bulk state.
In the present work, we studied the crystallization and polymorphic behavior of the CB in fresh cacao beans, aiming at relating them to germination behavior of cacao bean. CB are present in lipid bodies in fresh cacao beans and it comprises more than 50 % among three nutrients together with proteins and carbohydrates in dried matters of cacao nibs. It is hypothesized that the crystallization of CB in the lipid bodies at chilled temperatures may negatively affect the germination behavior of cacao beans, and this is one of the reasons why cacao tress can survive solely at tropical areas.
The results thus obtained were compared with CB in bulk state at the same experimental conditions, as crystallization behavior in emulsion droplet is different from bulk crystallization. 13 In order to monitor the occurrence of different polymorphic forms, we selected varied thermal treatments based on different cooling and heating rates. The influence of dynamic temperature variations on the polymorphic behavior of main TAGs of edible fats and oils was already analyzed by our group, 10,[14][15][16] and the results demonstrated that desired polymorphic forms with specific physicochemical properties may be obtained by tailoring appropriate dynamic thermal treatments.
We also compared crystallization temperatures of CB in fresh beans and bulk state. The observation of the crystallization behavior of CB in fresh cacao beans with differential scanning calorimetry (DSC) and synchrotron radiation X-ray diffraction (SR-XRD) may permit to relate 6 the necessary conditions of germination of cacao beans, which are strongly determined by its presence in oil-in-water emulsion droplets.
Fresh cacao beans were selected prior to fermentation, as enzymatic changes during fermentation involve obvious structural modifications, such as the re-deposition of lipids within cells, which may modify the crystallization behavior of CB. 1   Three independent measurements were made for each experiment (n = 3). Random uncertainty was estimated with a 95% threshold of reliability using the Student's method.
Samples of Dominican Republic fresh cacao beans and CB were subjected to varied dynamic thermal treatments based on the application of different cooling and heating rates. Thus, samples were cooled from 55 °C to -80 °C and reheated to 55 °C at the rates of 15, 2, 0.5 and 0.1 °C/min. DSC results were complemented and interpreted with X-ray diffraction experiments, using laboratory-scale or synchrotron radiation source, at the same experimental conditions. A 8 correction (described elsewhere 19 ) was applied for analyses with cooling or heating rates other than 2°C/min, since the calorimeter was calibrated at this rate.
DSC experiments based on cooling and heating of 2 °C/min were also conducted in fresh and dried cacao beans with other geographical origins (India, Colombia and Philippines).

Laboratory-Scale X-ray Diffraction
Laboratory-scale powder XRD experiments were performed by using a PANalytical X'Pert Pro Lindemann glass capillary, which was rotated about its axis during the experiment to minimize preferential orientations of the crystallites. The step size was 0.026° from 1° to 28° 2θ and the measuring time of 75 s per step. X'Pert HighScore software was used to process XRD data.

Synchrotron Radiation X-ray Diffraction
Synchrotron radiation X-ray diffraction (SR-XRD) experiments were conducted on beamline BL11-NCD-SWEET at the synchrotron ALBA (Cerdanyola del Vallès, Barcelona, Spain) at 12.4 9 keV. The sample-detector distance was 2.2 m. X-ray scattering data were collected on a Pilatus 1M detector with a pixel size of 172 × 172 μm 2 for the small-angle X-ray diffraction (SAXD) data and on a LX255-HS Rayonix detector with a pixel size of 44 × 44 mm 2 for the wide-angle X-ray diffraction (WAXD) data. The temperature of the sample was controlled by a Linkam stage. SR-XRD experiments were particularly required for thermal treatments based on high cooling and heating rates of 15 °C/min. However, SR-XRD patterns were also acquired for cooling-heating processes carried out at 2 and 0.5 °C/min. The sample was placed in an aluminum sample cell with a Kapton film window. The q-axis calibration was obtained by measuring silver behenate for SAXD and Cr2O3 for WAXD. The program pyFAI was used to integrate the 2D WAXD into the 1D data; the SAXD data were processed with in-house software.

Rhizogenesis experiments
Vietnam cacao beans were subjected to rhizogenesis experiments, which consisted of determining stem and root shooting when fresh beans remained several days at fixed temperatures. Then, fresh beans were washed to remove gel-like white pulp and set, under wet conditions, at fixed temperatures of 17, 20 and 32 °C for 15 days.

Polymorphic crystallization and transformation behavior
10 Figure 2 shows the polymorphic behavior of cocoa butter in bulk (left side in Figure 2) and fresh cacao beans (right side) determined when samples were cooled from 55 °C to -80 °C at a rate of 15 °C/min and reheated to 55 °C at the same rate.  Table 2), which, according to the SR-XRD data, corresponded to a concurrent crystallization of metastable forms I and II. In more detail, initial form II was firstly detected with the occurrence of SR-SAXD peak The same dynamic thermal treatment was applied to fresh cacao beans and significant differences were detected compared to the bulk CB case. Most important variations were related to the initial crystallization temperatures, as shown in Table 2. Thus, at approximately 12.9 °C, that is at a temperature almost 4 °C lower than that of bulk CB, an exothermic signal was observed On further cooling, the DSC thermogram showed the presence of a sharp exothermic phenomenon with peak top temperature at around 22 °C, which was due to the crystallization of the water contained in fresh cacao beans. Ice crystals melted at approximately 0 °C (peak top temperature), as shown in the corresponding DSC heating thermogram and, afterwards and similarly to bulk CB, Form II and I melted (onset temperature of 12.5 ± 1.8 °C), which was confirmed by the disappearance of SR-XRD peaks. By comparing the SR-XRD patterns of the two samples, one 16 may note significant differences in the relative intensity of the peaks. In more detail, form I peaks became more intense than those of form II in bulk CB, whereas the peaks intensity of the two forms became comparable in fresh cacao beans. This means that higher amount of metastable form I was obtained in bulk, and higher amount of more stable form II could be obtained in fresh beans at the same experimental conditions.
More complex polymorphic behavior was observed when the two samples were subjected to intermediate cooling/heating conditions of 2 °C/min. Figure 3 depicts the polymorphic behavior of bulk CB (left side) and fresh cacao beans (right side) when samples were cooled from 55 °C to -80 °C and reheated to °C at the mentioned rate.  The same behavior was monitored when lower rate of 0.5 °C/min was applied, as depicted in In more detail, in bulk state, concurrent crystallization of forms I and II was observed for cooling rates of 15, 2 and 0.5 °C/min. However, the amount of form II increased at the expense of form I as the cooling rate decreased, as confirmed by the relative intensity of SR-SAXD peaks in Figures   2, 3 and 4. In other words, form I predominated at 15°C/min, whereas form II dominated for cooling rates of 2 and 0.5 °C/min, as pointed out in Figure 6 (squares). More stable forms II, III and IV crystallized from the melt at lower cooling rate of 0.1 °C/min. As to the variation of heating rates and starting from form I and II crystals, they simply melted by quickly heating at 15 °C/min, 26 whereas further polymorphic transformation to more stable forms took place at lower rates. Then, form III and IV were reached at heating rates of 2 and 0.5 °C/min, respectively. More stable form V was obtained by heating crystals of form II, III and IV at the lowest rate of 0.1 °C/min. CB in fresh cacao beans exhibited a simpler polymorphic behavior than that of CB in bulk state, and more stable polymorphic forms were obtained at the same experimental conditions. Similarly to the bulk case, concurrent crystallization of form I and II was obtained, but with higher predominance of form II than in bulk state (see relative stability of SR-SAXD peaks in Figure 2).
Furthermore, no form I crystallized at 2 and 0.5 °C/min and only form II was present when cooling.
Finally, form III and IV were obtained at 0. In addition, the results showed that initial CB crystallization temperatures in fresh beans were lower by ~ 4 -6 °C than that of bulk at all experimental conditions. Furthermore, end crystallization temperatures were much lower in bulk than in fresh beans, so that ΔT corresponding to the whole crystallization process was much higher in bulk (~ 10 -19 °C), as summarized in Table 3. * This peak was so flat (due to low rate and lower intensity of DSC signals compared to bulk) that the onset and end Ts were not so easy to define.
In order to understand the decreased crystallization temperature and different polymorphic behavior of CB contained in fresh cacao beans, one may pay attention to its microstructure. As studied by Lopez et al. (1987) 1 with scanning electron microscopy techniques, most abundant cell types in cacao beans are small lipid/protein storage cells, which contain large number of lipid vacuoles or globules embedded in cytoplasm. That is, CB is contained in oil-in-water emulsion droplets (lipid bodies) of around 1-2 µm inside cacao beans (see Figure 1). Lipids in emulsion droplets crystallize and melt just like bulk lipids, but the kinetics of the processes may be different.
Although emulsified fats melt about the same temperature as fats in bulk, they crystallize at much lower temperature, and this temperature may decrease as droplets diameter decreases. 13 Recent work on thermodynamics and nucleation kinetics of lipid crystals in oil droplets was extensively reviewed by Ueno (2011), 20 McClements (2012) 21 and Povey (2014). 22 As carefully described by McClements, the difference in the formation of nuclei due to supercooling in oil-inwater emulsions and bulk may be due to the extremely small volume of the lipid phase within each droplet. In bulk oil, the formation of nuclei usually occurs by heterogeneous nucleation, because of the presence of impurities or other substances. However, in emulsion state, it may proceed through homogeneous nucleation, as the probability of finding catalytic impurities in a droplet is so small. Therefore, in order to observe nucleation and crystallization, one may apply a higher supercooling to a lipid in emulsion than to the same lipid in bulk state.
Regarding the different polymorphic behavior observed in CB bulk and CB contained in fresh cacao beans, one may consider that polymorphic transformation tends to be much faster in emulsified lipids than in bulk oil, although some factors may modify this behavior, such as particle size, lipid type and the use of particular surfactant systems. The higher rate of polymorphic transformation in emulsion state has been attributed to smaller crystal size in emulsified fats compared to bulk, but also interfacial phenomenon. 21

Thermal analysis of cacao beans with different geographical origins
We also determined the thermal behavior of fresh cacao beans with different geographical origins. Figure   The thermal behavior of fresh cacao beans having different geographical origins were essentially the same. As depicted in Figure 7a, DSC cooling curves consisted of two exothermic signals: a first one with Tonset between 15 and 20 °C due to CB crystallization, and another one between 20 and 30 °C, corresponding to ice formation. Regarding the subsequent heating step (Figure 7b), ice melted at around 0 °C, and Tonset of CB melting was observed within the temperature range from 12 to 17 °C. No significant differences were detected between DSC thermograms of fresh and dried cacao beans having the same geographical origin, except for the presence of water in fresh beans. Furthermore, endothermic CB melting peaks became flatter in some dried samples, although onset and end temperatures were similar to those of fresh beans (see Table 4).

Rhizogenesis experiments
The different crystallization behavior of emulsified CB in fresh cacao beans and CB in bulk state may explain germination conditions of cacao beans. We may assume that a condition for germination is that CB in cacao beans is in the liquid state, since enzymatic activity to convert CB to the nutrients for the germination of cacao bean is prohibited. In other words, no germination must occur when CB in intact cacao bean is in the solid state.
To demonstrate this, we conducted several rhizogenesis experiments by incubating fresh cacao beans under wet conditions at fixed and controlled temperatures of 17, 20 and 32 °C. Figure 8a shows resultant cacao beans after incubation for ten days at 32, 20 and 17 °C. Rooting was detected at 32 °C, whereas just stem shooting was observed at 20 °C and no significant changes occurred at 17 °C after ten days of incubation. The occurrence of stem and roots were noted just after 2 and 10 days of incubation at 32 °C, respectively, and roots grew as the days went by (see Figure 8b). At a working temperature of 20 °C, fresh beans did not experience any changes after two days of incubation, and stem emerged after 10 days. Finally, roots were developed after 15 days at 20 °C. These results proved that the occurrence of stem and rooting processes were promoted by increasing incubation temperatures, at which CB in fresh cacao beans remained in the liquid state. By contrast, at lower temperatures of 17 °C, which are close to onset crystallization temperature of CB in fresh beans at the experimental conditions analyzed (see Table 3), no rooting occurred. However, germination cannot occur below 18 °C due to CB crystallization. The fact that CB is in emulsion state in fresh cacao beans is the responsible for the lowering of Tc of CB compared to bulk state. As liquid state of CB is needed for cacao beans germination, growing environmental areas with temperatures higher than 16-17 °C are required. In other words, germination conditions of cacao beans are extended to low temperature environmental areas by lowering the Tc of CB in emulsion droplets than those in the bulk oil.

CONCLUSIONS
This study examined and compared the crystallization and polymorphic behavior of CB in fresh cacao beans and in bulk state at the same experimental conditions of varied cooling and heating rates. Simpler polymorphic behavior, based on the occurrence of more stable polymorphic forms, was observed in fresh cacao beans. Furthermore, CB in fresh cacao beans crystallized at significantly lower temperatures compared to bulk CB, due to the presence of CB 33 in the form of oil-in-water emulsion droplets. The thermal behavior of fresh cacao beans having different geographical origins was also analyzed, and no significant differences were detected between the different varieties. The results obtained in this work were consistent with rhizogenesis experiments at fixed temperatures, and permitted to relate the necessary conditions of germination of cacao beans at concrete growing environmental areas.