Interactions in interesterified palm and palm kernel oils mixtures . II – Microscopy and Differential Scanning Calorimetry

Fueron interesterificados en el laboratorio mezclas de aceite de palma (PO) y aceite de palmiste (PKO) en diferentes proporciones (100/0, 80/20, 60/40, 50/50, 40/60, 20/80 y 0/100) bajo condiciones predeterminadas (0.4% metoxido de sodio, 20 minutos, 100C). Las catorce muestras fueron caracterizadas antes y después de la interesterificación por Microscopía de Luz Polarizada y por Calorimetría Diferencial de Barrido (DSC). Los resultados mostraron el efecto de varios factores sobre la forma y anchura de los cristales. El área media de los cristales revela el aumento de tamaño de los mismos cuando aumenta la proporción de PKO, con valores que varían entre 2.7 x 10 μm para PO y 1.8 x 10 μm para PKO. Después de la interesterificación, la anchura de los cristales fue menor para las formulaciones de PO/PKO de 100/0, 80/20, 60/40 y 20/80, y fue mayor en las otras. La forma polimórfica β‘ se observo en la muestra de aceite de palma puro. Los resultados mostrados por las curvas de fusión, presentan valores de “onset” desde –19.6C para los picos correspondientes a los triglicéridos más insaturados, hasta 20.7C para los más saturados. Los valores mayores de entalpía de fusión de los picos más saturados del aceite de palma, es 38.7 J.g antes y 48.4 J.g después de la interesterificación, mostrando un grupo saturado más estable. De forma global, la interesterificación causa un incremento en el gradiente de cristalización y mejora la compatibilidad en las fracciones de PO/PKO.


INTRODUCTION
The utilization of chemical interesterification in fats and oils mixtures is very important and versatile, which allows the combination of different fats and oils properties, with final use in margarines and shortenings.The raw materials react in a catalyst presence at mean temperature of 100 o C, that promoting a random distribution of fatty acids bound to triacylglycerides, leading to the formation of new compounds with different physical properties (Allen, 1996).
Several instrumental techniques are used to monitoring the interesterification reaction, that include differential scanning calorimetry as an important tool to investigate transition of crystalline forms during fats melting (Zeitoun et al., 1993).When the fat is heated, it can exhibit multiple melting phases, such as each crystallization step represents a transition phase from a less stable polymorphic form to a more stable one (Schmidt et al., 1996).The peak temperature transition can be an important indicator of crystal polymorphic form, since the more stable form has a higher melting point (Zeitoun et al., 1993).Nassu (1994) studied a thermal behavior of deodorized Brazilian oils like soybean, cottonseed, palm, palm kernel and coconut, besides hydrogenated, fractioned and interesterified oils.In other study, Grimaldi (1998) characterized eight samples of hydrogenated fats, used in margarine productions.
Melting termograms, through differential scanning calorimetry supply valorous information on how the products melt in the mouth during mastication.The partial area values, identified under the melting peak (endothermic), is equivalent to the remaining solids percent at the selected temperature and this value is higher than the values determined through nuclear magnetic resonance (Ali and Dimick, 1994).Cebula and Smith (1991) utilized DSC in the investigation of influence of progressive changes on the formulation of cocoa butter substitutes and compounds with saturated triacylglycerides and other polar compounds, normally present in bakery fats, through calorimetric curves.DSC was considered as a sensible technique to shortening characterization, permitting a good control in comparing with similar thermal behavior and that present melting and crystallization at similar conditions.Changes in a thermal profile are attributed to differences in the triacylglycerides compositions of products.Deman et al. (1989) used DSC to peak temperature, enthalpy and crystallization temperature verification.In another study, Deman et al. (1991) many shortenings were characterized getting different melting curves.
Another technique utilized in a crystallization study is polarized light microscopy, that can distinguish between liquid and solid phases, because the crystals are anisotropic whereas liquid fat is isotropic (Rousseau et al., 1996).
Exams of randomized lard (chemically interesterified) with polarized light microscopy showed that its composition presented small spherulites rather than large ones in the native lard (Rousseau et al., 1996).Hurtová et al. (1996) accomplished random interesterification of sunflower and canola oils mixtures with fully hydrogenated oils.In these reactions, sodium hydroxide and sodium methoxide were used as catalysts.The evaluations were made with polarized light microscopy, where the crystalline structure was observed.The composition photos showed small crystal clusters, characteristics for beta prime form.The crystallization of original samples was very fast, promoting clusters with higher crystals, characteristics of beta form.Rousseau et al. (1996) used the polarized light microscopy in the visualization of butter and canola oils mixtures before and after interesterification.The addition of canola oil promoted a gradual aggregation of crystalline structure.The micro photos of butter oil before the reaction showed a dense network of spherulites, with measure varying from 10-25 µm in size.After the reaction, a crystal network was composed of spherulites of varied densities, measuring around 15 µm, with a lacy network of small crystals.
The objective of this work was to make an investigation of interesterified palm and palm kernel oils fractions and their characterization through differential scanning calorimetry and polarized light microscopy.Another objective was the visualized of eutectic systems between binary mixtures of palm and palm kernel oils.

Microscopy analysis of crystals
Slide preparationthe samples were melted at 60-70 o C to complete melting of crystals.The laminas to microscopy were put on the heating plate (Thermal Microscope Stage, TS-4 series, PHYSITEMP INSTRUMENTS INC.) adjusted at 50 o C, fitted with a bath (Thermo Bath, model TB-85, Shimadzu Corporation, Japan) during 5 minutes to temperature stabilization.The samples were put on the laminas with the aid of capillary tubes (1 drop ≅ 12mg) and dispersed with the cover.The crystallization was carried at 25 and 35 o C during 48 hours.
Crystal visualizationwith polarized light microscopy (Olympus System Microscope, model BX 50, Olympus America Inc.).Amplification of 40X, 100X and 200X, in accord to width of crystals.The visualization was done at constant temperature.

RESULTS AND DISCUSSION
The laminas, conditioned according to methods section, were measured and the crystal data are 364 Grasas y Aceites showed on Table I.The parameters available were mean area and diameter.
The reading temperature was based on previous experience.In some cases at 35 o C, the samples were completely melted.The interesterification promoted a decrease of crystal sizes of 100/0, 80/20 and 60/40 PO/PKO fractions, while that to other samples, the interesterifcation promoted an increase of crystal sizes, with exception of PKO, where the mean area was from 1.8 x 10 6 to 9.8 x 10 5 µm 2 .
Many factors influence lipid crystallization, like tempering method, interactions between components and crystallization time.The eutectic effect, visualized at PO/PKO fractions is another parameter that affects the crystallization rate.
The evaluation parameters, calculated through obtained termograms, were similar to those cited by Nassu (1994).The Table II shows melting enthalpy values of PO/PKO compositions before and after interesterification.
The melting termograms and images at 25 o C can be visualize in the next figures.
The interesterification of PO/PKO 100/0 fraction excluded the area below base line in the melting curve, related to an increase of crystallization rate.The process avoided the simultaneous melting and crystallization, observed through the portion below the base line and visualized at 100/0 fraction before interesterification (Nassu, 1994).
This behavior of simultaneous crystallization and melting was studied by Cebula and Smith (1991).At this work, the effect of different crystallization and melting rates in pure triacilglyceride termograms   (POS and POP) was studied.After fast POP crystallization, different heating rates showed many polimorphic transitions to the same original sample.
The PO/PKO 80/20 showed many peaks in the melting curves before interesterification, with similar characteristics after reaction, and small differences in more unsaturated region (0 o C).The complexity of    Grasas y Aceites termograms can suggest a high incompatibility between triacilglycerides of the samples.Other melting curves presented similar behavior, with only a large melting peak.
The palm kernel oil presented a single melting peak, due to a high concentration of trisaturated triacylglycerides.The interesterification reaction to palm kernel oil modified the crystalline morphology, with more structured crystals.Berger and Ong (1985) cited that after interesterification, the applicability of palm kernel oil was improved, due to its better plasticity, characteristics of beta prime crystals.
Kaiserberger (1989) accomplished the thermal characterization of many oils and fats and concluded that the melting range and curve profile are dependent of many combined effects like triacylglycerides composition, polymorphism and thermal history.The Figures 8 and 9 show the melting curve to pure triacylglycerides.
The mathematic model, calculated by multiple regression for two components mixtures, according to Hare (1974) is the following: Where Y is the estimated mean area or mean diameter of crystals.X 1 is palm oil proportion, X 2 is palm kernel oil proportion.X 2 + X 1 = 100.The R 2 is used to estimate the adjustment of the mathematical model for the to obtained data (Table III).
A decrease of absolute values of β 1.2 , after the interesterification proved the better compatibility of palm and palm kernel oils.

CONCLUSION
Palm and palm kernel fractions showed characteristics of eutectic system, mainly at 80/20 and 50/50 PO/PKO fractions.The chemical interesterification at 100 o C and 20 minutes with 0,4% of sodium methoxide minimized the incompatibility between the fractions and improved the plasticity.
Crystallization profile of interesterified samples showed a better compatibility between the oils, with a sharp and well-defined peaks.The mathematic model using the diameter and area data of crystals, proved the decrease of eutectic system after chemical interesterification.
Figure 1 Melting curves and images of PO-100 before (b) and after (a) chemical interesterification.
Figure 5 Melting curves and images of PO/PKO 40/60 before (b) and after (a) chemical interesterification.
Figure 3 Melting curves and images of PO/PKO 60/40 before (b) and after (a) chemical interesterification.

Figure 9
Figure 9Melting curves of pure triacilglycerides (POP and SOS).

Table I Statistical data to crystal parameters of PO/PKO mixtures at 25 o C and 35 o C before and after chemical interesterification
b -before interesterification; a-after interesterification.Mean of standard deviations observed at each temperature -± 10%.

Table III Interactions between PO/PKO mixtures before and after chemical interesterification. Mean area and diameter data
b -before interesterification; a-after interesterification