There is a growing consensus among authors regarding the importance of determining the minimum concentration of vegetable wax needed to form an organogel at room temperature (Hwang et al., 2012; Blake et al., 2014; Sagiri et al., 2015). The lowest concentration of a vegetable wax needed to structure an oil is named “critical concentration”. Vegetable waxes with the lowest critical concentrations have shown the best organogelation properties (Hwang et al., 2012; Hwang et al., 2013; Blake et al., 2014). The samples used to determine the critical concentration may be prepared and passed through the tempering process directly in the analysis vials. Oil structuring is determined visually when after cooling and rest time provided there is no gravitational flow of the solution (Hwang et al., 2012). In all the articles about vegetable wax organogelation, the concentrations used to produce organogel and determine the critical concentration belong to the group of natural numbers. Exceptions arise in cases where the critical concentrations are lower than 1%, for instance, sunflower wax was able to structure soybean oil at 0.5% w/w (Hwang et al., 2012).

Since the critical concentration was determined through the analysis of apparent solid behavior in a test tube, a second analysis was carried out to prove the oil structurating. This characterization is usually carried out in a rheometer, based on the small amplitude oscillation test (Lupi et al., 2012; Lupi et al., 2013; Blake et al., 2014) through controlled stress analysis using parallel plates or cone and plate geometries.

To understand organogel structuring and when it happens, a rheological analysis must be developed following a temperature program that represents organogelation tempering as close as possible. For instance, authors have performed the controlled stress analysis during a temperature ramp from between 70 and 95 °C to under 20 °C, at cooling rates from 1 to 5 °C/min (Lupi et al., 2012; Lupi et al., 2013).

At elevated temperatures, a solution is at its liquid state and its loss modulus (G’’) is higher than its storage modulus (G’). But during the process of cooling, a solution that changes from a liquid to solid-like state will present a temperature at which G’’ and G’ crossover. This point is considered the phase transition. So, it is implicit that a structured organogel at room temperature (20 °C) will present values of storage modulus (G’) higher than loss modulus (G’’). From the values of storage modulus and loss modulus, it is possible to calculate the phase transition angle (δ), which is the angle corresponding to the tangent of the ratio between loss modulus and storage modulus. Phase angle transition equal to 45 °C may also indicate the moment of phase transition. (Lupi et al., 2012).

Interesting rheological results were obtained by Lupi et al., (2012) and Lupi et al., (2013). In both works they observed that wax concentration is related to the organogelation temperature and the onset of crystallization temperature. Furthermore, the organogelation temperature was always at lower temperatures than the onset of crystallization temperature. These results are interesting because since the onset of crystallization temperature is the temperature at which the first crystals are considered to appear, crystal presence is mandatory for organogelation.

Crystallization is a generic term used to describe all the steps in the process to form a crystal. In a timeline sequence, crystal formation follows three basic steps, starting from supersaturation of the crystallizable compounds, nucleation and, then, crystal growth (Mullin, 1961; Hartel, 2001). Figure 1 presents a schematic representation of saturated triacylglycerol (TAG) at an elevated temperature, where TAG is a liquid state (Figure 1a). With temperature reduction, TAG molecules start to supersaturate and pack in organized systems that do not last long enough to form a crystal (Figure 1b). This state of supersaturation, where crystallizable molecules are not able to form crystals by themselves, is known as the “metastable state” (Mullin,1961). At these temperatures, crystallization is possible with external help, such as the presence of crystal seeds, agitation and mechanical shock (Mullin, 1961).

A temperature which is low enough to allow the formation of the first crystals is known as the “onset of crystallization temperature” (T_OC, Figure 1). Under T_OC the molecular packing can organize into stable crystalline structures, with a nucleus of bigger crystals (Figure 1c). Further temperature reduction may cause a higher degree of supersaturation, promoting crystal growth and new crystal formation (Figure 1d).

In a multi-component system, such as solutions of vegetable waxes in vegetable oil, supersaturation may be affected by the presence of different types of compounds (Mullin 1961). Therefore, nucleation may be assisted or avoided, depending on the characteristics of the compounds present. A negative influence on the onset of crystallization and gelation temperatures due to addition of cocoa butter to a solution of Myverol in olive oil was observed (Lupi et al., 2012). While the presence of hydrocarbons (6–7%) and free fatty alcohols (11–13%) in sunflower waxes seems to have improved itd organogelation capacity (Hwang et al., 2012; Blake et al., 2014).

The analysis of organogel and vegetable waxes in differential scanning calorimetry (DSC) is one of the most common analysis in the fiel. The term “differential scanning calorimetry” refers to a technique able to measure the movement of heat in (endothermic) and out (exothermic) of the sample (Rheingans and Mittemeijer, 2015). The methodology allows precise control of temperature and accurate determinations of phase transition temperatures, enthalpy and heat capacity. The most adequate protocol of analysis is one that similar to phase transition analysis, is able to represent the tempering used to prepare the organogel. A temperature program coinciding with a rheological analysis allows for comparisons (Lupi et al., 2012; Lupi et al., 2013). In fact, even when a rheological analysis is not carried out, authors have developed a thermal analysis in DSC with a temperature program similar to organogel tempering (Dassanayake et al., 2009; Öǧütcü and Yilmaz 2014; Öǧütcüand Yilmaz, 2015a; Öǧütcü and Yilmaz, 2015b).

The most common temperature program for determining the organogelation parameters in DSC is to increase the temperature of the organogel up to 100 °C and cool at nearly 0 °C, at a cooling rate from 1 to 5 °C/min (Lupi et al., 2012; Lupi et al., 2013). The sample cooling step in DSC allows for determining the onset of crystallization temperature (Figure 2, Onset), and other parameters, such as the temperature of the main peak of crystallization (Figure 2, Peak), endset of crystallization temperature (Figure 2, Endset) and the enthalpy of crystallization (Figure 2, Area under the curve) that could be used to characterize vegetable wax crystallization in oil (Aggarwal, 2000; Tan and Nehdi, 2015). The onset of crystallization temperature became the most important parameter once it was shown that no organogelation happens without the presence of crystals (Lupi et al., 2012).

Table 1 presents the onset of crystallization temperatures of different solutions of vegetable waxes in edible oil from the literature that were structured at room temperature. In Table 1 the influence of the wax and oil composition on the onset of crystallization temperature can be seen. In addition to the presence of crystals, a minimal amount of crystal is needed for oil structuring and this is why there are some differences between crystallization and organogelation temperatures (Lupi et al., 2012). The amount of crystals present in the organogel can be determined using laboratorial nuclear magnetic resonance as explained below.

The determination of solid fat content frequently follows an AOCS Official Method, which the most appropriate would be the Cd 16-93 (1997) for non-stabilizing fats and oils, used to analyze fats like cocoa butter substitutes and replacers with fast crystallization. This is a non-destructive method that uses Nuclear Magnetic Resonance (NMR) laboratorial equipment. The tempering process stipulated in the protocol is similar to organogel tempering, which allows comparison with other results.

The content of solids in organogels is in general smaller than the proportion of waxes added to the solution, which indicates that all the crystals present in the organogel are from the vegetable wax without further alterations in the oil (Dassanayake et al., 2009). It was also observed that organogelation rarely occurs in solutions with less than 3% solids (Table 1). Unfortunately, the authors who obtained organogel at a low vegetable wax concentration (≤ 1%) did not analyze the solid fatty content of the solutions at critical concentration (Hwang et al., 2012; Blake et al., 2014). X-ray diffraction analysis can be used to verify whether the crystals present in the bulk vegetable waxe and in the organogels are the same, corroborating with the previous idea that the crystals are only from the vegetable waxes.

X-ray diffraction is a rapid analysis that has been widely used to understand organogelation. However, the theory behind it is complex and deserves some attention. A good explanation about the x-ray diffraction (XRD) theory may be found in the work by Idziak (2012). What is important to understand here is that during crystallization, molecules are initially packed (Figure 3A) and form sub cells (Figure 3B), which are the structuring parts of the unit cells (Figure 3C and 3D, Mullin, 1961; Hartel, 2001; Idziak, 2012). A unit cell is a highly organized structure with 2 and 3-dimensional space configurations and is the fundamental building block of a crystal (Idziak, 2012). A crystal will have the same geometry as the unit cells and may be called cubic, tetragonal, orthorhombic, trigonal, hexagonal, monoclinic and triclinic, depending on the angle between plains of the 3D geometry faces (Figure 3E) and the distance between parallel lattices and the face under the analysis (Figure 3F, d-spacing). Figure 3F shows a representation of the x-ray of diffraction analysis, where d-spacing is shown as the distance between two lattices and obtained by applying Bragg’s law, as presented in equation (1):

where n is the order of reflection, λ is the wavelength of the incident x-rays, d is the interplanar distance and θ is the angle between lattice and incident x-ray. The most commonly used source of x-rays is copper (Cu), which emits radiation with a wavelength of 1.54 Å (Marangoni and Wesdorp, 2013).

Since vegetable wax could be used as an additive or trans and fully saturated fatty acid substitute in lipid based products, it would be beneficial for vegetable wax compound to present a sub cell structure configuration which is similar to lipid crystals. For instance, triacylglycerols may crystallize in α, β’ and β sub cell polymorphisms, which have hexagonal, orthorhombic and triclinic crystalline geometry, respectively (Sato, 2001; Dassanayake et al., 2009; Idziak, 2012). The capacity that triacylglycerols have to pack in several ways causes changes in the sub cell structure and, by consequence, alters the unit cell organization, what is known as polymorphism (Sato, 2001; Hartel, 2001). β’ is considered the optimal triacylglycerol polymorphism for milk fat-based food and substitutes due to its thermodynamic properties, stability and better texture and mouth-feel than other polymorphisms (Dassanayake et al., 2009; Hartel, 2001).

In x-ray diffraction analysis the sub cell packing may be characterized by the number of peaks and the distance between lattices (d), or plans of unit cells (Dassanayake et al., 2009; Idziak, 2012). For instance, triacylglycerol presents crystals at α configuration when one peak is detected with d-spacing at 4.15 Å, β’ when a main peak with d-spacing at 4.2 Å is present and a second peak at 3.8 Å and, β configuration presents one peak with d-spacing at 4.6 Å.

The relation between the number of peaks, d-spacing between lattices of vegetable waxes and organogels of vegetable waxes in edible oil is presented in Table 2. The authors have identified crystals either in the bulk vegetable waxes or in the organogel with an x-ray diffraction pattern close to triacylglycerols in the β’ polymorphic configuration (Table 2). This is an interesting result because it indicates the possibly good technological and functional properties of vegetable wax crystals. Furthermore, a similar x-ray diffraction pattern of bulk and organogel shows that crystals come exclusively from the neat vegetable wax (Dassanayake et al., 2009). In addition, crystals with β’ polymorphic configuration may have a needle-like or a platelet-like morphology that can be visualized using polarized light microscopy.

The morphological structure of a crystal may be seen under a microscope with polarized light. In agreement with XRD analysis, vegetable wax crystals present a needle-like morphology. It was observed from previous works that a high concentration of esters in the vegetable wax seems to be related to bigger and isolated needle-like crystals (Blake et al., 2014). Further, a tendency to form clusters in the presence of minor compounds was noticed (Blake et al., 2014). Figure 4 presents examples of flax and wheat straw wax crystals in canola oil at 5 and 6% (w/w), and the influence of the composition and concentration on crystal size, shape, density and morphology can be seen. Waxes from flax straw are rich in esters and aldehydes (del Río et al., 2013), which help the formation of crystals with homogeneous size and high density. Wheat straw waxes present important concentrations of β-diketones and primary alcohols (Racovita et al., 2016), a composition that is related to a less homogeneous crystal morphology at a lower concentration and the formation of huge crystals at higher concentrations.

Images from a polarized light microscope may be analyzed using specialized software to determine the number of crystals or the porosity (% area/area) present in the organogel (Blake et al., 2014). A free software able to do this analysis is the ImageJ^® (National Institute of Health, USA). It would be always necessary to avoid visual adjustments during the image analysis methodology to allow for better reproducibility of the results.

It is important to analyze organogel at the same concentration as the previous analysis for comparison and for better understanding of the relationship between morphology and oil structuring. Regarding oil structuring, one of the expected characteristics of the crystal is the capacity to entrap oil, maintaining it at a liquid state but immobilized. To evaluate the capacity of a determined group of crystals to hold oil, an analysis was developed called “oil binding capacity”.

The theory behind the oil binding capacity analysis is interesting because it would be able to determine the amount of oil entrapped within the crystals of an organogel. To measure this holding capacity, two methods have been used. One was proposed by Blake et al., (2014), where the general idea was to determine the amount of oil able to migrate from the structured organogel in 24 h. This is a homemade analysis that resembles the method for the determination of oil blooming (Matsuda et al., 2001) mixed with water-binding capacity (Lewicki et al., 1978). The authors transferred already structured organogel (100 g) to a funnel, which was placed over a graduated test tube. Then, the set was placed at 40 °C and the volume of oil which flowed from the sample to the test tube was measured periodically. In addition to interesting results, considering the fact that most analysis are done at room temperature, which is not necessarily a good organogelator, this method uses 40 °C due to varying melting behavior, and is isolated without possibility for comparison.

The other method used is centrifuge based and was initially proposed by Da Pieve et al., (2010). Some adaptations of the method have appeared elsewhere (Öǧütcü and Yilmaz, 2014; Öǧütcü and Yilmaz, 2015a; Öǧütcü and Yilmaz, 2015b), mainly related to the organogel tempering process before analysis. For instance, up to now, the best procedure for filling sample containers (Eppendorf tube of 1.5 mL) with organogel (~ 1 g) has been to transfer the solutions in their liquid state to the Eppendorf tubes and allow them to cool down to room temperature, therefore following a similar tempering procedure to other analyses. The results for oil binding capacity (OBC) from this last method are presented in Table 1. Vegetable wax concentration, solid fatty content and oil binding capacity are related and a total oil binding capacity was not necessary to structure an oil (Table 1).