The Effect of Cooling Rate on the Microstructure and Macroscopic Properties of Rice Bran Wax Oleogels.

The main purpose of this paper is to study the microstructure and macroscopic characteristics of rice bran wax (RBW) oleogels at a cooling rate of 1°C/min and 10°C/min by polarized light microscopy, X-ray diffraction, differential scanning calorimetry, texture analyzer, and micro rheometer. The oleogels of soybean oil were prepared by RBW in concentrations of 5%, 7.5%, 10%, 15% and 20% (wt/wt). The results of this study indicated that the concentration of RBW and cooling rates were affected by the crystal size and spatial distribution of these crystals. For the same RBW concentration, oleogels contained smaller crystals when cooled at 10°C/min compared to 1°C/min. And the oleogels obtained at a rate of 10°C/min exhibited a tighter crystal network, lower melting point, harder texture, and energy storage modulus. These results demonstrated the impact of cooling rate on the rheological behavior, nucleation, and crystallization process.

found that RBW oleogels did not mix well with air, so ice cream did not have a good shape 10 . Thus, there are certain limitations of RBW oleogels instead of solid fat 10 12 .
In addition, RBW crystals formed a dense crystal network in oleogels depending on the van der Waals interactions as gelator-gelator interacter. The network structure is strongly correlated to macroscopic. But the network structure can be modulated by the cooling process, which could affect the nucleation and crystallization kinetics. Several studies reported that a rapid cooling rate could decrease crystal length and network pore area fraction, increase the fractal dimension of the crystal network, and lead to an increase in the oil binding capacity 13 . Slower cooling allowed for the growth of larger and consistently shaped crystals, while faster cooling incongruently shaped small crystals. However, the effect of the cooling rate on the microstructure and macroscopic properties of RBW oleogels is still incomplete.
Therefore, the objective of this study was to investigate the effect of cooling rate and RBW concentration on the microstructure domain size and shape , macroscopic properties, texture characteristics, thermal properties, rheological properties, and microrheological information of RBW oleogels crystals. The results will help us to under-
Hubei, China . Soybean oil was heated at 90 and RBW was evenly dispersed in soybean oil at 5 levels 5 , 7.5 , 10 , 15 , and 20 w/w to prepare the RBW oleogels. The mixture was stirred at 90 for 30 min using a magnetic stirring apparatus Model EM300T, Labotech Inc., Berlin, Germany at 50 g until completely dissolved. Then, the mixture was cooled down to 20 at different rates 10 /min and 1 / min and kept for 24 h before observation 14 .

Analysis of the fatty acids and fatty alcohols of RBW
A mixture of 20 mg of RBW with 20 mL of 30 KOH in isopropanol was placed in a 100 mL flask. The mixture was refluxed for 6 h in an oil bath at 100 . Then, the isopropanol was evaporated under reduced pressure until the residue was completely dry. 50 mL of ethyl acetate was added to this residue under stirring at 50 for 2 h. The mixture was filtered, giving a filtrate and a solid residue. The solid residue was washed with ethyl acetate 3 20 mL . All filtrates fatty alcohols included were collected and dried over anhydrous sodium sulfate. Fatty alcohols were identified by high-temperature GC 15 .
The solid residue was further washed with ethyl acetate 3 20 mL , and the solid portion was acidified with 20 mL of 30 HCl for 1 h at 50 . 20 mL distilled water was added, and fatty acids were extracted with ethyl acetate 3 20 mL . The combined extract was washed with water to neutral pH, and the ethyl acetate layer was then dried over anhydrous sodium sulfate. The solvent was removed in a vacuum rotary evaporator, and fatty acids were obtained 15 . Fatty acids was converted to fatty acid methyl esters by heating with 2 mL of 5 methanolic sulfuric acid at 70 for 2 h for GC analysis 16 . The compositions of fatty acids and fatty alcohols of RBW were listed in Table 1.

RBW crystal morphology
The crystallization of RBW oleogels was observed by Leica DM4000M polarizing microscope Leica Microsystem, Wetzlar, Germany . Oleogels of different concentrations 5 uL were placed on a glass microscope. The slides were heated at 90 for 30 min to erase the crystal memory, then cooled to 20 at different rates 10 /min or 1 / min by a temperature-controlled microscope stage Linkam Scientific Instruments, Surrey, U.K. .

RBW crystal structure
The X-ray diffractometer XRD D8 Advance, Bruker, Germany was operated at 40 kV and 40 mA with Cu Kα radiation λ 0.154 nm . For testing, samples were melted at 90 and then cooled to 20 at different rates 10 /min or 1 /min . The samples were kept at 20 for 24 h before testing. Jade 6 software was used to analyze the X-ray diffraction image of oleogels to calculate the grain size of oleogels, and to analyze the physical properties of oleogels by comparing the size of the grain.

RBW oleogels crystallization and melting
Thermal behavior of RBW oleogels was determined by differential scanning calorimeter DSC 200 F3, Netzsch, Germany . The oleogels sample is accurately weighed 5 mg into the pan of DSC. The samples were heated from 20 to 90 at 10 /min and held for 5 min 17 . Then the sample was cooled to 20 at a different rate 10 /min or 1 / min and reheated to 90 at 5 /min.

RBW oleogels texture characteristics
Prepared RBW oleogels were placed in a petri dish with 90 mm diameter for testing. The texture Analyser TA-XT plus Stable Micro Systems, UK was used to detect the hardness, cohesiveness, gumminess, chewiness, and resilience of the oleogels. The sample was extruded with the cylinder probe P/50 at a rate of 1 mm/s and a compression "-" , Not detected.
ratio of 50 .

RBW oleogels microrheology
The RBW oleogels were placed in a sample bottle, equilibrated at 30 for 1 h, and then placed in a microrheometer Rheolaser Master, Formulaction inc., France . The test was carried out at 30 and the test mode was fully characterized. The G' and G'' values were obtained by software Rheosoft Master using EI Elasticity Index and MVI Macroscopic Viscosity Index . G' represents the storage modulus and G'' represents the loss modulus of the RBW oleogels. When G' G'', the sample mainly undergoes elastic deformation, so the sample is in a solid state; when G' G'', the sample mainly undergoes viscous deformation, so the sample is in a liquid state. When G' ≈ G'', the sample is semi-solid.

Statistical analysis
The samples were analyzed in triplicate and the results were expressed as mean standard deviation. A one-way ANOVA analysis of variance was performed to test if statistically significant differences were existed. The equality of variances were verified using Levene s test prior to usage of Tukey s test to compare the mean values at p 0.05 significance level.

Crystal morphology
Crystal morphology of RBW oleogels the concentrations of RBW were 5 , 7.5 , 10 , 15 and 20 wt/wt prepared at a rate of 10 /min and 1 /min were shown in Fig.  1. Micrographs show that these crystals displayed as dendritic. The morphology of RBW crystals in SBO contrasted with those previously reported where the appearance of RBW described as needle-like and fibrous 3, 6 . But our results were similar to RBW-rice bran oil oleogels 18 . The morphology characteristic of RBW was an ideal state for oleogels because these crystals could capture a greater amount of liquid oil than other crystalline forms 6,18 .
It can be seen from Fig. 1 A, C, E, G, I , with the increase of the content of RBW, the crystals in the oleogels were larger, the relative motion space between the crystals was smaller, and the network structure was more closely constructed. For the same concentration of RBW, the crystal network formed by the slow cooling rate 1 /min was incompact. The length of the crystal is more than 200 µm, which weakens the binding ability of liquid oil. So the crystal network is very fragile and instability. But the crystal network formed by the fast cooling rate 10 /min is more uniform and dense. The crystals interact well, forming a tight three-dimensional network and strong gel properties. This result was consistent with the previous re-searches. Dassanayake reported that when the olive oleogels with the same RBW content under different cooling rates compared to the organic gel network crystal, the smaller cooling rate of the organic gel formed the crystal network is larger and more space 8 . The reason may be that the RBW crystal in the slow cooling rate has been fully grown compared with rapid crystallization. During the cooling process of 10 /min, the ambient temperature drops too fast and the crystal does not have enough time to grow. Thus, the size of the crystal is less than 200 µm.
RBW crystals have longer morphology 20-50 µm 19 , which is an ideal characteristic for gel formation 20 . Combine the results of Fig. 1, we can conclude that at least 20 content of RBW is needed for slow crystallization to form the ideal crystal morphology, while only 10 of RBW is needed for fast crystallization. As expected, a faster cooling rate results in an earlier onset of crystallization and the formation of the relatively smaller crystals than a slower cooling rate 21 . In principle, a higher thermodynamic drive related to a lower cooling rate affects the nucleation rate, resulting in the formation of finer crystals. However, a slower cooling rate causes the primary nucleus to grow into a relatively larger crystal 22 . Therefore, we can see that we need to use a fast cooling rate to achieve a better gel effect with the minimum amount of RBW. This also provides a reference for industrialized production of RBW oleogels.

Crystal structure
XRD diagrams of oleogels provided the information about crystal morphology. The polymorphism of oleogel samples was shown in Fig. 2. Two diffraction peaks were observed in the diffraction patterns of the oleogel samples, which were 4.1 Å, and 3.7 Å. They were corresponding to the forms of wax crystals 23,24 . The very weak signal at 4.6 Å indicated the β form of triglyceride crystals, which reflects triglycerides with poor crystallization. Similar peaks β form had been observed for other wax oleogel samples such as beeswax, candelilla, carnauba, and sunflower wax 25 . Irrespective of RBW concentration, all oleogels exhibited similar diffraction. Some researchers also indicated that the effect of wax concentration on the crystal form is less compared with other factors such as molecular composition, preparation conditions e.g. stirring or cooling rate , and storage conditions 3,26,27 . In addition, the XRD patterns were less influenced by the types and compositions of the triglycerides 3 .
The peak intensity of oleogels crystallized at 10 /min was stronger than the oleogels crystallized at 1 /min Fig.  2 . Previous studies have shown that the short spacing patterns were significantly more intensely than the long spacing patterns 6 . Generally speaking, the contrast of the peak intensity in X-ray diffraction image is related to the strong anisotropy of the crystal growth rate perpendicular  to and parallel to the layered plane. If the growth rate parallel to the layered plane is slower than that perpendicular to the layered plane, needle-like or sheet-like thin crystals will be formed, showing a weaker long-distance type and a stronger short-distance type 6 . This property can explain the difference of peaks in X-ray diffraction image between fast and slow cooling rate crystallized for RBW crystals 28 . This means that the content of RBW and the cooling rate are not affected by the crystal form but affected by the nanostructure. Figure 3 showed the domain size of RBW crystals and triglyceride crystals crystallized at 1 /min and 10 /min based on the d001 long spacing. The crystallite domain size was 8.21-80.36 nm 2.81-72.55 nm between 5 and 20 wt RBW at 1 /min 10 /min cooling rate, which is larger than the triglyceride crystallites. It can be seen that at the same crystallization rate, the domain sizes of RBW and triglyceride crystals increased with the increase of RBW concentration. These results may be due to the . These cooling rate-dependent differences of domain sizes were interpreted as faster rates of nucleation more crystallographic mismatches and branching arising from the fast cooling rate.

Thermal behavior
The cooling and heating thermograms of the different concentrations of RBW oleogels after cooling at 1 and 10 / min, and then melting at 5 /min were shown in Fig. 4.  And the melting temperature T m , crystallization temperature T c , melting enthalpy ΔH m and crystallization enthalpy ΔH c were summarized in Table 2 to characterize the melting and crystallization behavior of RBW oleogels. Independent of the cooling rate used, the T m , T c , and enthalpy were increased as the RBW concentration increased. The T m and T c of 5 RBW oleogels crystallized at 1 /min increased from 65.4 and 57.5 to 71.8 and 69.4 in the oleogels with 20 RBW. The oleogels with higher RBW concentrations had the higher ΔH m and ΔH c values at different cooling rates Table 2 . The hysteresis was also observed in the thermograms for 5-20 RBW dispersion in SBO. This phenomenon was consistent with the result of Toro-Vazquez et al. that more gel was developed as the concentration of the gelator increased 29 . The cooling rate changed the crystallization behavior of RBW oleogles Fig. 4 . When the cooling rate was increased from 1 to 10 /min of the oleogels prepared by 5-20 RBW, the T m and T c of RBW oleogles were moved from 65.4-71.8 and 57.5-69.4 to 62.8-71.6 and 57.3-67.5 . For a given RBW concentration [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] , the ΔH c was increased from 0.3 24.4 to 2.1 27.9 J/g Table 2 . This means that when the cooling rate is increased, the T m and the T c of RBW oleogles are moved to a lower temperature region and an increase of ΔH c . A similar result was reported by Pérez-Monterroza in the crystallization of beeswax and avocado oil 30 . In principle, higher thermodynamic driving forces associated with lower cooling rates will affect the nucleation rate, resulting in the formation of finer crystals. At the same time, slower cooling rates will cause the pronuclei to grow into larger crystals 21 . This was in agreement with the polarized light microphotographs Fig. 1 . Thus, RBW had less time to be crystallized at 10 /min than 1 /min, requiring a lower crystallization temperature to achieve the crystal formation. However, the cooling rate effect on ΔH m was different at each RBW concentration. This result might be related to the high temperature 20 during isothermal gelation of oleogles. Toro-Vazquez indicated that the other minor components nalkanes presented in RBW might develop a mix of molecular packing with the subsequent effect on melting enthalpy 29 .

Texture analysis
The texture-profile analysis TPA results were shown in Fig. 5. Hardness indicated the compactness of the gel network and the strength of the oleogels. The content of RBW is a major factor in changing the physical properties of the oleogels. Under the same cooling rate, the hardness force of the oleogels increased as the concentration of RBW increased. Besides, a significant difference between the samples that crystallized at 10 /min and 1 /min was existed. When the RBW concentrations were above 5 the critical concentration , the hardness was at least twice higher than that at a lower cooling rate. These results were in agreement with the other studies, which reported that the hardness enhanced with a faster cooling rate 13,31 .
The hardness values of 20 RBW oleogels cooling at 10 / min were similar to the hydrogenated coconut oil 381.14 N . Cohesiveness indicated the ability of the oleogels to resist the second deformation after the first deformation. The cooling rate did not significantly affect the cohesiveness of oleogels. In the case of gumminess, the RBW oleogels cooling at 10 /min was higher than that at 1 /min. And the value of gumminess increased with the RBW content increased due to their hard texture. These results showed that the cooling rate played the main role in the texture of oleogels.

Rheological measurements
The G' LVP and G'' LVP profiles of RBW oleogels that formed under high 10 /min and low 1 /min cooling rates were shown in Fig. 6. It is indicated that the G' values were higher than G'' in all RBW oleogels and the values of G' and G'' increased with the increasing concentration of RBW. In all cases, the G' value was higher than G'', showing more elastic properties. When the RBW at an equivalent concentration, the G' values cooled at 10 /min were larger than 1 /min. In general, the G' is usually associated with the  amount or the size of crystals. A higher difference between G' and G'' indicates a stronger gel and a more stable network 32 . These results were consistent with that the smaller crystal size and the more compact crystal morphology were presented in the RBW oleogels developed at 10 / min than 1 /min Fig. 1 . The thermodynamic driving force for RBW gel formation increased faster led to that the RBW molecules had less time to organize at the 10 /min cooling rate. Consequently, the smaller crystal size and stronger network in the RBW oleogels crystallized at 10 / min than 1 /min. Patel et al. explained from the fact that the formation of finer crystals promoted stronger network formation due to the higher crystal-crystal interactions probably due to an increase in the total effective surface area of crystals 22 . Therefore, we can conclude that the rapidly cooling RBW oleogels have better rheological properties, which is conducive to the development of oleogel products.

Conclusion
In conclusion, the crystallization characteristics of RBW oleogels crystallized at 10 /min and 1 /min rate were thoroughly investigated. The results show that there is a significant difference between RBW oleogels crystallized at different cooling rates. It can be concluded that the RBW crystals crystallized at 1 /min is larger than 10 /min at the same concentration of RBW in SBO. Due to the influence of the size of crystals, the thermal properties are also markedly different, and the slow-crystallized oleogels melt at a higher temperature and lower hardness than the rapidly crystallized oleogels.

Conflicts of Interest
The authors declare no competing financial interest.