Refined, bleached, and deodorized superolein (POoIV64) was obtained from PGEO Sdn. Bhd. in Johor, Malaysia. The PBA gelator was obtained from Sakamoto Yakuhin Kogyo Co., Ltd., Osaka, Japan. It is a permitted food additive in Japan, which is commercially known as CV-1L, and contains fatty acids which comprise a mixture of stearic acid, oleic acid and behenic acid ( Saw et al. , 2023 ). Food-grade SFW with a melting point of 77 °C was obtained from Shri Balaji Driers in Karnataka, India. Fully hydrogenated palm-based monoacylglycerol (MGHO), consisting of 40.9% palmitic acid and 56.8% stearic acid, was sourced from Ecolex Sdn. Bhd., Selangor, Malaysia.

Fatty acid composition was analysed in following with the boron trifluoride method according to ISO 12966-2:2011 , using a 430-GC gas chromatography (Bruker, Germany) ( Saw et al. , 2023 ). Triacylglycerol composition was analyzed using the AOCS Official Methods Ce 5c-89 and Ce 5c-93 ( AOCS, 2017a ; AOCS, 2017b ) ( Saw et al. , 2023 ).

Oleogels were made following a preliminary investigation which determined that the best gelator concentrations were 3% w/w for PBA and SFW and 8% for MGHO ( Saw et al. , 2020 ). Oleogels were prepared in 200-g batches by heating the mixture to 90 °C in an oven for one hour to fully dissolve the gelator. The mixtures were put into their respective containers for various analyses.

SFC was determined using a minispec mq20 pulsed-NMR from Bruker, Germany. The NMR tubes, measuring 10.0 mm in diameter, 1.0 mm in thickness, and 180 mm in height, were filled with 3 cm melted samples in height. After being left at room temperature for an hour, the tubes were transferred to incubators, set at temperatures of 5, 15 and 25 °C. SFC was measured on days 1, 2 and 3, with day 0 being the day of preparation.

The molten gel was placed in polypropylene containers with a 5.5 cm internal diameter and 3.5 cm height in incubators at the storage temperatures. Gel strength was measured using a texture analyzer (TA.XT plus, UK) equipped with a 500-g load cell. The hardness of the oleogel was measured using a cylindrical probe (2 mm diameter) at 1 mm/s and a maximum penetration depth of 8 mm. This method was modified from ( Bot et al. , 2009 ).

The stabilities of the oleogels were analyzed using a centrifugal stability analyzer, LUMiFuge Model LF111 (LUM Ltd., Berlin, Germany). The sample was placed in a LUMiFuge tube ((PC10 mm with a PP stopper) and stored at room temperature for an hour before being transferred to incubators at 5, 15, and 25 °C. The equipment was preconditioned to the storage temperature before loading the cells into the centrifuge. The spinning speed was set to 4000 rpm for 4 hours, under the respective storage conditions. The analyses were conducted on days 1, 2, and 3. The system continuously recorded near-infrared transmission profile during the process. The data was integrated using LUMiFuge® SEPVie 5.1 software. The percentage of oil-released was calculated by comparing the oil-released height after the centrifugation to the initial sample height.

A Rigaku TTRAX III X-ray diffractometer (XRD) (Rigaku, Japan) was used to measure the polymorphism of the oleogel. The oleogel was melted at 90 °C to destroy the crystals’ history. The melted oleogel was placed into the XRD sample pans and stored in an incubator for a day. The samples were analyzed at 1 to 30° 2θ using a Cu source-X-ray tube at 40 kV and 200 mA at 0.5 °/min.

The morphology of the oleogels was studied using a Leica DM4500 P LED polarized light microscope (Germany). The microscope was equipped with a LINKAM LTS120 temperature controller, a LINKAM T95 system controller and a JULABO F250 recirculating cooler. The oleogel sample was placed onto a glass slide and pressed with a glass cover slip to ensure thin layer of well-distributed sample. The microscope images were captured at magnifications of 10, 20 and 50x in a standard bright field configuration.

Analysis of variance using Minitab 16.2 was employed to investigate the effect preparation day on oil released, SFC and hardness of the oleogels.

Figure 1 shows the SFC of oleogels from PBA, SFW and MGHO gelators at 5, 15 and 25 ºC from day 1 to day 3. As expected, the SFC increased significantly with decreasing storage temperature due to the increase in crystal formation at lower temperature. An extremely high percentage of SFC (55%) was observed at 5 °C for all oleogels on day 2 and day 3, which initiated with much lower SFC. Table 1 shows that superolein contains high-melting triacylglycerols, such as POP and SOS, with a total saturation content of 40.35%. This finding suggests that these high-melting triacylglycerols in superolein crystallized and reached equilibrium at an SFC of approximately 55%.

At 15 ºC, PBA oleogels showed the lowest SFC (2.5%) during the 3-days storage. SFW oleogels demonstrated similar SFC on day 1 as the PBA oleogels. However, the SFC was gradually increased from 1.93 to 5.22 and 6.77% on days 2 and 3, respectively. This result aligns with previous findings that indicated the slow crystallization of superolein at 15 ºC storage with SFW; whereas PBA showed crystallization inhibition ( Saw et al. , 2023 ).

In contrast, a stagnant SFC (7.5%) trend was observed in MGHO oleogels stored at 15 ºC, indicating a similar amount of crystals present during the 3-day storage. This result did not show the same trend as the results previously reported in Saw et al. (2023) , which showed a drastic increase in Gʹ _LVR and Gʹʹ _LVR during the 3-day storage period. However, the thermal analysis results could explain these phenomena, as the melting properties indicated a possible crystal rearrangement during storage, which did not influence the total amount of crystals, thus showing a constant SFC ( Saw et al. , 2023 ). Omar et al. (2015) reported similar observations, in which RBD palm oil clusters became more spherulitic with increasing size but SFC still remained the same throughout a 1-week storage period. The crystal rearrangement was due to the slow crystallization behavior of palm oil products, providing similar effect to post-hardening.

At 25 ºC, PBA and SFW oleogels showed much lower SFC (2%) than the MGHO oleogels (7%), indicating the presence of more crystals in the MGHO oleogel. At 25 ºC, some high melting TAGs in the superolein interact with the MGHO molecules to form more crystals, thus leading to a higher SFC in MGHO oleogels. Interestingly, MGHO oleogels showed very similar SFC when stored at 15 and 25 ºC, meaning that the difference in the storage temperature did not alter the total amount of crystals formed. More analyses are required to explain this observation.

The hardness levels of superolein oleogels are illustrated in Figure 2 . The oleogels at 5 ºC showed the highest hardness, ranging from 310 to 440 g, but the hardness reduced very drastically in stages to approximately 1.5-23 g at 15 ºC and to 0.8-1.6 g at 25 ºC. At 15 ºC, PBA oleogels showed a significantly lower hardness of 1.5 g, whereas SFW and MGHO oleogels showed relatively higher hardness levels during the 3-day storage period. A drastic increases in hardness were observed in SFW and MGHO oleogels during the 3 days of storage, which was in line with the rheological results ( Saw et al. , 2023 ). This was also due to the slow crystallization behavior of superolein at 15 ºC.

Figure 3 shows the XRD diffractograms of the superolein oleogels on day 1. In both the SAXS and WAXS region, PBA oleogels did not show clear crystalline peaks for oleogels stored at 15 or 25 ºC. This finding indicated that no crystalline particles were involved in the structuring of PBA oleogels, as only an amorphous peak at approximately 4.5 Å was observed in the WAXS region for PBA oleogels. Pradhan et al. (2014) also reported the detection of this single peak at approximately 20º(2Ө) in palm oil-based organogels, which suggested the amorphous nature of the organogel. Bot et al. (2009) reported that two peaks associated with liquid phases for a self-assembly organogel, which was similar to those of the PBA oleogels stored at 15 and 25 ºC. This finding indicated that the organogelation event that took place in the formation of PBA oleogels also via self-assembly.

At 5 ºC, a few crystalline peaks were observed at WAXS and SAXS, indicating the presence of crystalline particles in the oleogel system due to the crystallization of the superolein. Figure 3 shows that 3 wide-angle peaks were present: the peak that appeared at d = 4.61 Å indicated the detection of β polymorphic form crystals; whereas the peaks detected at d = 4.31 Å and 3.89 Å were due to the presence of the βʹ polymorphic form of crystals ( D’Souza et al. , 1990 ; deMan, 1992 ; Peyronel and Marangoni, 2013 ).

Figure 3B shows how the XRD patterns for SFW oleogels displayed a combination of amorphous and crystalline behavior. Three persistent peaks were detected at 4.60 Å, 4.15 Å and 3.74 Å for SFW peaks stored at all storage temperatures. Peaks with exactly the same d- spacing were also reported by Öğütcü et al. (2015) in which wax oleogels from beeswax and rice bran wax also produced peaks at similar WAXS regions, indicating the presence of the βʹ polymorphic form of crystals. Tavernier et al. (2017) also reported similar peaks which represented βʹ crystals associated with high-melting wax. This explains the detection of these peaks in almost all the literature concerning wax oleogels. In contrast, the peak detected at 4.60 Å could also indicate the presence of β polymorphs of crystal ( AOCS, 2017c ). However, this peak was just a broad peak at storage temperature of 15 and 25 ºC, which indicated an amorphous scattering contributed by the superolein ( Bot et al. , 2009 ; Da Pieve et al. , 2011 ). In contrast, the peak at d- spacing of 4.60 Å for 5 ºC storage was sharp, which indicated the presence of β polymorphs of crystals at the low temperature. These observations indicated that SFW oleogels consist mainly of βʹ polymorphs. Additionally, peaks at 4.42 Å and 3.89 Å were detected in PBA and SFW oleogels stored at 5 °C, indicating βʹ polymorphic crystals due to the crystallization of the superolein. This shows that palm superolein crystallization did not interfere with the gel structure presence in both PBA and SFW oleogels. On the other hand, this observation also suggests that superolein is fully melted at temperatures above 15 °C.

MGHO oleogels showed a complicated pattern with a combination of amorphous and crystalline behaviour. Unlike PBA and SFW oleogels, MGHO oleogels did not show a consistent trend when stored at different temperatures, indicating that the crystal packing in MGHO oleogels was different when stored at different temperatures. At 25 ºC, a major peak at 4.58 Å, and some minor peaks at 4.38 Å, 3.93 Å, and 3.78 Å were observed. Da Pieve et al. (2011) reported a very similar X-ray diffraction pattern in cod liver oil monoglyceride oleogel. These peaks are due to the in-plane ordering of monoglyceride aliphatic chains into the β-phase. This makes sense as the peak at 4.58 Å was sharp, different from those amorphous peaks detected in SFW and PBA oleogels at 15 and 25 ºC.

At 15 ºC, only some small peaks at 4.57 Å, 4.21 Å and 3.92 Å were observed. The intensity of the 4.57 Å peak was lower than that at 25 ºC, which revealed a decrease in the β polymorphic form crystals. The detection of very-low-intensity peaks at 4.21 Å and 3.92 Å indicated the presence of a low amount of βʹ crystals. This finding indicates that the in-plane ordering of the MGHO aliphatic chains is predominantly in triclinic configuration with some crystals packed in an orthorhombic configuration. At 5 ºC, MGHO oleogels showed a similar XRD pattern to SFW and PBA oleogels, indicating that the crystal polymorphism in all these oleogels was mainly contributed by the superolein crystals at 5 ºC.

The molecules of the MGHO interacted very closely with superolein molecules due to their high solubility in the superolein, thus displaying different interactions at different temperatures. Their interactions could be associated with the crystallization properties of TAGs in the superolein. At 25 ºC, the superolein remained liquid, thus resulting in minimal interactions. During this period, the MGHO molecules self-assemble into inverse lamellae, forming α-crystals (hexagonal packing) before transition to sub-α crystals (orthorhombic chain packing) ( Chen and Terentjev, 2009 ). At low temperatures, the higher melting TAGs in superolein interacted with the MGHO molecules, packing themselves into crystals with different polymorphism compared to the oleogels stored at 25 ºC. This observation was different from the findings reported by Da Pieve et al. (2011) , whereby no diffraction difference was detected when XRD patterns were recorded at 4 and 20 ºC in a cod liver oil oleogel system. This could be due to the properties of the oil used, as superolein oil consists of some saturated components, similar to those in MGHO, thus affecting the packing of molecules in crystal formation. The measured diffraction values for the PBA, SFW and MGHO oleogels are summarized in Table 2 .

According to Peyronel and Marangoni (2013) , the Bragg peak position in the SAXS region characterizes the longitudinal packing of fat molecules, indicating the size of bilayers or lamellae formed by stacking (001) planes. In the SAXS region, single, broad, weak peaks were observed for oleogels stored at 15 and 25 ºC, indicating no obvious longitudinal packing and only liquid/amorphous phases in the PBA oleogel system. At 5 ºC, a peak of 47.71 Å corresponded to 2L of the (001) plane, showing lamellae of approximately 47.71 Å ( Da Pieve et al. , 2011 ). Weaker peaks at 33.90 Å and 17.11 Å corresponded to higher-order reflections of the same repeating distance.

At 5 ºC, the first peak observed in SFW oleogels was at 47.60 Å, followed by a higher-order reflection at 33.86 Å, which corresponded to a 2L lamellae of the (001) planes. A consistent broad peak at 23.5 Å across all temperatures reflected the formation of a lamellae layer by the SFW. In MGHO, a single sharp peak in the SAXS region which increased with the decrease in storage temperature, with d- spacing values increasing from 55.31 Å to 57.02 Å and 59.04, when the temperature was reduced from 25 , to 15 and 5 ºC, respectively. These findings indicated the formation of a thicker lamellae layer with a reduction in storage temperature.

Figure 4 illustrates the morphology of the superolein oleogels on day 1 of storage. Only day 1 images were discussed due to the lack of noticeable differences over time. Obviously, the gel network present in PBA oleogels was a non-crystalline structure, formed via the self-assembly of the PBA molecules, creating a self-supporting gel network to entrap the superolein. Under bright-field microscopy, the oleogels displayed a tiny worm-like entangled network at 15 and 25 ºC, aligning with XRD results, which showed only an amorphous peak for PBA oleogels. At 5 ºC, the worm-like structure was not noticeable, but many spherulitic crystals were visible. These crystals were arranged in a regular manner, indicating that the crystals were separated from one another by the “invisible” non-crystalline gel structure, which was less visible under bright-field microscopy. This confirms the crystallization of the higher melting fraction in the superolein within the PBA gel at 5 ºC.

From the microscope images of SFW oleogels illustrated in Figure 4D-F , fewer crystals were visible at lower temperatures, which was contrary to expectations from SFC results. This counter-intuitive observation is due to the limitations of bright-field microscopy, whereby the actual presence of crystals cannot be clearly seen due to the presence of thick background crystals. The gel structure was more visible at higher temperatures when the oil was liquid, allowing better light transmission ( Microscopy, 2013 ).

The crystals shown in the microphotographs of MGHO oleogels stored at 5 ºC were less visible compared to those stored at 15 and 25 ºC ( Figure 4G-I ). The actual quantity of crystals could not be reflected on the bright-field microscopy for the same reason as discussed previously. In comparison, the gel network in the oleogel at 5 ºC was more compact and arranged in a more uniform order compared to the oleogels stored at higher temperatures. Some voids between the crystals were observe in the MGHO oleogels stored at 15 and 25 ºC. Overall, the superolein MGHO oleogels exhibited needle-like structures at all storage temperatures. This finding is similar to other studies that involved the use of monoglyceride (MG) gelators ( Bin Sintang et al. , 2017 ; Da Pieve et al. , 2010 ; Kesselman and Shimoni, 2007 ).

The oil-released percentages reflecting oleogel stability are summarized in Figure 5 . At 5 °C, the oil released significantly increased with a longer preparation day for PBA superolein oleogels from 5.0 to 7.5%. For SFW oleogel, only 4.5% oil release was observed after completing the storage duration. For MGHO oleogel, the oil release for MGHO oleogels wsd significantly higher than those with PBA and SFW oleogels, ranging from 8.1 to 14.3%.

At 15 °C, SFW oleogels were completely stable during storage. A low amount of oilrelease of 3.6 to 6.8% was observed for PBA oleogel. At 25 °C, PBA oleogels released more oil compared to the amounts released at lower temperatures. For MGHO oleogel, the oil released significantly increased with higher temperatures. The oil release was the highest at 25 °C, whereby almost 50% of the oil was released out from the structure.

In general, the centrifugal force applied in this analysis was able to separate the free liquid oil that was bound to the crystal structure ( Kanagaratnam et al. , 2013 ). These results indicated that SFW gelator had the highest capacity to hold liquids within its structure. The gelator was very different in composition compared to the liquid oil, consisting of mainly esters of fatty acids with fatty alcohols ( Carelli et al. , 2002 ). The gelator was able to form a homogenous and continuous structure capable of holding the oil within it.

MGHO gelator was not effective in forming a strong structure to hold the liquid oil even when a high dosage of 8%w/w gelator was used. This is probably due to the composition and structure of the MGHO gelator, with fatty acid chain lengths of C16 and C18, which are very similar to the superolein. At low temperatures, the MGHO molecules and the higher saturation component in superolein underwent nucleation and aggregated into larger crystals. Voids were formed between the large crystals without a continuous structure ( Figure 4 ), in which MGHO appeared as large dendritic crystals in the superolein without forming a tight network to hold the liquid oil in its structure. These voids filled with the less saturated liquid oil, easily released from the structure when centrifugal force was applied. A similar explanation was reported by Patel et al. (2015) , as bee wax and fruit wax formed loose entanglements of large crystals that could sustain lower magnitudes of stress. Thus, these oleogels had lower values of dynamic moduli in the linear response region compared to the other oleogels from natural waxes, such as SFW and carnauba wax. Additionally, MGHO oleogels present as β-crystalline phase exhibit large crystal aggregation which may also affect their oil binding capacity ( Bin Sintang et al. , 2017 ), thus making them least stable compared to the other oleogels.

Interestingly, MGHO gelators showed very high hardness but still exhibited very poor stability. A study conducted by Razul et al. (2014) indicated that harder surfaces retain less oil than softer surfaces, meaning that harder surfaces tend to have lower oil-binding capacities, which was in-line with our findings. At 25 ºC, the gelator molecules were highly soluble in the liquid oil due to their similarity in fatty acid composition. The gelator was not able to form a strong structure, which resulted in a very high amount of oil released.