The melting profiles of oil samples were determined by differential scanning calorimetry (DSC) using a DSC Q2500 according to the method Cj1-94 (AOCS, 2009).

The AV, PV and p-AV were determined according to methods 5a-40 Cd, 8b-90 and p 2.4 (AOCS, 1997), respectively.

The oxidative stability was determined in terms of induction period (IP) using the Rancimat method (Olajide et al., 2020). 3 Grams of sample with and without added antioxidants were carefully weighed into Rancimat tubes and subjected to accelerated oxidation at 100 °C under an air flow of 20 L/h.

For convenience, infant formula liquid milk is also popular in the market. Most papers only tested the oxidative stability of HMFS itself, but did not evaluate the oxidation stability of HMFS in emulsion. HMFS-in-water emulsions (500 g) were prepared as mixtures of purified HMFS, Tween 80 and a phosphate buffer solution (pH 7.0) according to methods described by Chen et al. (2020). These samples were divided into experimental groups (0.02% antioxidants added) and a control group (no antioxidant added). Particle size distributions of the emulsions were measured by Mastersizer 2000 (triplicate, at room temperature) according to the method by Zou et al., (2012a). The average emulsion droplet size 14.90±0.76 nm was similar to HMF, and there was no visible significant change in the state of the emulsions over the course of the oven test. The emulsions were held in an oven at 63 ± 1 °C and the induction period of the samples was dependent on the oil reaching a peroxide value of 80 meq O₂/kg oil (Shi et al., 2017; Olajide et al., 2020).

Higher temperature decreases the viscosity of substrates and increase mass transfer, which in turn reduce reaction time to equilibrium, but also enhance the rate of occurrence of acyl-migration and other negative impacts, such as fat oxidation. Previous studies show that substrate could be dissolved at low temperature in solvent system, which partitions in a more stable environment (Esteban et al., 2011; Wei et al., 2015). However, the addition of solvents is harmful to the environment and/or economy. Solvent-free is a much safer, much more environmentally-friendly and economical industrial application. Thus, all the experiments in this study were based on a solvent-free system.

The reaction accelerated as temperature increased from 50 to 55 °C and the C52 content increased with it (Figure 2A). However, the C52 content, when above 55 °C, changed only slightly. At 55 °C, the C52 content was 45.4% and PA content at the sn-2 position was 75.2% at the end of the 3-h reaction. Compared to other temperatures, the products at 50 °C were characterized by higher PA content (i.e. 77.0%) at the sn-2 position; while the C52 content was only 39.8%, which may be due to lower catalytic efficiency. Moreover, higher temperature could decrease the sn-2 PA content significantly and might cause adverse consequences on enzyme properties and so, 55 °C was selected as the most ideal reaction temperature.

According to the reaction formula, OPO can be synthesized under ideal conditions by maintaining the ratio of the PPP-rich fraction to ethyl oleate at 1:2. However, the interesteri- fication reaction is reversible, and the feedback regulation of the product will inhibit the reaction. To obtain the highest OPO yield, the effect of substrate ratio was compared in this experiment. From Figure 2B, the C52 content in the enzymatic products at the substrate ratios of 1:2, 1:4, 1:6, 1:8, 1:10 and 1:12 were 14.7, 25.7, 35.1, 41.7, 44.1 and 44.4%, respectively. The C52 content between the molar ratios of 1:10 and 1:12 were nearly identical, indicating that increasing the amount of ethyl oleate continuously would not necessarily improve the OPO yield. At the same time, using high amounts of ethyl oleate would mean a limitation for industrial production of OPO-rich HMFS. In addition, no significant difference in the sn-2 PA content with all the molar ratios tested indicated that the amount of ethyl oleate had very little influence on the acyl migration reaction. Thus, to improve catalytic and economic efficiency, an optimum molar ratio of 1:10 between the PPP-rich fraction and ethyl oleate is required.

Enzyme load is related to reaction rate. Higher enzyme load will accelerate the reaction rate and improve the incorporation rate of the acyl donor in the interesterification reaction. When the enzyme load was less than 10%, the content in C52 increased as enzyme load increased and reached its highest point (43.8%) at 10%, but later decreased as enzyme load increased (Figure 2C). Meanwhile, the content in sn-2 PA decreased slowly and continuously as the enzyme load increased. The sn-2 PA content was only 71.8% when the enzyme load reached 12%. These findings are understandable because the water content in the Lipozyme RM IM may have promoted the amount of DAG produced from TAGs, thereby enlarging the possibility of acyl-migration. An enzyme load of 10% (w/w) generated the C52 content (43.8%) and sn-2 PA content (73.9%) after 3 h, showing preferable yield and catalytic efficiency.

Researchers have found that the water content in the synthetic reaction of TAG favors the content in DAG, which is due to hydrolytic side reactions (Zou et al., 2012b). Moreover, the presence of water on the surface of lipase is important for maintaining its activity and the flexibility of its protein structure. With increased water content in the system, the yields of C52 and sn-2 PA decreased consistently (Figure 2D). The content in C52 was 44.9% in the system without water, which was 3.6% higher than that in the system with 10% water content. Similarly, the content in sn-2 PA was 77.1% in the system without water, which was 7.8% higher than that in the system with 10% water content. A similar study was also reported in a study by Liu et al., (2020). The results indicated that high moisture in the reaction system can accelerate the hydrolysis of TAGs to DAGs and cause more acyl-transfer, reducing the content in sn-2 PA. It has been speculated that there are some hydrophilic groups on the surface of Lipozyme RM IM itself, which maintains its activity. As a result, the reaction was carried out in the absence of water.

Overall, the optimal OPO synthetic conditions were as follows: temperature, 55 °C; substrate molar ratio, 1:10 (PPP-rich fraction/ethyl oleate); enzyme load, 10 wt %; water content, none; reaction time, 3 h. Under these conditions, using molecular distillation, the composition of fatty acid and acyl glycerol of the OPO-HMFS are shown in Table 2. The OA and PPP contents in the OPO-HMFS were 53.3 and 2.9%, respectively, showing that OA was effectively incorporated into the substrate. A relatively small amount of PA on the TAGs migrated from the sn-2 to sn-1, 3 positions, since 75.3% of PA can be observed at the sn-2 position of OPO-HMFS.

The melting curves of OPO-HMFS, Final-HMFS and palm stearin were determined by DSC. A lower melting temperature was observed for the OPO-HMFS product (18.1 °C) compared to palm stearin (57.6 °C), which contributed to the high content in OA and low content in PA in the product. The melting temperatures of both OPO-HMFS and Final- HMFS (14.3 °C) were lower than the normal human body temperature (about 37.5 °C) depicting that they are suitable to be used as fat base materials in infant formulas. The melting range of HMFS is important for its digestion and absorption. The melting range of Final-HMFS was relatively narrower than that of OPO-HMFS (Figure 3), showing that the content in the high melting point component in OPO-HMFS was higher than that of Final-HMFS.

Table 4 summarizes the basic properties of palm stearin, OPO-HMFS and Final-HMFS. TAGs hydrolyze to release free fatty acids during heating or the action of lipase, which influences the stability of oil. AV can be used to indicate the degree of oil hydrolytic rancidity and refining. The undetectable AV of OPO-HMFS showed that molecular distillation was an effective method to remove free fatty acid or fatty acid ethyl ester. The AV of the Final-HMFS was only 0.3 mg KOH/g, indicating that the selected oil blended with OPO-HMFS was of good product quality.

PV and p-AV were used to evaluate the degree of oil oxidation and deterioration. PV represents the main products of oil oxidation, like hydroperoxides, while p-AV represents the small molecular compounds such as aldehydes and ketones produced by further decomposition of peroxides. High PV and p-AV indicate high oxidation of oil samples. From the Table 4, the PV and p-AV of OPO-HMFS (1.1 meq O₂/kg oil and 0.9, respectively) were quite low compared with palm stearin (3.8 meq O₂/kg oil and 3.2, respectively), thus implying the purification process had also removed the majority of peroxides. The PV (3.4 meq O₂/kg oil) and p-AV (3.0) in Final-HMFS was higher than that in OPO-HMFS but it was still within an acceptable range.

The rancimat assay characterizes the oxidative stability of oils. The oxidative stability indexes of palm stearin, OPO-HMFS and Final-HMFS were measured by the Rancimat test and expressed as IP (Table 4). Higher IP reflects a longer shelf-life of products. The IP value of the OPO-HMFS (4.1 h) and Final-HMFS (6.3 h) were lower compared to palm stearin (16.8 h). For one thing, USFAs in HMFS were more easily oxidized than SFAs in palm stearin; for another thing, the reason may be due to the loss in tocopherols and β-carotene in the process of refining OPO-HMFS by molecular distillation, thus reducing antioxidant capacity. The decrease in oxidative stability after molecular distillation is in agreement with several similar studies (Nielsen et al., 2006; Qin et al., 2014). Fat oxidative rancidity is due to the long-term storage of HMFS under unfavorable conditions, which usually requires some protection from antioxidants.

The efficacy of β-carotene, L-AP, VE and TBHQ (tert-butylhydroquinone) as antioxidants was evaluated using the Rancimat test at 100 °C, and concentrations at 0.02% (w/w) under air saturation conditions. The results were expressed as induction period (IP) corresponding to the oxidative stability of OPO-HMFS (Figure 4). The IPs of the blank, β-carotene, L-AP, VE and TBHQ were 3.8, 5.6, 7.8, 8.1, and 12.7 h, respectively. This indicates that the antioxidant activities of the antioxidants decreased as follows: TBHQ > VE ≈ L-AP > β-carotene. Although TBHQ has strong antioxidant activity compared to other antioxidants, it is a synthetic antioxidant. The existence of a double bond in β-carotene can prevent oil from being oxidized, but the effect is not profound. VE, which contains hydroxyl groups, can inhibit lipid oxidation by capturing free radicals, which is a natural antioxidant and a nutritional component contained in vegetable oil. L-AP is the only unnatural antioxidant allowed to be added to baby food in China. In some instances, combined antioxidants have greater antioxidant activities than their individual effects. The IP of the mixture of VE and L-AP (at a weight ratio of 1:1) is 9.8 h, which is higher than that of VE or L-AP alone, indicating that L-AP has a synergistic effect with VE. When VE and L-AP are used together, L-AP could regenerate the phenoxyl radical of VE back to active VE. The synergistic effect of VE and L-AP cannot only enhance the oxidative stability of OPO-HMFS, but also reduce cost because L-AP is low-priced.

HMFS are widely used in emulsions because of low water solubility. Emulsion can be used as an excellent carrier for wrapping, protecting and transporting HMFS, so that it can reach the designated position in the gastrointestinal tract digestion process (Hu, et al., 2003). Nevertheless, the oxidation of HMFS in emulsion is inevitable. In this study, PV was used to evaluate the oxidative stability of OPO-HMFS-Emulsion during the oven test at 63 ± 1, for 21 days. As shown in Figure 5, the PV of the blank group was 2.7±0.5 meq/kg on the first day, which then began to accelerate until it reached a maximum of 118.6±1.9 meq/kg after 21days of storage. In contrast, the PV of HMFS containing the TBHQ, VE, L-AP, mixture of VE and L-AP increased slightly until day 9, and then gradually increase dynamically with storage time. The changes in PV in the samples spiked with TBHQ, VE and L-AP showed that the addition of antioxidants was better at preventing oxidation of the emulsions and they were 20.4, 70.2, and 80.5 meq/kg on the last day, respectively. This indicates that the antioxidant activities of the antioxidants in emulsions decreased in the following order: TBHQ > VE > L-AP. Similarly, the results from Figure 5 showed that the mixture of VE and L-AP resulted in a synergistic antioxidant effect. Besides, the OPO-HMFS-Emulsion oxidative stability of L-AP and VE at a ratio of 2:3 was greater than that of ratio 1:3. Although increasing the proportion of L-AP in mixed antioxidants can improve the oxidative stability of OPO-HMFS emulsion, the proportion of VE in it should be kept at a higher level because VE also plays an important role in improving immunity and promoting the growth of infants.