Physicochemical Characterization of Edible Insect Oils: Insights Physicochemical Characterization of Edible Insect Oils: Insights into Fatty Acid Composition, Thermal Behavior and Quality into Fatty Acid Composition, Thermal Behavior and Quality Parameters Parameters

Edible insects are emerging as a sustainable food source due to their high nutritional value. This study employed the three-phase partitioning (TPP) method to extract oils from sago worm ( Rhynchophorus ferrugineus ), superworm ( Zophobas morio ), and cricket ( Gryllus campestris ). The study evaluated and compared the physicochemical properties of these insect oils, including iodine value (IV), peroxide value (PV), p -anisidine value (p-AV), acid value (AV), refractive index (RI), thermal behavior, and fatty acid compositions. Results revealed that the sago worm exhibited the highest oil yield (47.8 ± 2.2%) compared to the superworm (23.8 ± 1.4%) and cricket (6.4 ± 0.6%). Cricket oil (CO) displayed the highest IV (85.62 ± 0.35 g/100 g), PV (50.50 ± 1.10 mEq/kg), and p-AV (4.66 ± 0.45), while AV and RI were the highest in sago worm oil (SWO) (8.23 ± 0.09 mg KOH/g) and superworm oil (SO) (1.4655 ± 0.00), respectively. The melting and crystallization pro ﬁ les of these insect oils varied. Palmitic acid (C16:0) was the predominant fatty acid in all insect oils. CO exhibited the highest content of unsaturated fatty acids (51%), followed by SO (42%) and SWO (39%). This study highlights the potential applications of insect oils in various industries.


Introduction
A s the global population continues its upward trajectory, the exploration of alternative food sources has drawn significant attention across numerous countries.Edible insects have therefore emerged as an alternative food source to alleviate hunger and improve malnutrition.Among the myriad of edible insects, sago worm (Rhynchophorus ferrugineus), superworm (Zophobas morio), and cricket (Acheta domesticus) have gained prominence recently.These insects represent just a fraction of the more than 2000 species of edible insects, which are categorized into 18 different orders, including Orthopterans, Coleopterans, Lepidopterans, Isopterans, and Hymenopterans (Tang et al., 2019).
Edible insects are rich in nutritional content, encompassing proteins, fats, minerals, vitamins, and calories.However, the nutritional content of these insects differs based on factors like their developmental stage, insect species, diet, and environment (Phuah, Chong, et al., 2022).Typically, the pupa, larvae, and adult forms of edible insects are preferred for consumption, and among these, the sago worm, superworm, and cricket have carved a niche as notable choices.
In developing countries, particularly in South Asia, malnutrition and food insecurity pose challenges, especially among children who struggle to access conventional meats (Tao & Li, 2018).Edible insects, owing to their ready availability and nutrient richness, have become a primary food source for these vulnerable populations.The Food and Agriculture Organization (FAO) vigorously promotes insect consumption due to its numerous health advantages and its positive impacts on the environment, economy, and society (Van Huis et al., 2013).Insects require less land, produce fewer greenhouse gases, and can be raised on organic waste, presenting a compelling contrast to traditional livestock farming.However, despite their well-documented nutritional benefits, cultural acceptance and food safety concerns continue to impede the widespread adoption of insect-based diets.
Insects offer two primary macronutrients namely protein and fat.Research conducted by Tuhumury (2021) reveals that Coleoptera larvae and adults contain protein levels ranging from 23% to 66%, Hemiptera from 42% to 74%, while Lepidoptera pupae and larvae exhibit protein contents ranging from 14% to 68% (Tuhumury, 2021).Beyond protein, insects are abundant sources of mono and polyunsaturated fatty acids, closely resembling those fats and oils found in meat and fish.It is also noteworthy that edible insects can significantly contribute to the human diet by supplying essential minerals and vitamins required for optimal health (Phuah, Chong, et al., 2022).
In this study, we employed the three-phase partitioning (TPP) method to extract oils from three distinct edible insect species namely sago worm, superworm, and cricket.The novelty of this research lies in the comprehensive evaluation and comparison of the physicochemical properties of these insect oils extracted using TPP technique, including iodine value (IV), peroxide value (PV), p-anisidine value (p-AV), acid value (AV), refractive index (RI), and fatty acid composition.Additionally, our study investigated the melting and crystallization profiles of these insect oils, providing insights into their thermal behaviors, which can be crucial for various food applications.To the best of our knowledge, this is the first report of the melting and crystallization profiles of insect oils derived sago worm, superworm, and cricket from using TPP method, offering valuable perspectives on their potential applications in the food industry and beyond.Moreover, we analyzed the fatty acid composition of these oils, highlighting the presence of both saturated and unsaturated fatty acids in all insect oils.These findings emphasize the physicochemical properties and nutritional potential of these oils for human consumption.

Preparation of insect samples
Sago worm (R. ferrugineus) was sourced from a local farmer in Sarawak, Malaysia.Superworm (Z.morio) and cricket (A.domesticus) were purchased from local shops in Shopee Malaysia.All insects were dried for 24 h at 60 C using a drying oven to remove moisture.Once dried, they were ground into a powder using a grinder, and the resulting insect powder was stored in a freezer until further use.

Extraction of oils
Insect oils were extracted using the three-phase partitioning (TPP) method (Laroche et al., 2019).Distilled water (60 mL) was added to a beaker containing 10 g of dried insect powder.The mixture was adjusted to pH 4.5 using 0.1 M HCl.Subsequently, 20 g of ammonium sulfate and 60 mL of tert-butanol were added, and the mixture was stirred for 1 h at 35 C using a magnetic stirrer.After resting for 1 h at room temperature, the mixture was centrifuged at 6000 rpm for 5 min.A rotary evaporator was employed to remove tert-butanol from the collected supernatants.The extracted oil was then centrifuged under the same conditions to remove impurities or unwanted substances.The oil samples were stored in a freezer at À20 C until analysis.The extraction yield of oil was calculated using Eq. ( 1

Determination of quality attributes of insect oils
In this study, we analyzed the quality parameters of the insect oils, including iodine value (IV), peroxide value (PV), p-anisidine value (p-AV) and acid value (AV).The IV of insect oils was determined using the Wijs method in accordance with AOAC Official Method 993.20.The PV of insect oils was determined using the acid-base titration method according to AOAC Official Method 965.33.The p-AV of insect oils was determined based on AOCS Official Method Cd 18-90.The AV of insect oils was determined by the acid-base titration method according to AOAC Official Method 940.28.The refractive index (RI) of insect oils was determined using a PAL-3 Digital Pocket Refractometer (Atago, Japan) following AOCS official method Tp 1a-64.

Fatty acid composition of insect oils
For the preparation of fatty acid methyl esters (FAMEs), 100 mg of oil sample was dissolved in 5 mL of hexane and 250 mL of 0.5 M methanolic sodium methoxide solution before vortexing min (Soo et al., 2020).The mixture was then filtered through a 0.22 mm nylon syringe filter.Approximately 1 mL of the filtered top layer of FAME was transferred into a vial, and the vial was filled up to 1.5 mL using hexane before analysis using GC-MS (GCMS-QP2010 plus) (Shimadzu Corporation, Japan) with a split injector.Separations were performed using a fused silica capillary column (30 m Â 0.25 mm Â 0.25 mm film thickness).The column oven temperature and injection temperature were set to 140 C and 250 C, respectively.Initially, the oven temperature was held at 140 C for 10 min before being increased to 250 C at a rate of 7 C/min, with a hold time of 10 min (Moigradean et al., 2013).Helium gas was used as the carrier gas with a column flow rate of 2 mL/min, purge flow rate of 3 mL/min, and a split ratio of 1:30.For mass spectrometry (MS), the ion source temperature and interface temperature were set to 210 C and 255 C, respectively, with a 3-min solvent cut time.The event time, scan speed, and MS spectra range were set to 0.50 s, 1000, and 40e500 m/z, respectively.The NIST Mass Spectral Library was used to analyze and identify the FAME peaks of oils by comparing mass spectra and retention times.

Melting and crystallization profiles of insect oils
Differential Scanning Calorimetry (DSC) (Mettler Toledo DSC823c, Switzerland) was used to analyze the crystallization and melting properties of insect oils.Nitrogen gas was supplied to the system at a flow rate of 60 mL/min.Approximately 5e6 mg of oil sample was weighed into a 20 mL aluminum crucible and sealed with a pierced aluminum lid using a crucible sealing press.An empty and sealed aluminum crucible served as a reference.The software was programmed to heat the oil sample from 25 C to 70 C at a rate of 5 C/min and maintain it at 70 C for 10 min.Subsequently, the oil was cooled from 70 C to À30 C at 5 C/min and held at À30 C for 10 min.The oil sample was then heated back to 70 C at a rate of 5 C/min and held for another 10 min (Soo et al., 2020).Crystallization and melting curves were then analyzed using Star-e Mettler Toledo analytical software.

Statistical analysis
All experiments in this study were conducted in triplicates, and the data were presented as mean ± standard deviation (SD).One-way analysis of variance (ANOVA) was performed using JMP software to analyze the results.The significant difference between oil samples was determined using Tukey's test at a significance level of 5% (p < 0.05).

Extraction yield
The extraction yield of edible insect oils was assessed and compared among three different species namely sago worm, superworm, and Cricket.These oils were labeled as sago worm oil (SWO), superworm oil (SO), and cricket oil (CO), respectively.The extraction yield of SWO was found to be significantly higher than that of SO and CO (p < 0.05), as indicated in Table 1.The difference in extraction yield can be attributed to the inherent variations in fat content among edible insects.Typically, the fat content in edible insects ranges from 10% to 60% on a dry basis, with triacylglycerols (TAGs) being the predominant fat component, followed by phospholipids and others (Kou rimsk a & Ad amkov a, 2016).Furthermore, it is observed that larvae and pupae tend to possess higher fat content compared to adults of the same species (Rumbos & Athanassiou, 2021).Previous literature has indicated that various factors, including diet, gender, size, habitat, geographic region, and developmental stage, could influence the fat content of edible insects (Rumbos & Athanassiou, 2021;Yap et al., 2023).For instance, Magara and colleagues (2021) highlighted a wide range of fat content in edible crickets, ranging from 4.3% to 33.4%, with Gryllus bimaculatus and Acheta domesticus among the species with the highest fat content.Additionally, previous studies have indicated that Z. morio adult fat content is significantly lower when compared to larvae stage, aligning with the general pattern where larvae tend to exhibit higher fat content than their adult counterparts (Oonincx & Dierenfeld, 2012;Rumbos & Athanassiou, 2021).

Quality attributes of insect oils
Iodine value (IV) serves as an important indicator of the degree of unsaturation and the number of double bonds present in oils.A higher IV signifies a higher level of unsaturation, which, in turn, indicates a greater potential for oxidation.Our results showed that CO exhibited the highest IV, while SWO displayed the lowest IV (Table 1).The highest IV of CO could be explained by the high concentrations of unsaturated fatty acids, particularly oleic and linoleic acids as evidenced from Table 2.A study by Paul et al. (2017) also revealed that CO exhibited a high IV of 93.2, comparable to or even superior to some conventional vegetable oils such as olive oil and palm oil (O'brien, 2009;Phuah, Yap, et al., 2022).On the other hand, SWO showed a relatively low IV owing to the dominance of saturated and monounsaturated fatty acids, making it suitable to be used as cooking oil because of its high oxidative stability.
In addition to IV, the peroxide value (PV) was determined to assess the quality of the insect oils.Table 1 reveals that CO exhibited the highest PV, followed by SWO and SO.The PV serves as a crucial indicator of oil quality, with lower values signifying superior oil quality due to reduced oxidation and rancidity.Oxidation occurs when oils are exposed to oxygen, resulting in the formation of hydroperoxides as primary oxidation products before further breaking down into low-molecular-weight off-flavor compounds (Shahidi & Hossain, 2022).Among the three insect oils, CO demonstrated the highest PV, indicative of the highest level of lipid oxidation.The rate of oxidation in oil is influenced by factors such as temperature, light exposure, oxygen concentration, as well as the composition of fatty acids and acylglycerol species present in the oil (Shahidi & Zhong, 2010;Yun & Jeonghee, 2012).Unsaturation of fatty acid plays an important role in oxidation, with highly unsaturated fatty acids being more susceptible to oxidation.For instance, linoleic acid is approximately 40 times more reactive to oxidation compared to oleic acid (Yun & Jeonghee, 2012).Consequently, CO showed the highest PV in the present study.
The p-anisidine value (p-AV) was used to evaluate the extent of secondary lipid oxidation in insect oils.A low p-AV value indicates a high oil quality with minimal oxidation and vice versa.Typically, an acceptable p-AV value for edible oils is 8 (Alimentarius, 1999).As depicted in Table 1, CO 1.469 a-c indicates the significant difference at p < 0.05.Data with different superscripts within the same column indicates significant difference between samples.SWO ¼ sago worm oil; SO ¼ superworm oil; CO ¼ cricket oil.*Sources: (Alimentarius, 1999;Lai et al., 2020;Phuah, Yap, et al., 2022).(Alimentarius, 1999;Lai et al., 2020;Phuah, Yap, et al., 2022).exhibited the highest p-AV, indicating the presence of a higher quantity of secondary lipid oxidation products and a stronger odor was detected compared to SWO and SO.The secondary oxidation products primarily consist of volatile aldehydes and ketones.The observed trend in p-AV aligns with the results obtained for IV and PV, where CO demonstrated the highest degree of unsaturation and primary lipid oxidation.Consequently, unstable primary oxidation products in CO are prone to further oxidation, resulting in the formation of secondary oxidation products.In contrast, SO, which contains primarily saturated fatty acids, exhibited the lowest p-AV compared to the other insect oils due to its less susceptible towards oxidation.The acid value (AV) was examined to assess the formation of free fatty acids in oil due to hydrolytic breakdown.Lower AV values indicate a lower concentration of free fatty acids in the oil, rendering the oil less susceptible to rancidity.In this study, the AV was higher compared to Codex specification ( 0.6 mg KOH/g) and it followed SWO > SO > CO (Table 1).The observation could be explained by the hydrolysis of oil that takes place when water interacts with the oil during the extraction process, resulting in the formation of free fatty acids.Therefore, additional purification steps may be required to eliminate these free fatty acids.
The refractive index (RI) is a parameter that correlates with molecular weight, fatty acid chain length, degree of unsaturation, and degree of conjugation (Amos-Tautua & Onigbinde, 2013; Rahman et al., 2023).Our findings indicated that SO possessed the highest RI (1.4655 ± 0.00), followed by CO (1.4630 ± 0.00) and lastly SWO (1.4535 ± 0.00).Importantly, all RI values obtained for these samples fall within the established standard range (1.40e1.47)recommended for most edible oils (Alimentarius, 1999; Amos-Tautua & Onigbinde, 2013; Rahman et al., 2023).The lower RI in SWO could be attributed to its relatively lower unsaturated fatty acid content when compared to SO and CO.

Fatty acid compositions of insect oils
The fatty acid composition of the insect oils was analyzed and summarized in Table 2. Palmitic (C16:0) and oleic acids (C18:1) were identified to be the major fatty acids in all insect oil samples, followed by myristic (C14:0) and linoleic acid (C18:2).Our study highlighted that CO displayed the highest unsaturated fatty acid content (~51%) particularly linoleic acid (21.2%), followed by SO and SWO.These findings are consistent with a previous study that reported a similar fatty acid composition in CO, with approximately 57% of unsaturated fatty acids, where linoleic acid was the predominant species (Laroche et al., 2019).However, the authors mentioned that different extraction techniques could lead to insect oils with distinct fatty acid profiles (Laroche et al., 2019).CO can be employed as a functional oil owing to its significant linoleic acid content, which offers numerous health benefits such as lowering risk of cardiovascular diseases and other related complications (Marangoni et al., 2020).
On the other hand, our study revealed that SWO was abundant in both palmitic and oleic acids, constituting over 80% of the total fatty acid content.The low degree of unsaturation in SWO, as revealed by the fatty acid composition analysis, was consistent with the previously reported low IV (Table 1).Contrary to the findings of Chinarak et al. (2022), our investigation did not detect any presence of linoleic and stearic acids (Chinarak et al., 2022).However, another similar study conducted Chinarak et al. (2020) reported relatively low levels of linoleic and stearic acid (<1%) in SWO obtained from three different farms (Chinarak et al., 2020).These discrepancies may be linked to factors such as geographical locations, dietary habits, and the developmental stage of the insects (Phuah, Chong, et al., 2022;Yap et al., 2023).
This current study also demonstrated that SO exhibited a well-balanced fatty acid profile, consisting of medium-chain, long-chain, saturated, and polyunsaturated fatty acids.In contrast with previous studies, palmitic acid (C16:0) appeared to be the primary fatty acid species in this study, comprising nearly 40% of the total fatty acids.However, previous studies reported that oleic acid was the most prevalent fatty acid, followed by palmitic acid (Ad amkov a et al., 2017;Barroso et al., 2014).It was postulated that the variations in the extraction technique could account for the differences in fatty acid compositions in SO.

Thermal behavior of insect oils
The melting and crystallization behaviors of insect oils were analyzed in this study to gain insights into their functional and sensory properties, which is essential for their potential applications in the food industry.Fig. 1a illustrates the crystallization curve of these insect oils (SWO, SO and CO).In SWO, the crystallization curve displayed two distinct crystallization peaks, indicating the coexistence of two phases at specific temperatures (A cry,1 ¼ À15.7 C; A cry,2 ¼ 1.8 C).The higher temperature region in the crystallization profile indicates the stearin fraction, while the lower temperature region represents the olein fraction (Ong et al., 2019;Tan & Che Man, 2000).The presence of two crystallization peaks may be ascribed to the two major TAG groups with varying degrees of unsaturation, specifically (a) high-melting-point TAGs (saturated TAGs) and (b) low-melting-point TAGs (unsaturated TAGs).Therefore, oil fractionation can be employed for the separation of these two fractions, thereby expanding the potential food applications of SWO, such as producing super olein.The crystallization curve of CO exhibited a major exotherm peak (C cry,1 ) at 5.9 C and a minor shoulder peak (C cry,2 ) at À4.1 C whereas SO showed two overlapping exothermic peaks.Tan and Che Man proposed that the existence of shoulder peaks (transition peaks) might be attributed to polymorphic transformation of TAGs in oil samples (Tan & Che Man, 2000).
Fig. 1b Compares the melting curve of insect oil samples.In SWO, a major endothermic peak and a small distinct endothermic peak were observed, while three peaks with the last two peaks merging were noticed in CO.Meanwhile, SO displayed two endothermic regions: the higher region featured two minor fusion peaks, while the lower region comprised two major endothermic peaks.Our study also demonstrated that both SO and CO had considerably lower melting onset temperatures, approximately ranging from À5 C to À7 C, compared to SWO.This observation suggests that both SO and CO contained a higher proportion of low-melting temperature TAGs, which aligns with the high concentration of unsaturated fatty acids (Table 2).However, further investigation of the TAG composition of the insect oils may be needed.

Conclusion
The present study explored the physicochemical properties, fatty acid compositions and thermal behaviors (melting and crystallization profiles) of insect oils obtained from sago worm, superworm and cricket using TPP extraction technique.Our study revealed that the extraction yield of SWO was significantly higher than the others due to inherent variations in fat content among insect species.CO exhibited the highest IV, indicating higher degree of unsaturation and heightened oxidation potential as evidenced by high lipid oxidation indexes namely PV and p-AV.Nevertheless, CO can serve as an excellent source of functional oil owing to the high concentration of linoleic acid.In terms of AV, all insect oil samples exceeded Codex specifications ( 0.6 mg KOH/g), implying the possibility of hydrolytic reactions occurring during the extraction process.Therefore, subsequent purification steps are required.Our study also demonstrated that SWO had a low IV and was predominantly composed of saturated and monounsaturated fatty acids, making it well-suited for use as a stable cooking oil.SO exhibited a balanced fatty acid profile with varying chain lengths, providing versatility for different food applications.Thermal analysis disclosed differences in crystallization and melting profiles among the insect oils.This investigation underscored the feasibility of oil fractionation for SWO, enabling the production of a highly stable super olein with enhanced transparency when stored at low temperatures.CO and SO displayed lower melting onset temperatures, likely due to higher concentrations of low-melting-point TAGs.In summary, different edible insect oils exhibit distinct physicochemical properties owing to variations in fatty acid composition.These variations have implications for the quality, stability, and potential applications of these oils in the food industry.

Table 1 .
Extraction yield, Iodine value, peroxide value, p-anisidine value, acid value, and refractive index of insect oils.

Table 2 .
Fatty acid composition of three different insect oils.-b indicates the significant difference at p < 0.05.Data with different superscripts within the same column indicates significant difference between samples.ND ¼ not detected; SWO ¼ sago worm oil; SO ¼ superworm oil; CO ¼ cricket oil.*Sources: a