Analysis of Physicochemical Properties, Lipid Composition, and Oxidative Stability of Cashew Nut Kernel Oil

Cashew nut kernel oil (CNKO) is an important oil source from tropical crops. The lipid species, composition, and relative content of CNKO were revealed using ultra high performance liquid chromatography time-of-flight tandem mass spectrometry (UPLC-TOF-MS/MS), and the physicochemical properties, functional group structure, and oxidation stability of CNKO at different pressing temperatures were characterized using a near infrared analyzer and other methods. The results showed that CNKO mainly consisted of oleic acid (60.87 ± 0.06%), linoleic acid (17.33 ± 0.28%), stearic acid (10.93 ± 0.31%), and palmitic acid (9.85 ± 0.04%), and a highly unsaturated fatty acid (78.46 ± 0.35%). In addition, 141 lipids, including 102 glycerides and 39 phospholipids, were identified in CNKO. The pressing temperature had a significant effect on the physicochemical properties of cashew kernels, such as acid value, iodine value, and peroxide value, but the change in value was small. The increase in pressing temperature did not lead to changes in the functional group structure of CNKO, but decreased the induction time of CNKO, resulting in a decrease in their oxidative stability. It provided basic data support to guide subsequent cashew kernel processing, quality evaluation, and functional studies.


Introduction
The cashew nut is a genus of plants of the genus Dicotyledonea, order Sapotaceae, family Lacertidae, and genus Cashew [1]. Currently, the countries with relatively large cashew cultivation areas in the world include India, Brazil, Vietnam, Côte d'Ivoire, Mozambique, and Tanzania, whose total output accounts for more than 60% globally. According to the statistics of the world food and agriculture organization (FAO), it can be seen that the total global production of cashew nuts (in shell) was up to 4,093,000 tons in 2018 and 3,960,700 tons in 2019. The total cashew nut production (in 2019) in Africa, represented by Côte d'Ivoire and Mozambique, was 2,334,400 tons, accounting for 60% of the world's total production. The total production in Asia, represented by India, was 1,474,900 tons, accounting for 37%, and the total production in the Americas, represented by Brazil, was 151,400 tons, accounting for 3%.
Cashew nut kernels (CNKs) are the kernels of shelled cashew nuts after dehulling, which are mainly composed of 47.0% fat, 21.1% protein, 4.6-11.2% starch, 2.4-8.7% sugar, and other components, as well as a variety of amino acids, vitamins, and trace elements, such as phosphorus, iron, and calcium [1]. CNK processing mainly produces primary products, and deep processing products are less common, such as charbroiled cashew nuts, seaweed cashew nuts, salt-baked cashew nuts, cashew oil microcapsules [2], defatted

Physicochemical Analysis of Cashew Nut Kernel Oil
The determination of acid values was made with reference to GB5009.229-2016 [12], and the determination of iodine values was made with reference to GBT5532-2008 [13].
The peroxide value was determined by referring to the method of Akbar et al. [14] with appropriate modifications. A total of 0.02 mg of oil (m) was dissolved in 2 mL of chloroform/methanol (7:3), 0.05 mL of ammonium thiocyanate (0.3 g/mL) was added as sample solution 1, and the absorbance of sample solution 1 at 501 nm was E 0 . A total of 0.05 mL of ferrous chloride was added to sample solution 1, and the reaction was left for 5 min after vortex shaking to obtain sample solution 2, and the absorbance of sample solution 2 was at 501 nm. The absorbance of sample solution 2 at 501 nm was E 2 . Without adding oil, the absorbance at 501 nm was E 1 , and the peroxide value (POV) = (E 2 − (E 0 + E 1 ))/(55.82 × m).
The specific extinction coefficient was determined by referring to the method of HST57-2017 and Yakindra et al. [15] with appropriate modifications. A total of 0.25 g of oil sample was put into a test tube, dissolved by adding 5 mL of isooctane, and then diluted to 25 mL by adding isooctane. The concentration of the sample solution was ω (g/100 mL), and the oil sample was placed in the automatic refractometer (XFZGY-3000, Xiamen Xiongfa Instrument Co., Xiamen, China) for measurement. Using isooctane as the reference, the absorbance of the sample solution at 232 nm was A 232 , and the corresponding specific extinction coefficient K 232 = A 232 /ω. The absorbance of the sample solution at 270 nm was A 270 , and the corresponding specific extinction coefficient K 270 = A 270 /ω.

Determination of Fatty Acid Composition of Cashew Nut Kernel Oil
The fatty acid composition in cashew nut kernel oil was determined by gas chromatography (GC-MS) (LC-30A, Shimadzu, Kyoto, Japan) coupled with the potassium hydroxide methylation method. The relevant parameters were referred to in Liu et al. [16].

Determination of Lipids of Cashew Nut Kernel Oil
The lipid composition in CNKO oil was performed by a Shimadzu UPLC LC-30A system (LC-30A liquid chromatograph, Shimadzu Corporation, Kyoto, Japan) equipped with a Phenomenex Kinete C18 column (100 × 2.1 mm, 2.6 µm), and the relevant parameters were referred to in Liu et al. [16].

Determination of Fourier Transform Infrared Spectroscopy of Cashew Nut Kernel Oil
Referring to the method of Yakindra et al. [15] with some modifications, oil droplets were placed on the plane sensitive surface of the crystal and then placed in an infrared spectrometer (Thermo Nicolet iN10, ThermoFisher Scientific, Greenville, SC, USA) for measurement. The infrared spectra were collected in the range of 500-4000 cm −1 with a resolution of 4 cm −1 and 64 scans, and the infrared spectral data were collected with the OMSNIC software (Thermo Nicolet iN10, ThermoFisher Scientific, Greenville, SC, USA) with an average of 3 parallel acquisitions per sample. The average spectrum was used as the sample spectrum, and the infrared spectral curve was plotted using Origin software (2021, OriginLab Corporation, Northampton, UK).

Determination of Oxidative Stability of Cashew Nut Kernel Oil
The oxidative stability of the oil was determined by referring to the method of Xia et al. [17] with some modifications. A 3.5 g oil sample was weighed into a test tube, which was placed in an oil oxidation stability tester (Rancimat743, Swiss Aptar China Ltd., Hong Kong, China) at 110 • C with an air flow rate of 20 L/h. The induction time was recorded from the inflection point of the conductivity curve and the results were recorded in hours.

Data Processing and Analysis
All samples were measured 3 times in parallel. LipidView software (v2.0, ABSciex, Concord, ON, Canada) was used to undertake qualitative analysis of shotgun-MS data. In the process of data analysis, the relevant parameters of the software were set as follows: the mass tolerance was 0.5, the minimum% intensity was 1, the minimum signal-to-noise ratio was 10, the average flow injection spectrum from the top was 30% TIC, and the total double bond was ≤12. OriginPro (2021, OriginLab Corporation, Northampton, UK), SIMCA (14.1, Sartorius Lab Instruments GmbH & Co. KG, Goettingen, Germany), and Photoshop (2022, Adobe Systems Incorporated, San Jose, CA, USA) were used for plotting, data processing, and statistical analysis.
The lipid composition of CNKO was comprehensively profiled using UPLC-TOF-MS/MS, and information on the precise relative molecular masses of lipids, isotopic distribution, and secondary mass spectrometry cleavage fragments were obtained in composite scanning mode. The lipids of CNKO were identified as shown in Tables 1 and 2 and Figure 1. As shown in Figure 1A  As shown in Table 1, the total number of carbon atoms in the fatty acid side chains of lipids in CNKO was 16-32,   As shown in Figure 1C, it could be seen that the content of each glyceride in CNKO was ranked as TG > DG > EtherTG > OxTG > DGCC > EtherDG > DGGA, where TG had (617.97 ± 60.02) mg/g and DG had (7.82 ± 1.28) mg/g. As shown in Figure 1D, it could be seen that the content of each phosphate ester in CNKO was ranked as PC> PI > PE > EtherPC > LPC > PG > LPE > LPI > EtherPE > EtherPI, where PC had (8.32 ± 0.41) µg/g and PI had (5.28 ± 0.29) µg/g.

Physicochemical Properties of Cashew Nut Kernel Oil
The CNKO obtained at different pressing temperatures was slightly yellow in color, without obvious precipitation, with a slight aroma of cashew nut kernel, and its physicochemical properties are shown in Table 3. As can be seen from Table 3, the acid value of cashew nut oil was (0.41-0.53) mg/g < 4 mg/g as national standard and the peroxide value was (0.036-0.118) g/100 g < 0.25 g/100 g as national standard, all of which satisfied GB 2716-2018 "National Standard for Food Safety Vegetable Oil" [18]. The specific extinction coefficient at 232 nm was related to the primary and secondary stages of oil oxidation, while the specific extinction coefficient at 270 nm was related to the secondary stages of oil oxidation [19,20]. The K232 and K270 of CNKO were (1.0-1.2) and (0.05-0.12), respectively, indicating that the cashew nut kernel oil obtained using the pressing method contains only a very small amount of hydroperoxides and was not easily acidified.
The results of the significance analysis showed that there were different degrees of influence of pressing temperature on acid value, iodine value, peroxide value, refractive index, and specific extinction coefficient. The differences in acid value were not significant at temperatures greater than 100 • C. The differences in peroxide value were not significant at 140 • C and 160 • C, and significant at other temperatures (100 • C, 120 • C, 160 • C, 180 • C). The differences in iodine value were not significant at 120 and 140 • C, and significant at other temperatures. The differences in the refractive index were not significant at 160 • C and 180 • C, and the differences in the refractive index were not significant at 100 • C, 120 • C, 140 • C, and 200 • C, and significant between the two groups. Although the results of the significance analysis showed that the relevant indexes of the oils obtained at different temperatures were affected, the value fluctuated less, indicating that the physicochemical properties of CNKO were relatively stable.

Near-Infrared Spectral Characteristics of Cashew Nut Kernel Oil
The Fourier NIR spectra of CNKO are shown in Figure 2. Figure 2A represents the NIR spectra of cashew nut kernel oil at different pressing temperatures, and Figure 2B represents the NIR spectra of different types of oils. 1233.88 cm −1 , the position of this absorption band changed with the structure of the compound molecule due to the vibrational coupling effect and weak intensity, so it was not meaningful in the structure identification. The peak at 1164.63 cm −1 corresponded to the C-O stretching vibration in alcohols, which could be used to distinguish between primary, secondary, and tertiary alcohols, and CNKO might be the C-O stretching vibration of tertiary alcohols. The peak at 720.30 cm −1 corresponded to a swinging vibration in the -CH2 plane and had more than four methylene-linked structures. As shown in Figure 2B, it can be seen that the NIR spectra of camellia seed oil, macadamia nut oil, pitaya seed oil, and cashew nut kernel oil had similar peak shapes, peak positions, and number of characteristic peaks, indicating that the functional groups of the oils had similar structures. However, the peak signal intensities of different oils and fats were different, probably due to differences in the number of functional groups, such as differences in the composition of the oils [21].

Analysis of Oxidative Stability of Cashew Nut Kernel Oil
Oxidative stability could be a good predictor of the oxidation reaction of oils. The auto-oxidation of oils was divided into the induction phase as well as the oxidation phase, and the length of time required from the induction phase to the oxidation phase could reflect the ability of oils to resist auto-oxidation, which was the oxidation stability of oils [22,23]. The induction time could indirectly reflect the size of the oxidative stability of oils. The longer the induction period was extended, the better the oxidative stability of oils, and vice versa, the worse their oxidative stability.
As shown in Figure 3, the oxidative stability indices of CNKO at different pressing temperatures were in the interval of 9.3-10.2 h with an error of no more than 1 h. Meanwhile, the oxidative stability indices began to show a decreasing trend when the pressing temperature was greater than 120 °C. The results of the significance analysis showed that the induction time of CNKO was not significant between 100 °C and 120 °C, and between 140 °C, 160 °C, and 180 °C. The difference between 200 °C and other temperatures was As shown in Figure 2A, it can be seen that the peak at 3005.43 cm −1 corresponds to the -CH 3 antisymmetric stretching vibration, at 2930.41 cm −1 corresponds to the -CH 2 antisymmetric stretching vibration, at 2855.40 cm −1 corresponds to the -CH 2 symmetric stretching vibration, and at 1744.57 cm −1 corresponds to the C=O stretching vibration. This could be used to identify ketones, aldehydes, acids, esters, and anhydrides. The possibility that CNKO had an anhydride structure was ruled out because the anhydride would have a double peak due to vibrational coupling. Although 1658.01 cm −1 corresponded to the C=C stretching vibration, 2-4 peaks due to benzene ring skeleton vibration were not found near 1600 cm −1 and 1500 cm −1 , so the possibility of the presence of an aromatic ring structure in CNKO was excluded. The peak at 1461.81 cm −1 corresponded to the -CH 3 asymmetric deformation vibration and at 1375.25 cm −1 corresponded to the -CH 3 symmetric deformation vibration. Although it corresponded to the C-C stretching vibration at 1233.88 cm −1 , the position of this absorption band changed with the structure of the compound molecule due to the vibrational coupling effect and weak intensity, so it was not meaningful in the structure identification. The peak at 1164.63 cm −1 corresponded to the C-O stretching vibration in alcohols, which could be used to distinguish between primary, secondary, and tertiary alcohols, and CNKO might be the C-O stretching vibration of tertiary alcohols. The peak at 720.30 cm −1 corresponded to a swinging vibration in the -CH 2 plane and had more than four methylene-linked structures. As shown in Figure 2B, it can be seen that the NIR spectra of camellia seed oil, macadamia nut oil, pitaya seed oil, and cashew nut kernel oil had similar peak shapes, peak positions, and number of characteristic peaks, indicating that the functional groups of the oils had similar structures. However, the peak signal intensities of different oils and fats were different, probably due to differences in the number of functional groups, such as differences in the composition of the oils [21].

Analysis of Oxidative Stability of Cashew Nut Kernel Oil
Oxidative stability could be a good predictor of the oxidation reaction of oils. The autooxidation of oils was divided into the induction phase as well as the oxidation phase, and the length of time required from the induction phase to the oxidation phase could reflect the ability of oils to resist auto-oxidation, which was the oxidation stability of oils [22,23]. The induction time could indirectly reflect the size of the oxidative stability of oils. The longer the induction period was extended, the better the oxidative stability of oils, and vice versa, the worse their oxidative stability.
As shown in Figure 3, the oxidative stability indices of CNKO at different pressing temperatures were in the interval of 9.3-10.2 h with an error of no more than 1 h. Meanwhile, the oxidative stability indices began to show a decreasing trend when the pressing temperature was greater than 120 • C. The results of the significance analysis showed that the induction time of CNKO was not significant between 100 • C and 120 • C, and between 140 • C, 160 • C, and 180 • C. The difference between 200 • C and other temperatures was significant. It could be inferred that the oxidation rate of CNKO accelerated, and the induction time decreased as the extraction temperature increased, and the oxidative stability decreased.

Discussion
Cashew nut kernel oil accounted for approximately 47% of the cashew nut kernel content, which was higher than the oil content of avocado (8-29%) [24,25], olive (18-24%) [26], and camellia seed (14-27%) [27], etc., and lower than the oil content of macadamia nut (70-79%) [28]. It had a higher iodine value compared to macadamia nut oil and camellia seed oil. CNKO was typically characterized by a high oleic acid content of 60%, while camellia seed oil was high in α -linolenic acid and macadamia nut oil contained lauric and myristic acids [29]. Similar to olive oil, macadamia nut oil, and dragon fruit seed oil, the lipid composition in cashew nut kernel oil also consisted mainly of glycerides and phospholipids, but cashew nut kernel oil was rich in 39 phospholipid components, which was higher than the 16 reported for pitaya seed oil [16] and much less than the 172 reported for soybean, 109 for peanut, and 351 for sesame [30]. This stems from the fact these studies analyzed all phospholipid species in the fruit, whereas the present study analyzed the lipids in the oil.
Phospholipids are the main components of the cell membranes of animal and plant cells and play an important role in maintaining the physiological activity of biological membranes and the normal metabolism of the organism. They have important functions in antioxidation and delaying aging [31], regulating blood lipids and protecting the liver [32,33], and in enhancing the immunity of the organism [34,35], making them an excellent functional lipid.
Physicochemical properties and oxidative stability were important indicators of the quality of oils [22,24]. The iodine value of CNKO was (75-80) g/100 g, indicating that CNKO is a non-drying oil. The acid value was (0.41-0.53) mg/g, indicating that the content of free fatty acids in CNKO is low. The peroxide value was (0.036-0.118) g/100 g, indicating that there are less oxidation products in the oil, and the results of NIR (near-infrared) analysis indicated that the pressing temperature had no effect on the functional group structure of the oil. The above results indicate that different pressing has less effect on the quality of CNKO. In contrast, Li et al. [36] reported that there was a difference in the conclusion that the pressing process had a significant effect on the acid value and peroxide value of sesame, linseed, and perilla violet oils, and the reason for the difference might be due to the fact that the cashew nut kernels used in this study were directly pressed without roasting, which produced less antioxidant substances, such as nigrosine-like substances, due to the Merad reaction.
In addition, Michae et al. [37] showed that the oxidative stability of canola oil, olive oil, corn oil, soybean oil, sunflower oil, and flaxseed oil were 14.4 h, 19.9 h, 12.8 h, 10.9 h, 7.9 h, and 1 h, respectively, indicating that the oxidative stability of CNKO was worse than that of canola oil, olive oil, and corn oil, comparable to that of soybean oil, and better than that of sunflower oil and flaxseed oil. The oil had a high unsaturated fatty acid content and a fast oxidation rate, [38] while CNKO had an unsaturated fatty acid content up to 78%,; therefore, the oxidation of CNKO should be avoided during processing, storage, and transportation.

Conclusions
In this study, 141 lipids, including 102 glycerides and 39 phospholipids, were isolated and identified from cashew nut kernel oil using high performance liquid chromatography time-of-flight tandem mass spectrometry. Cashew nut kernel oil was a high oleic acid oil. The glycerol esters were mainly composed of DG, EtherDG, DGCC, DGGA, TG, EtherTG, and OxTG, and the phospholipids were mainly composed of LPC, LPE, LPI, PC, EtherPC, PE, EtherPE, PG, PI, and EtherPI. The total number of carbon atoms in the side chains of fatty acids in cashew nut kernel oil mass was 16-62, with a double bond number of 0-7. With the increase in pressing temperature, the functional group structure of cashew nut kernel oil was not changed, although the iodine valence, peroxide value, and the specific racemization coefficient increased, decreasing the induction time and reducing the oxidative stability of the cashew nut kernel oil.