Comprehensive Evaluation of the Nutritional Quality of Stored Watermelon Seed Oils

Abstract

The appropriate use of food industry by-products such as watermelon seeds could reduce the problem of food waste, following the “zero waste” concept. Research in recent years suggests that these unused waste products could be a source of nutrients and bioactive compounds. Accordingly, the present study aimed to evaluate the nutritional potential and selected quality parameters of watermelon seed oils. Four commercial oils (three unrefined cold-pressed and one refined pressed) and one self-extracted oil were considered. The oils were analyzed over three months of storage after opening/extraction to determine their fatty acid (FA) composition and distribution, hydrolytic and oxidative stability, and selected health indices. Linoleic acid was the predominant FA, ranging from 52.9% (refined oil) to 62.2% (self-extracted oil). Refined oil demonstrated superior oxidative stability, with the lowest acid value (AV) and peroxide value (PV) throughout the storage period, adhering to the Codex Alimentarius standards. Unrefined oils, particularly WO₃, showed significantly higher AVs and PVs after storage, indicating greater susceptibility to hydrolytic and oxidative changes. Health indices were favorable for all oils, with self-extracted oil exhibiting the highest health-promoting index (7.07) and hypocholesterolemic/hypercholesterolemic ratio (7.18). Oxidative stability showed that self-extracted oil had significantly higher stability (76.6 min) than other tested oils, despite having the highest PUFA content. In turn, refinement has a significant effect on the AVs and PVs and the oxidative stability of oil, achieving the lowest PUFA level (53.61%). These results emphasize the potential of watermelon seed oil as a health-promoting product and emphasize the role of production and storage conditions in maintaining its quality.

1. Introduction

Watermelon (Citrullus lanatus) is one of the most commonly consumed species of the Cucurbitaceae family. This fruit is very popular due to its sweet taste, low energy value, and high water content [1]. Watermelon is consumed around the world, but its availability and consumption patterns vary by region. In tropical and subtropical areas such as China, Iran, Turkey, India, and Brazil, watermelon is widely available for most of the year due to the favorable growing conditions. In temperate climates, however, watermelon remains a seasonal fruit, typically consumed during the summer months when local harvests take place [2]. According to FAO data [3], global watermelon production reached 160.5 million tons in 2023.

Watermelon is typically consumed as fresh fruit or processed into other products, such as juices, nectars, jellies, concentrates, and jams, while the seeds and rind are usually discarded as waste. By utilizing these by-products, both environmental and economic benefits can be achieved, promoting sustainable practices and a circular economy in line with the concept of “zero” or “less” waste [4]. Consequently, an increasing amount of research is focusing on the valuable properties of watermelon seeds and rind and their potential for further use. Studies suggest that watermelon rind may be a source of bioactive compounds, such as phenolic acids, lycopene, and citrulline [5,6], or a source of pectin [7].

The literature indicates that watermelon seeds are highly nutritious. They are a source of protein, unsaturated fatty acids, vitamins C and B, and significant amounts of minerals, primarily phosphorus, magnesium, manganese, and zinc, as well as potassium and calcium [8,9,10]. They also contain bioactive compounds such as phenolic compounds and flavonoids [10,11]. Moreover, studies highlight their antimicrobial, antifungal, antidiabetic, and antihyperlipidemic properties [1,12,13]. The nutritional value of watermelon seeds is further enhanced by their favorable amino acid profile. Among the amino acids, watermelon seed proteins contain the highest amounts of glutamic acid and arginine and are also a source of tryptophan and lysine [14,15]. In Arab and Asian regions, roasted and salted watermelon seeds are consumed as snacks [16]. They are also used as a topping for bread, cakes, confectionery, sweets, and snacks [17]. Fermented seeds have also been used as a flavor enhancer in sauces and soups [18].

Watermelon seed oil is amber, and its taste and aroma are described as distinctive, resembling that of nuts [19]. It is a good source of essential fatty acids, thiamine, riboflavin, vitamins A and E, and minerals, mainly iron, magnesium, copper, phosphorus, potassium, and manganese [20]. Additionally, it contains antioxidants, including tocopherols, polyphenolic compounds, and carotenoids [21]. Its unique characteristics, combined with its high nutritional value and limited availability, solidify its status as a specialty oil. Understanding the properties of watermelon seed oil, which could potentially support disease prevention and overall well-being, is crucial for realizing its full potential in health, nutrition, and sustainable applications. In turn, investigating how storage conditions influence its quality can point to practical production and storage challenges, such as the need to prevent oxidation and preserve its health-promoting properties. Such insights are important for optimizing the use of such oils as sustainable and functional food products.

Given the health and economic benefits of the significant amount of unused watermelon seeds, this study aimed to examine the nutritional quality of watermelon seed oils, including their composition and distribution of fatty acids, their oxidative and hydrolytic stability, and their health-promoting indices. The study focused on four commercial oils: three unrefined cold-pressed watermelon seed oils and one refined watermelon seed oil. Additionally, oil extracted from the ‘Sugar Baby’ watermelon variety was analyzed. The research also investigated how the type of oil and the storage time affect the quality and the oxidative stability of the oil.

2. Materials and Methods

2.1. Materials

Four commercial watermelon seed oils (three unrefined cold-pressed oils obtained via pressing watermelon seeds after removing their husks and one refined pressed oil) and a self-extracted oil (assigned as WO_ext) from watermelon seeds (the ‘Sugar Baby’ variety) were tested in this study. Commercial oils were purchased in Poland within their shelf life and tested before expiration. The origin of the products was as follows: Great Britain (oil assigned as WO₁), Africa (oil assigned as WO₂), Poland (oil assigned as WO₃), and Italy (refined oil assigned as WO_ref). The Warsaw Agricultural and Food Wholesale Market S.A acquired the watermelon seeds.

The analyses were carried out directly after the purchase of commercial oils and after the oil extraction process. The measurements were repeated every four weeks for the next three months. According to the manufacturer’s recommendations, the oils were stored in a cool and dark place before further tests.

2.2. Methods

2.2.1. Oil Extraction from Watermelon Seeds

Washed and sun-dried watermelon seeds were ground in a laboratory mill. Ground seeds (40 g) were extracted with 100 mL of hexane at room temperature for 120 min using a magnetic stirrer. Next, the samples were centrifuged for 20 min at 5000 rpm. Extracts were evaporated to dry at 60 °C on a rotary evaporator and then purged under a nitrogen stream to remove residual solvent.

2.2.2. Fatty Acid Composition Analysis

The fatty acid (FA) composition of the tested oils was determined using gas chromatography according to the method described by Bryś et al. [22]. A YL6100 GC Clarity gas chromatograph (Young Lin Bldg., Anyang, Hogye-dong, Republic of Korea), equipped with a flame ionization detector and a BPX-70 capillary column (SGE Analytical Science, Milton Keynes, UK), was used. Nitrogen served as a carrier gas for the methyl esters prepared according to EN ISO 5509:2001 [23]. The initial column temperature was set at 70 °C for 30 s, then increased at rates of 15 °C/min, 1.1 °C/min, and 30 °C/min within the ranges of 70–160 °C, 160–200 °C, and 200–225 °C, respectively. Upon reaching 200 °C, the temperature was maintained for 12 min. The injector and detector were set to 225 °C and 250 °C, respectively. The percentage abundance of each FA was subsequently determined [22].

2.2.3. Health Indices of Oils

The FA composition was used to calculate the health indices of tested oils. The atherogenicity index (AI) and thrombogenicity index (TI) were obtained from Equations (1) and (2), respectively [24]; the health-promoting index (HPI) and hypocholesterolemic/hypercholesterolemic ratio (h/H) were obtained from Equations (3) and (4) [25,26]:

$$\mathrm{A}\mathrm{I}={\displaystyle \frac{\mathrm{C}12:0+\left(4\times \mathrm{C}14:0\right)+\mathrm{C}16:0}{\sum UFA}}$$

(1)

$$\mathrm{T}\mathrm{I}={\displaystyle \frac{\mathrm{C}14:0+\mathrm{C}16:0+\mathrm{C}18:0}{0.5\times \sum MUFA+\left(0.5\times \sum n-6PUFA\right)+\left(3\times \sum n-3PUFA\right)+\frac{n-3}{n-6}}}$$

(2)

$$\mathrm{H}\mathrm{P}\mathrm{I}={\displaystyle \frac{\sum UFA}{\mathrm{C}12:0+\left(4\times \mathrm{C}14:0\right)+\mathrm{C}16:0}}$$

(3)

$$\mathrm{h}/\mathrm{H}={\displaystyle \frac{cis-\mathrm{C}18:1+\sum PUFA}{\mathrm{C}12:0+\mathrm{C}14:0+\mathrm{C}16:0}}$$

(4)

2.2.4. Fatty Acid Distribution Analysis

In order to define the positional distribution of FAs in the sn-2 and sn-1,3 positions of the TAG molecule, hydrolysis in the presence of pancreatic lipase was applied. Our previous studies described the enzymatic hydrolysis method in detail [27].

Based on the compositions of the isolated sn-2 monoacylglycerols (MAGs) and the starting TAGs, the composition of the FAs in the sn-1,3 positions was determined.

2.2.5. Hydrolytic Stability Determination

The physicochemical quality of tested oils was determined based on the lipid values. To evaluate the degree of hydrolytic changes, the acid values (AV) were determined according to the AOCS method (AOCS Official Method Te 1a-64) [28]. The peroxide content, expressed as the peroxide value (PV), was tested according to the AOCS method (AOCS Cd 8b-90) [29] using an automatic titrator TitraLab AT1000 Series (HACH LANGE, Wroclaw, Poland).

2.2.6. Oxidative Stability Determination

The oxidative stability was determined using a differential scanning calorimeter (DSC Q20P, TA Instruments, New Castle, DE, USA) coupled with a high-pressure cell (series DSC Pressure Cell, TA Instruments). The experiment was performed at a constant temperature of 120 °C under ~1400 kPa oxygen pressure. The procedure was described in our previous studies [30]. The PDSC oxidation time (τ_max) was determined based on the maximum rate of heat flow, with an accuracy of 0.005.

2.3. Statistical Analysis

All oil samples were analyzed in three replicates and the obtained results are presented as mean ± SD. Statistica version 13.3. software (StatSoft, Krakow, Poland) was used to statistically analyze the results. The homogeneous groups were divided using a two-way analysis of variance (ANOVA) and the Tukey test at a significance level of p-value ≤ 0.05.

3. Results and Discussion

3.1. Fatty Acid Composition

Fruit seed oils are mainly composed of triacylglycerols. Therefore, the FA composition and distribution in TAG molecules are representative properties of the lipid fraction of fruit seeds.

Table 1. Fatty acid composition [%] and health indices of tested watermelon seed oils (WO) before and after three months of storage at 4 °C.

Composition After Opening/Extraction [%]						Composition After 3 Months of Storage After Opening/Extraction [%]
Fatty Acid	WO1	WO2	WO3	WOref	WOext	WO1	WO2	WO3	WOref	WOext
C14:0	0.26 ± 0.01	0.25 ± 0.04	0.07 ± 0.01	0.25 ± 0.01	0.07 ± 0.01	0.27 ± 0.01	0.24 ± 0.01	0.06 ± 0.01	0.25 ± 0.01	0.07 ± 0.01
C16:0	11.79 ± 0.06	11.43 ± 0.29	12.45 ± 0.21	12.18 ± 0.01	11.00 ± 0.05	11.68 ± 0.03	11.42 ± 0.10	12.06 ± 0.12	11.89 ± 0.07	11.07 ± 0.32
C16:1	0.32 ± 0.01	0.15 ± 0.01	0.10 ± 0.01	0.16 ± 0.01	0.10 ± 0.01	0.32 ± 0.01	0.16 ± 0.01	0.10 ± 0.01	0.16 ± 0.01	0.10 ± 0.01
C17:0	0.07 ± 0.01	0.07 ± 0.01	0.09 ± 0.01	0.08 ± 0.01	0.14 ± 0.01	0.07 ± 0.01	0.07 ± 0.01	0.09 ± 0.01	0.08 ± 0.01	0.13 ± 0.01
C17:1	0.04 ± 0.01	0.04 ± 0.01	0.01 ± 0.01	0.04 ± 0.01	0.03 ± 0.01	0.04 ± 0.01	0.04 ± 0.01	0.01 ± 0.01	0.04 ± 0.01	0.02 ± 0.01
C18:0	4.42 ± 0.02	4.97 ± 0.07	12.52 ± 0.21	4.85 ± 0.07	8.54 ± 0.01	4.32 ± 0.01	5.01 ± 0.02	12.00 ± 0.03	4.76 ± 0.03	8.42 ± 0.09
C18:1 n-9	27.02 ± 0.02	25.23 ± 0.05	14.20 ± 0.28	28.18 ± 0.02	16.99 ± 0.02	26.76 ± 0.02	25.22 ± 0.07	13.70 ± 0.04	28.11 ± 0.02	16.90 ± 0.06
C18:2 n-6	54.58 ± 0.03	56.20 ± 0.27	58.56 ± 0.58	52.90 ± 0.06	62.21 ± 0.01	55.01 ± 0.02	56.28 ± 0.02	60.04 ± 0.13	53.23 ± 0.10	62.34 ± 0.14
C18:3 n-3	0.29 ± 0.01	0.40 ± 0.02	0.38 ± 0.01	0.42 ± 0.01	0.20 ± 0.01	0.31 ± 0.01	0.40 ± 0.01	0.39 ± 0.01	0.43 ± 0.01	0.22 ± 0.01
C20:0	0.36 ± 0.01	0.38 ± 0.02	0.57 ± 0.02	0.33 ± 0.01	0.43 ± 0.01	0.36 ± 0.01	0.35 ± 0.01	0.53 ± 0.01	0.34 ± 0.01	0.43 ± 0.02
C20:1 n-9	0.25 ± 0.01	0.22 ± 0.01	0.10 ± 0.01	0.20 ± 0.01	0.14 ± 0.01	0.25 ± 0.01	0.22 ± 0.01	0.10 ± 0.01	0.23 ± 0.02	0.12 ± 0.01
C20:3 n-3	0.25 ± 0.01	0.27 ± 0.02	0.13 ± 0.01	0.29 ± 0.01	0.10 ± 0.01	0.24 ± 0.01	0.25 ± 0.02	0.11 ± 0.01	0.31 ± 0.01	0.12 ± 0.01
C24:1	-	-	0.74 ± 0.02	-	-	-	-	0.71 ± 0.04	-	-
other	0.35 ± 0.01	0.39 ± 0.07	0.08 ± 0.01	0.13 ± 0.01	0.06 ± 0.01	0.38 ± 0.02	0.35 ± 0.02	0.08 ± 0.02	0.17 ± 0.01	0.07 ± 0.01
Σ SFA	16.91 ± 0.04 aA	17.10 ± 0.27 aAB	25.70 ± 0.31 bD	17.68 ± 0.07 aB	20.19 ± 0.03 aC	16.70 ± 0.04 aA	17.09 ± 0.09 aAB	24.74 ± 0.15 aD	17.32 ± 0.10 aB	20.12 ± 0.21 aC
Σ MUFA	27.62 ± 0.02 aD	25.64 ± 0.07 aC	15.15 ± 0.27 aA	28.58 ± 0.03 aE	17.25 ± 0.02 aB	27.37 ± 0.02 aD	25.64 ± 0.08 aC	14.63 ± 0.02 aA	28.55 ± 0.01 aE	17.13 ± 0.06 aB
Σ PUFA	55.12 ± 0.03 aB	56.87 ± 0.28 aC	59.07 ± 0.57 aD	53.61 ± 0.06 aA	62.51 ± 0.02 aE	55.56 ± 0.02 aB	56.92 ± 0.03 aC	60.54 ± 0.13 bD	53.97 ± 0.11 aA	62.67 ± 0.15 aE
Health indices
AI	0.16	0.15	0.17	0.16	0.14	0.15	0.15	0.16	0.16	0.14
TI	0.39	0.39	0.65	0.40	0.48	0.38	0.39	0.62	0.39	0.48
HPI	6.45	6.64	5.83	6.24	7.07	6.50	6.67	6.11	6.40	7.03
h/H	6.82	7.03	5.85	6.58	7.18	6.89	7.04	6.13	6.76	7.14

WO₁–WO₃—unrefined commercial oils; WO_ref—refined commercial oil; WO_ext—self-extracted oil; MUFA—monounsaturated fatty acids; PUFA—polyunsaturated fatty acids; SFA—saturated fatty acids; AI—atherogenic index; TI—thrombogenic index; HPI—health promoting index; h/H—hypocholesterolemic/hypercholesteremic index. Data are presented as mean values followed by standard deviation (±SD). Results marked with the same lower-case letters (a, b) do not statistically significantly differ within the same type of oil (before and after storage) at the level of α = 0.05. Results marked with the same upper-case letters (A–E) do not statistically significantly differ within the same type of oil at the level of α = 0.05.

presents the FA composition of the tested oils before and after three months of storage.

Considering the average content of saturated fatty acids, it should be noted that, during storage, WO₁ exhibited the lowest (~17%) and WO₃ exhibited the highest (~26%) SFA content. In each of the analyzed oils, palmitic acid and stearic acid had the highest percentage of the total SFAs. The oils were characterized by a higher MUFA content than SFA, except for WO₃, which showed an inverse relationship. The results regarding monounsaturated fatty acids show that, after storage, the lowest MUFA content was recorded for WO₃, while WO_ref had the highest MUFA content. In each analyzed oil, oleic acid was the predominant MUFA. Watermelon seed oils were rich sources of PUFAs. Linoleic acid (C18:2 n-6) was the predominant PUFA in the oils analyzed in this study. WO_ref exhibited the lowest (52.90%), and the WO_ext had the highest (62.21%), PUFA content.

A statistical analysis investigating the effect of storage time on the obtained SFA, MUFA, and PUFA levels indicated that storage period only had a significant impact on FA content for WO₃.

The identified composition of FAs in the oils analyzed in this study was typical of watermelon seed oils. However, it can be concluded that the oil production method and the refining process significantly affect the FA profile. Differences in the FA contents between commercial oils may also result from the varying quality of the raw materials used for oil production. Krygier et al. [31] demonstrated that rapeseed oil obtained from damaged, broken, and contaminated seeds was characterized by a lower oleic acid content and a higher linoleic acid content. Similar conclusions were drawn in the present studies. The significantly lower share of C18:1 n-9 and higher share of C18:2 n-6 for WO₃ than for the other tested oils may indicate that WO₃ was obtained from seeds of inferior quality to those used in other commercial oils.

Białek et al. [32] determined the FA composition of cold-pressed watermelon seed oil purchased from a grocery store. The PUFA, SFA, and MUFA contents were 68.00%, 18.30%, and 12.40%, respectively. These results are different from those obtained in our study, as all tested WOs contained less PUFAs and more MUFAs compared to the results obtained by Białek et al. [32]. Ouassor et al. [20] obtained oil from watermelon seeds of two varieties using cold-pressing, chemical extraction, and ultrasound-assisted extraction. For cold-pressed watermelon seed oil obtained using the first/second variety, the following contents were obtained: 17.69/18.48% SFA of 14.95/19.30% MUFA of 67.36 and PUFA of 62.22% PUFA. For the oil extracted from watermelon seeds of the first (second) variety, the results were 17.49/19.37% SFA, 14.92/16.80% MUFA, and 67.59/63.83% PUFA.

Dietary indices for assessing the risk of cardiovascular disease, such as the index of atherogenicity (AI) and the index of thrombogenicity (TI), can be calculated using fatty acid profiles. Oils recommended for consumption should have low atherogenicity (AI < 1.0) and low thrombogenicity (TI < 0.5), while also exhibiting a high health-promoting index (HPI) and a high hypocholesterolemic/hypercholesterolemic ratio (h/H). The consumption of products with low AI is correlated with a reduction in total cholesterol and low-density lipoprotein cholesterol in human blood plasma, whereas the consumption of products with lower TI and a higher h/H ratio may be beneficial in the prevention of cardiovascular heart disease [26,30,33].

Considering the results obtained for the studied oils (Table 1), it can be stated that they have favorable nutritional and health value, except WO₃, which was characterized by an increased TI index (0.65). It should be underlined that the omega-6 to omega-3 ratio for the tested oils is very poor as a result of the high linoleic fatty acid content and low percentage of omega-3 FAs.

The results showed that even a three-month storage period did not significantly change the values of the health indicators of the tested oils. The AI values obtained were comparable to those reported by Ulbricht and Southgate for olive oil (0.14) [34] and those reported by Ying et al. for borage oil (0.18) [35]. Górska et al. [36] reported even lower AI values for oils from blackcurrant, strawberry, and cranberry seeds (0.098, 0.057, and 0.065, respectively). Additionally, the TI values determined in their study were significantly lower, not exceeding 0.13. Ratusz et al. [37] also obtained TI values of around 0.1 for cold-pressed camelina oils.

Self-extracted watermelon seed oil was characterized by the highest HPI and h/H indices (7.07 and 7.18, respectively), while WO₃ had the lowest values (5.83 and 5.85, respectively). The HPI index is currently mainly used in research on dairy products and varies between 0.16 and 0.68 [24]. Wirkowska et al. [25] obtained higher HPI values for sea buckthorn and rosehip oils (4.161–21.467) than for dairy products. In turn, Ratusz et al., for camelian [37], and Siol et al. [30], for pomegranate cold-pressed oils, obtained higher h/H values that were more than two times higher.

3.2. Fatty Acid Distribution

In the composition of vegetable fats, there are, on average, 5 to 15 fatty acids. Their acid residues can occupy external (sn-1,3) or internal (sn-2) positions in TAG molecules. Under the influence of the action of a regiospecific enzyme, e.g., pancreatic lipase, it is possible to determine the FA content in these positions. The external positions of pancreatic lipase cannot be distinguished; therefore, they are assumed to be equivalent. If the FA share at sn-2 is below 33%, its distribution is mainly in the external TAG positions [30,38].

Table 2. Fatty acid composition of the outer (sn-1,3) and inner (sn-2) triacylglycerols (TAGs) positions of the tested watermelon seed oils.

Fatty Acid	Composition [%]
		WO1	WO2	WO3	WOref	WOext
C16:0	TAG	11.79 ± 0.04 b	11.43 ± 0.04 b	12.45 ± 0.04 b	12.18 ± 0.04 b	11.00 ± 0.04 b
	sn-2	12.18 ± 0.66 ab	10.34 ± 0.66 ab	15.68 ± 0.66 ab	12.68 ± 0.66 ab	9.65 ± 0.66 ab
	sn-1,3	11.60 ± 0.33 b	11.98 ± 0.33 b	10.84 ± 0.33 b	11.93 ± 0.33 b	9.35 ± 0.33 b
C18:0	TAG	4.42 ± 0.01 bc	4.97 ± 0.01 bc	12.52 ± 0.01 bc	4.85 ± 0.01 bc	8.54 ± 0.01 bc
	sn-2	5.70 ± 0.13 a	5.95 ± 0.13 a	9.19 ± 0.13 a	6.92 ± 0.13 a	6.92 ± 0.13 a
	sn-1,3	3.78 ± 0.07 b	4.48 ± 0.07 b	14.19 ± 0.07 b	3.81 ± 0.07 b	9.35 ± 0.07 b
C18:1 n-9	TAG	27.02 ± 0.06 c	25.23 ± 0.06 c	14.20 ± 0.06 c	28.18 ± 0.06 c	16.99 ± 0.06 c
	sn-2	27.26 ± 1.29 c	28.71 ± 1.29 c	23.95 ± 1.29 c	31.22 ± 1.29 c	18.87 ± 1.29 c
	sn-1,3	26.90 ± 0.65 c	23.49 ± 0.65 c	9.32 ± 0.65 c	26.66 ± 0.65 c	16.05 ± 0.65 c
C18:2 n-6	TAG	54.58 ± 0.06 b	56.20 ± 0.06 b	58.56 ± 0.06 b	52.90 ± 0.06 b	62.21 ± 0.06 b
	sn-2	51.85 ± 1.35 b	51.99 ± 1.35 b	39.00 ± 1.35 b	44.89 ± 1.35 b	61.27 ± 1.35 b
	sn-1,3	55.95 ± 0.68 b	58.30 ± 0.68 b	68.34 ± 0.68 b	56.91 ± 0.68 b	62.68 ± 0.68 b

WO₁–WO₃—unrefined commercial oils; WO_ref—refined commercial oil; WO_ext—self-extracted oil; The different lower-case letters indicate significantly different values (p ≤ 0.05). Data are presented as mean values followed by standard deviation (±SD).

shows the FA composition of the outer and inner TAG positions of the tested watermelon seed oils and Figure 1 presents the contents of the main FA share in the sn-2 position of TAGs.

Watermelon seed oils contained the most linoleic acid in the internal position (from 39% for WO₃ to ~62% for WO_ext), but the share of linoleic acid in the sn-2 position of TAGs was less than 33%. The second most abundant FA in the internal position was oleic acid (from ~19% for WO_ext to ~32% for WO_ref), and its share in the sn-2 position of TAGs was above 33.3% for all tested oils. SFAs were present in the lowest amount in the sn-2 position and their share in the internal position of TAGs varied depending on the type of oil used; it was more than 33.3% for WO₁ and WO_ref and less than 33.3% for WO_ext. The share of palmitic acid in the sn-2 position was around 30% for WO₂ and ~42% for WO₃, in comparison to that of stearic acid, which was ~40 and ~25% for these oils, respectively.

There is limited information in the literature on the distribution of FAs in watermelon seed oil. Yao et al. [39], for self-extracted watermelon seed oil, obtained similar results for C18:2 (32.40%) to the contents reported for WO_ext in this study. Moreover, they claimed that SFA contents in the sn-2 position were much lower than the unsaturated FA contents. These findings were only consistent for WO_ext. In turn, Montesano et al. [40] determined the distribution of FAs present in self-extracted pumpkin seed oil, which belongs to the same family—Cucurbitaceae. The findings for pumpkin seed oil differ from the results of the present study on watermelon seed oils. In pumpkin seed oil, SFAs were confined to the outer TAG positions, whereas in watermelon seed oils, they were distributed across both the inner and outer positions. If SFAs occur in the internal sn-2 position, then the absorption of fats must take place because the saturated monoacylglycerols formed after hydrolysis have a fairly high absorption coefficient. An unfavorable phenomenon is that SFAs occur in the external positions sn-1,3. SFAs released from the external positions are absorbed into the bloodstream much less efficiently. They bind with free calcium ions, causing the formation of insoluble calcium salts. These salts are removed from the body with the feces. As a result, calcium deficiency in the body may occur [23,30]. Therefore, it can be stated that watermelon seed oil is more digestible than pumpkin seed oil, as the SFAs occupy the middle TAG position. It is also less prone to oxidation, since the PUFAs (primarily linoleic acid) are mainly located in the external TAG positions.

3.3. Determination of Acid Value

The acid value (AV) indicates the degree of freshness of the fats. It describes the degree of hydrolysis of the tested oils. It is also a measure of the free fatty acids content. The Codex Alimentarius specifies the requirement that the AV of cold-pressed vegetable oils does not exceed 4 mg KOH/g fat, while for refined vegetable oils the limit is 0.6 mg KOH/g fat [41].

The average AVs of the studied oils, obtained during the testing period, met the requirements specified by Codex Alimentarius (Figure 2). It should also be noted that the lowest average AV (0.29 mg KOH/g) was reported for refined watermelon seed oil (WO_ref). This oil exhibited the highest hydrolytic stability compared to the other oils. Moreover, its AV was not affected by the storage process. The low AV for WO_ref indicates the freshness of the tested oil. The presented results are consistent with the literature data for refined oils, as both free fatty acids and primary oxidation products are removed from the oil when subjected to the refining process, as reflected in its low AV values [42,43].

The quality of cold-pressed oil depends mainly on the quality of the seeds. The most important factors influencing the quality of seeds include, among others, the degree of their maturity, the content of damaged seeds, the content of other impurities, the moisture content of the seeds, and the storage conditions [44]. The quality of cold-pressed oil is also influenced by the hygienic conditions during processing, the conditions and parameters of the pressing, the filtration process, and the storage conditions of the finished product. Therefore, the quality of such oils can be quite diverse [45]. This is confirmed by the AVs obtained for the cold-pressed oils tested in this study. WO₁ and WO₂ consistently showed low AVs across all storage dates. The greatest degree of hydrolytic changes was observed in the case of WO₃ which exhibited AVs more than twice as high at every stage than those of other cold-pressed oils. The higher AV of WO₃ (1.96 mg KOH/g) may indicate that the oil was obtained from partially damaged seeds or that the pressing took place in undesired conditions. Oil extracted from damaged seeds undergoes hydrolysis. A greater degree of damage stimulates the activity of lipolytic enzymes, which is reflected in an increase in the AV [46]. An effect of storage time on the AV of cold-pressed oil was also observed, with an increase in the average AV for all tested samples following three months of storage after opening. An increase in the average AV was noted for all tested cold-pressed oils following three months of storage after opening, particularly in the case of WO₃.

Poornima et al. [21] extracted watermelon seed oil through cold-pressing, reporting an AV of 2.80 mg KOH/g fat. Cheikhyoussef et al. [47] observed a significantly lower AV of 1.63 mg KOH/g fat for cold-pressed watermelon seed oil. Ying et al. [35] analyzed cold-pressed oils available in grocery stores, noting an AV of 1.15 mg KOH/g fat for watermelon seed oil. In the present study, unrefined cold-pressed watermelon seed oils exhibited AVs ranging from 0.84 to 1.96 mg KOH/g fat at the beginning of the testing period. These results are consistent with the findings of other researchers, with slight variations, likely due to differences in seed quality, processing hygiene, or pressing parameters.

The WO_ext had a higher AV than cold-pressed oils, which is probably related to the hydrolytic changes that may have occurred as a result of heating the oil during the evaporation of the solvent used in the extraction process. The degree of hydrolysis of this oil may also have been influenced by the quality of the seeds used for oil extraction [46]. However, it is worth noting that the AV value did not depend on the storage time.

Athar et al. [48] extracted watermelon seed oil using a Soxhlet apparatus with the same solvent as used in the present study, resulting in AV of 2.88 mg KOH/g fat. Egbuonu et al. [49], employing the Soxhlet method with petroleum ether, reported a notably higher AV of 6.10 mg KOH/g fat. Ogunwole [50] found an even higher AV of 10.10 mg KOH/g fat for watermelon seed oil extracted with hexane. By contrast, the ‘Sugar Baby’ variety extracted in the present study exhibited an AV of 1.07 mg KOH/g fat. The significantly higher AVs reported by Egbuonu et al. [49] and Ogunwole [50] likely reflect differences in the seed quality or extraction methods.

3.4. Determination of Peroxide Value

The peroxide value (PV) indicates the degree of the fat’s rancidity, determines the degree of oxidative changes occurring in fats, and is proportional to the content of the primary fat oxidation products, i.e., peroxides. The fatty acid composition plays a crucial role in determining the rate of oxidative changes in oils, as oils with a high PUFA content oxidize more rapidly [51]. Internal factors affecting the rate of oxidation include the presence of natural pro-oxidants and antioxidants in the oil, while external factors that are important to the oxidation process include the conditions and parameters of the technological processes used, access to oxygen and light, and the storage temperature of the finished product [37,52]. The Codex Alimentarius informs that the maximum PV of cold-pressed vegetable oils is 15 mEq O₂/kg fat. In contrast, for refined vegetable oils, this result should not exceed 10 mEq O₂/kg fat [53].

The average PV of the studied oils, obtained during the three-month storage period, met the requirements specified by the Codex Alimentarius (Figure 3), except for WO₃, which met the requirement only at the beginning of the experimental period. WO₃, with the highest PV among the tested oils (16.64 mEq O₂/kg fat), was probably obtained from damaged or poor-quality seeds, or the pressing process was carried out in inappropriate conditions. The fat in damaged seeds oxidizes much faster [31], which may explain the high PV for this oil obtained in the first month of the study. The increase in the PV of cold-pressed oils may also result from the presence of pro-oxidant substances in the oils [43]. For commercial oils, the first determinations were made immediately after purchase. However, we do not know how long the oil was stored from the moment of production to the moment of purchase. In the case of oil extracted from watermelon seeds of the ‘Sugar Baby’ variety, the first determinations of PV were made for freshly obtained oil. It might seem that the initially determined PV would be very low. However, the results obtained for WO_ext were very similar to those obtained for WO₁ and WO₂. The increased PV may be due to the effect of oxidation processes related to extraction or may result from a lack of sufficient protection against oxidation immediately after the extraction process [54]. It could also be assumed that the seeds used for the extraction process were not of the best quality.

Cheikhyoussef et al. [47] obtained watermelon seed oil in laboratory conditions using the cold-pressing method and reported a PV of 2.98 mEq O₂/kg fat. Ouassor et al. [20] determined the PV for cold-pressed watermelon seed oil derived from two varieties, finding values of 3.8 and 4.0 mEq O₂/kg fat. Białek et al. [32] analyzed cold-pressed watermelon seed oil purchased from a grocery store, reporting a PV of 10.7 mEq O₂/kg fat. In the present study, cold-pressed oils from unrefined watermelon seeds had PVs ranging from 5.39 to 12.41 mEq O₂/kg fat immediately after purchase. Comparing the above results, it can be seen that the values determined for directly pressed oils under laboratory conditions are lower than those determined for commercially pressed oils. This can be explained by the fact that, during the storage process, oxidative changes occur in commercial oils. The differences in the obtained results may also be a consequence of variations in seed quality, which strongly affect the properties of the oils.

Oyeleke et al. [55] used the Soxhlet extraction method with petroleum ether as the solvent, obtaining a PV of 2.8 mEq O₂/kg fat for tested watermelon seed oil, much lower than the values reported by Francis et al. [56], who used hexane and reported ~12 mEq O₂/kg fat. The PV of WO_ext (6.24 meq O₂/kg fat) was higher than the value obtained by Francis et al. [56], but lower than that obtained by Oyeleke et al. [55]. The differences in the obtained results may be due to variations in the quality of the raw material, different parameters during the extraction process, or different degrees of protection against oxidation after the extraction process was completed.

As mentioned earlier, the FA composition of the oil is an important factor influencing the oxidation processes. The determined PUFA contents ranged from about 54% for WO_ref to about 62% for WO_ext. A lower PUFA content may, to some extent, influence the lowest PV observed for WO_ref. Furthermore, although the storage time negatively affected the PV of all tested oils, the WO_ref exhibited the smallest increase, demonstrating greater stability compared to other oils. This suggests that WO_ref is less prone to oxidation and could be more suitable for longer storage periods under similar conditions. The initially low PV of the WO_ref (1.96 mEq O₂/kg fat) was probably due to the fact that the refining process removes primary oxidation products. It could also be suggested that the seeds used to produce the oil were probably of good quality and that the conditions and parameters of the pressing process were appropriate.

3.5. Oxidative Stability, Obtained via Pressure Differential Scanning Calorimetry (PDSC)—Isothermal Measurements

Lipid oxidation determines the food products’ final quality and nutritional properties because it is the main reaction responsible for their degradation. Oxidative stability is an important quality indicator in edible oils and fats [30]. In food chemistry, pressure differential scanning calorimetry (PDSC) is a convenient, reproducible, and fast method that can be used to determine the oxidative stability of fats and oils [57]. The PDSC oxidation time (τ_max) is determined based on the maximum oxidation rate.

The obtained values for the PDSC oxidation time of individual oils are presented in Figure 4. The results obtained at the beginning of the testing period showed the shortest average τ_max of 23.88 min for WO₁ and the longest (76.55 min) for WO_ext. The same trend was observed during storage. A significant effect of oil type on the PDSC oxidation time was observed. Furthermore, the storage period has an impact on the oxidation time. For WO₂ and WO_ref, an especially significant difference was observed in the obtained τ_max values between the beginning and the end of the testing period.

The rate of oxidation is strongly influenced by the quality of the raw material used to produce the oil, since adverse changes in the finished product often begin in the seeds. The high initial oxidation level of oil extracted from low-quality seeds shortens its shelf life. In addition, the processing conditions and parameters play a key role. Factors such as exposure to light and oxygen, and high temperatures during storage, accelerate oxidation [37,58]. The rate of oxidation is also affected by the composition of the fatty acids and the presence of components of the non-glycerol fraction with pro- and antioxidant properties [43].

Throughout the storage period, self-extracted oil from the ‘Sugar Baby’ variety showed the longest τ_max. During the storage process, the value of the average τ_max for this oil decreased slightly. This indicates very good stability regarding the oxidation of the given oil. Comparing the results obtained for unrefined pressed oils, quite large differences could be noted. The WO₃ was characterized by a fairly long oxidation induction time of 49.59 min in the first month of the tests. The average τ_max for WO₂ was 36.36 min, while for the WO₁, this value was only 23.88 min. Of the cold-pressed oils, WO₃ showed the best oxidative stability. WO_ref also had a relatively long τ_max, comparable to that of WO₃. This prolonged oxidation induction time may be due to the removal of pro-oxidant compounds during refinement or the low initial oxidation level of the refined oil.

The high PUFA contents (above 50%) explain the oxidation processes occurring in the tested oils and the decreasing values of τ_max. Additionally, linoleic acid was located in the external position of the TAGs, which is in accordance with the literature reports indicating that PUFAs located in the sn-1,3 TAG position reduce the oxidative stability of oils [59].

There is a lack of data in the literature regarding the PDSC oxidation time of watermelon seed oil. Piasecka et al. [60] analyzed self-extracted blackberry, chokeberry, and raspberry seed oils. The high-pressure differential scanning colorimetry method determined the oxidative stability of these oils. The same conditions were used for determination as were used for the oils tested in this paper, i.e., a temperature of 120 °C and a pressure of 1400 kPa. The average τ_max for the tested cold-pressed oils was 41.20 min for chokeberry seed oil and 56.02 min for raspberry seed oil. The highest value of 90.29 min was reported for blackberry seed oil. The studied self-extracted watermelon seed oil is not as oxidatively stable as blackberry seed oil but is more stable than chokeberry seed and raspberry seed oils.

The tested commercial cold-pressed watermelon seed oils have a higher τ_max than the fruit seed oils mentioned above. They may be considered more stable at 120 °C than the pomegranate seed oils studied by Siol et al. [30], with an induction time below 2.5 min but lower than that of the pumpkin seed oil examined in the research of Symoniuk et al. [61]. In contrast, the τ_max of commercial cold-pressed pumpkin seed oil (under similar conditions) was, on average, 71.05 min. The similar oxidation induction times of commercial watermelon and pumpkin seed oils may be because watermelon and pumpkin come from the same family—Cucurbitaceae.

4. Conclusions

This study showed that the production method and storage conditions could affect the fatty acid composition, oxidative stability, and overall quality of watermelon seed oils. The analyzed fatty acid composition corresponded to the profile commonly reported in the literature for this kind of oil. The higher PUFA contents observed in cold-pressed oils, particularly linoleic acid, increased their susceptibility to oxidation when occupying the outer TAG positions. In contrast, refined oil showed better resistance to oxidative changes, reflecting the removal of pro-oxidant compounds during the refining process. However, all oils experienced increase in peroxide and acid number values over time, indicating that oxidative and hydrolytic processes occur during storage.

The variations in oil quality observed across different samples can be attributed to factors such as seed quality, processing practices, and production/storage conditions. Moreover, the presence of antioxidant and pro-oxidant substances in the oil affects the extent to which it undergoes oxidative changes. Further studies are required to identify the bioactive compounds in watermelon seed oil and to understand their interaction mechanisms under different storage conditions. The research should focus on optimizing storage conditions to minimize oxidative degradation, as well as improving oil production methods to increase natural antioxidant content, which could further improve product quality and extend shelf life.