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Innovative functional mayonnaise formulations with watermelon seeds oil: evaluation of quality parameters and storage stability

  • Eatemad M. El-Sayed , Essam Mohamed Elsebaie ORCID logo EMAIL logo , Shaymaa A. Hozifa , Eman M. Abo-Zaid , Marwa Fawzi Ahmed El-Farsy , Dalia M. El-Mesiry , Rehab A. Shehata , Aisha S. M. Fageer , Hala Ali Yousef Shaat and Suzan S. Ibraheim
Published/Copyright: November 27, 2025
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Open Chemistry
From the journal Open Chemistry Volume 23 Issue 1

Abstract

This study investigates the novel application of watermelon seed oil (WM-SO) as functional oil in both egg-based and eggless mayonnaise, assessing its impact on physicochemical, nutritional, and oxidative stability parameters. WM-SO exhibited high quality, characterized by low free fatty acids (1.21 ± 0.03 %), peroxide value (0.98 ± 0.05 meq O2/kg), and rich unsaturated fatty acids, notably linoleic acid (63.70 %) and oleic acid (16.87 %). Incorporating WM-SO significantly enhanced mayonnaise bioactivity: total phenolic content increased by ∼11–14 %, reaching 109.76 ± 2.90 mg GAE/g in egg-based and 97.42 ± 1.65 mg GAE/g in eggless formulations, while total carotenoids rose to 4.63 ± 0.38 mg/g and 4.29 ± 0.41 mg/g, respectively. Radical scavenging activity (%RSA) improved by ∼51 % in egg-based (from 32.59 % to 49.11 %) and ∼39 % in eggless samples (from 45.27 % to 62.88 %). WM-SO also reduced viscosity (e.g., from 1,486 to 1,131 cP in egg-based), decreased water activity (from 0.83 to 0.80 in eggless), and slightly lowered fat content. Particle size analyses showed reduced D90 (from 143.26 to 12.50 µm in egg-based), indicating finer emulsions with better stability. Sensory evaluation confirmed high acceptability (scores ∼8.5–8.6). During 30 days of storage, WM-SO samples exhibited lower peroxide values (1.05 meq O2/kg vs. 1.18 in control), reduced free fatty acid increase (∼9 % less in egg-based), and sustained higher RSA. These results position WM-SO as a promising, health-enhancing, and sustainable ingredient for stable, bioactive-enriched mayonnaise formulations.

Keywords: agro-waste; antioxidants; DPPH; fatty acids; oil; quality

1 Introduction

Mayonnaise is one of the most widely consumed condiments globally, formulated as a high-fat oil-in-water emulsion containing 60–80 % vegetable oil and egg yolk [1]. It is produced by emulsifying oil with ingredients like starchy paste, egg, vinegar, mustard, and various spices. In recent years, efforts to enhance its nutritional profile have led to the substitution of conventional oils with those rich in polyunsaturated fatty acids. However, while these healthier fats offer added nutritional benefits, they also introduce challenges related to increased susceptibility to lipid oxidation [2].

Mayonnaise, with its high oil content, is inherently prone to lipid oxidation, which can lead to rancidity and a shortened shelf life. To combat this, food manufacturers have traditionally relied on synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and ethylenediaminetetraacetic acid (EDTA). While effective, these additives have come under increasing scrutiny due to growing concerns about their potential toxic and carcinogenic effects when used beyond recommended limits [3]. As a result, the food industry is shifting toward more natural, clean-label alternatives. This shift has sparked widespread interest in both replacing synthetic antioxidants and reformulating mayonnaise with functional oils rich in natural antioxidants. Recent research reflects this trend, with several studies demonstrating that replacing conventional oils like soybean oil with alternatives such as roselle seed oil [4], black cumin oil [5], pumpkin seeds oils [6], flaxseed oil [7], and Sacha Inchi oil [8] not only enhances oxidative stability but also boosts the nutritional value of the final product. This evolution in formulation underscores a broader movement toward healthier, more transparent food systems that prioritize both functionality and consumer trust. Also, Akcicek et al. [9] demonstrated that incorporating carotenoid-rich tomato seed oil into mayonnaise not only enhanced antioxidant activity but also improved color and sensory appeal.

Among the promising substitutes is watermelon seed oil (WM-SO), nutritionally rich oil extracted from the seeds of Citrullus lanatus, an agricultural by-product often discarded during processing. Utilization of agro-industrial by-products in food systems improves the product functionality and reduces environmental stress. Their transformation to value-added ingredients also reduces organic waste from agro-industrial processes, often associated with greenhouse gas emissions and pollution [10]. The valorization is also in line with circular economy concepts and the production of sustainable food [11]. Watermelon seed oil is characterized by a high content of polyunsaturated fatty acids, particularly linoleic acid (up to 64 %), along with significant levels of tocopherols, sterols, and other bioactive compounds known for their antioxidant and anti-inflammatory properties [12], 13]. These properties not only make WSO a potential candidate for enhancing the nutritional profile of mayonnaise but also offer the added benefit of improving oxidative stability – an essential factor in determining the shelf life and sensory quality of emulsified products.

This study presents the first attempt to formulate both eggless and egg-based mayonnaise using WM-SO, investigating its influence on key quality parameters. Specifically, we evaluate the impact of WM-SO substitution on physicochemical properties, oxidative stability, and sensory attributes in comparison to a conventional oil-based control. This work addresses a pressing challenge in food innovation developing healthier, functional condiments that align with sustainability objectives.

2 Materials and methods

2.1 Materials

In the summer of 2024, ripe fruits of the Giza 1 watermelon (C. lanatus) were harvested from a private Nubaria field in Beheira, Egypt. Ingredients used for the mayonnaise (sugar, eggs, salt, soybean oil, apple vinegar, starch, and mustard flour) were purchased at El-Oruba Mall in Tanta, Egypt. The plate count agar (PCA) and potato dextrose agar (PDA) media were acquired from Oxoid in the United Kingdom. All chemical reagents, of analytical grade, were sourced from Sigma-Aldrich in Steinheim, Germany.

2.2 Watermelon seed powder (WM-SP) preparation

Immediately after harvesting, the watermelon was peeled, and seeds were manually extracted from the pulp, cleaned, and rinsed to eliminate any residue. Damaged seeds were discarded, and the intact ones were dried in a hot-air oven at 40 °C for 24 h. These were then milled (using a Moulinex AR1101) into a fine powder, passed through a 100-mesh sieve, and stored at −20 °C until analysis.

2.3 Extraction of WM-SO

WM-SP (40 g) were put in a 1 L dark flask, mixed with n-hexane (1:5, w/v), and left to stir at 30 r/min for 24 h at room temperature. The resulting mixture went through filtration with a Büchner funnel and a Whatman (NO.4) filter paper. In order to minimize any remaining solvent, the solvent was extracted under vacuum using an EYELA N–1210BV rotary evaporator (Narita, Japan) and then exposed to nitrogen until there was no discernible solvent odor. The obtained WMSO was kept in dark-colored bottles at 4 °C till testing [14].

2.4 Chemical characteristics of WM-SO measurement

Free fatty acids (% as oleic acid), acid value (mgKOH/g oil), peroxide value (mEq O2/Kg oil), saponification value (mgKOH/g oil), and iodine value (g I2/100 g oil) were tested with approved techniques of Idris et al. [15].

2.5 Pigments in WM-SO determination

Using a spectrophotometric technique, the amount of carotenoid (mg equivalent of β-carotene/g oil) and chlorophyll (mg of chlorophyll/kg) was measured. After dissolving the WM-SO in 1 % hexane, absorbance measurements were made at 670 nm for chlorophyll and 470 nm for carotenoid [16]. The process described in AOCS [17] was followed in order to estimate the total tocopherols in WM-SO with HPLC.

2.6 Polyphenols determinations

WM-SO (2.5 g) was fully dissolved in n-hexane (3 mL) and extracted with an equal amount of methanol (80 %). After 5 min of shaking at 2,500 rpm, the mixture was centrifuged for 10 minutes at 4,863×g. Three repetitions of the procedure were made, combining the extracts. The total phenolic content in the combined extract (WM-SO methanolic extract) has been measured using the technique developed by Elsebaie, Essa [18].

2.7 Physical characteristics of WM-SO measurement

The pH was determined using a HAANA HI903 PH meter (Steinheim, Germany), as previously defined by AOAC (2020). The Abbe refractometer was used to measure the refractive index at 25 °C in accordance with ASTM [19].The color of WM-SO was determined using a Lovibond-PFX195 colorimeter (Rhône, France).

2.8 Fatty acid composition

The WM-SO (1 mL) was methylated using 0.5 mL of 20 mg/mL sodium hydroxide in methanol in accordance with Boyapati et al. [20]. Fatty acids were then measured via an Agilent 7880 A gas chromatography apparatus (California, USA) fitted with a DB/FFAP column. The temperatures of the injection and detection were both 210 °C. After being maintained at 210 °C for 9 min, the temperature of the column was raised to 230 °C at a speed of 293.13 K/min and maintained for 10 minutes. The total volume of injection was 2 μL, and the carrying gas nitrogen flow rate was configured at 1.5 mL/min with an 80:1 splitting rate. The fatty acid profile was quantified as the percentage of each individual peak relative to the total chromatographic peak area.

2.9 Mayonnaise preparation

The control mayonnaise was formulated based on the method described by Elsebaie et al. [21], using a mixture of soybean oil (82.2 %), egg yolk (7.27 %), vinegar (8.72 %), salt (0.73 %), mustard powder (0.36 %), sugar (0.36 %), and white pepper (0.35 %). For the egg-free version, whey protein concentrate 80 % (NZWPI895 Caldic, Fonterra, USA) was incorporated as the primary emulsifier at 5 %. To create the starchy base, water (20 ml) and starch (1 gm) were heated together at 75 °C for 2 min with continuous stirring, then sweetened and seasoned with sugar, salt, and vinegar. The protein component was whipped for 2 min before incorporating the dry components (salt, sugar, spices, and mustard flour) along with vinegar and mixed for an additional minute. Finally, soybean oil (or its replacement with watermelon seed oil) and the starchy paste were blended in for 2 min, and then scraped slowly for one more minute. The finished product was packed into PET jars and stored at ambient temperature for further shelf-life evaluation.

2.10 Mayonnaise properties

2.10.1 Physicochemical properties

The pH of the samples was measured using a digital HAANA HI904 m pH meter (Köln, Germany), which was calibrated with standard buffer solutions at pH 4.0 and pH 7.0. Water activity was determined using a digital AQUALAB instrument (AquaLab, California, USA), following calibration with a certified reference standard. According to Akhtar, Masoodi [22], an LDVD–III–Brookfield viscometer, serial 67,854 (Boston, USA), was used for determining viscosity. Moisture, lipid, and ash contents of the mayonnaise were analyzed following the guidelines of AOAC [23].

Acid value was estimated wherein 1 g portion of mayonnaise was dissolved in 100 mL of hot, neutralized ethanol and treated with 1 mL of phenolphthalein. This solution was then titrated with a standardized alkali until a persistent faint pink endpoint was achieved [23]. Following AOAC [23] guidelines, we measured free fatty acids by extracting a weighed sample with 20 mL of benzene and then transferring 2 mL of this extract into a new flask. To this, we added 10 mL of benzene, 5 mL of ethanol, and a few drops of phenolphthalein. Finally, titration was carried out using 0.02 N KOH until the solution acquired a pale pink tinge. For peroxide value assessment, 5 g of mayonnaise was combined with 30 mL of a 3:2 acetic acid–chloroform mix, followed by 0.5 mL saturated potassium iodide and 30 mL distilled water. Iodine liberated within the solution was titrated against 0.1 N sodium thiosulfate until the yellow coloration nearly vanished.

2.10.2 Bioactive and antioxidant activity

2.10.2.1 Total phenol content and antioxidant activity

After dissolving 1 g of mayonnaise in 10 mL of 80 % methanol at 40 °C for 4 h, the mixture was centrifuged at 9,000 rpm for 5 min at 10 °C (HR-26LABOAO, Tianjin, China) and filtered through a nylon filter with a pore size of 0.2 mm. The resulting extracts were subjected to a measurement of their total phenolic content and DPPH radical scavenging activity. The Folin-Ciocalteu reagent was employed to determine the total phenolic content, with a few adjustments [24]. 5 mL of 10 % v/v of the Folin-Ciocalteu solution was combined with 1 mL of each methanol extract, and the mixture was left in the dark for 10 min. Thereafter, two mL of a 7.5 % w/v Na2CO3 solution were added, and it was left in the dark for 50 minutes. The UV–Vis spectrophotometer was used to measure the mixtures’ absorbance at 765 nm in comparison to a blank sample. The calibration equation for gallic acid with R 2  = 0.95 was y = 0.0111x – 0.0148, where y is absorbance and x is concentration of gallic acid in mg/ml. the final results were expressed as mg gallic acid equivalents/g.

Antioxidant capacity was evaluated by the % DPPH radical scavenging method [25]: Briefly, two mL of DPPH reagent in methanol, along with two mL of methanol and either 0.1 mL of distilled water or the sample solution, were combined. Following a vortexing process that lasted for approximately 10 seconds, the mixture was then allowed to remain in the dark for 20 minutes. Finally, the absorbance of the mixture was measured using a Cary 60 UV–Vis spectrophotometer (Analytik Jena, Germany) at a wavelength of 517 nm. The radical scavenging activity (RSA%) of DPPH was determined via Equation No. 1.

(1) RSA  % =   C o n t r o l   a b s o r b a n c e − S a m p l e   a b s o r b a n c e   C o n t r o l   a b s o r b a n c e × 100

2.10.2.2 Total carotenoids determination

Total carotenoids amount was estimated according to Rodriguez-Amaya, Kimura [26]. To extract the carotenoids, about 3 g of the sample was mixed with 15 ml of acetone: petroleum ether (2:3). This process was repeated until the sample had lost its color. Subsequently, the extract was placed into a separatory funnel for the purpose of separating two phases, namely acetone and petroleum ether. The phase that contained the carotenoid was taken out in a volumetric vessel, and then the absorbance was measured at 452 nm using a Cary 60 UV–Vis spectrophotometer (Analytik Jena, Germany). The carotenoid content was calculated from equation No. 2, and the data were reported as mg per 100 g.

(2) Total carotenoids content  mg / g sample = A × V   mL × 10   A IC   1 % × P   g

Where: A = absorbance; V = total volume of extract; P = sample weight; A I C m 1 %  = 2,592 (absorption coefficient of β–carotene in petroleum ether).

2.10.3 Colour measurement

The colour of the mayonnaise samples was assessed with a ColorFlex-XE device from Hunter Lab, located in Reston, USA. The CIE values that were determined were L*, a*, and b*, where the a* values varied between −100 (greenness) and +100 (redness), and the values of b* along the range of −100 (blueness) to +100 (yellowness), In contrast, the L* values, which represented the lightness scale, varied between 0 (black) and 100 (white).

2.10.4 Particle size distribution

The particle size distribution of mayonnaise was determined following the approach outlined by Sanei et al. [27]. Measurements were performed using a Malvern Mastersizer 3,000 Laser Scattering Particle Size Analyzer. Samples were diluted with water at a 1:10 ratio and analyzed twice at 25 °C, within a size range of 0.01–3,000 μm. The results reported the average particle size and distribution, represented by D10, D50, and D90, corresponding to the particle sizes below which 10 %, 50 %, and 90 % of the sample volume fall, respectively.

2.10.5 Optical microscopy

The microstructure of different mayonnaise samples was examined using an Olympus BX54 light microscope fitted with a digital camera. A small portion of each mayonnaise sample was placed on a glass slide, covered with a coverslip, and viewed under 100 × magnification, following the method of Rojas-Martin et al. [28]. Images capturing the microstructure were recorded via the digital camera attached to the microscope.

2.10.6 Sensory evaluation

Sensory analysis of the produced mayonnaise included the application of a nine-point hedonic scale such that 9 indicated ’like extremely’ while 1 indicated ‘dislike extremely’. The samples were randomly coded and served from transparent plastic cups. The panellists were instructed to wash their mouth with warm water and consume a typical cracker between samples after following the protocol of Elsebaie et al. [21]. Twenty semi-trained panellists consisting of 10 males and 10 females aged between 24 and 49 years participated in the analysis of color, flavor, texture, and overall acceptability. The panellists received a short training session before testing such that each panellist could well discuss and articulate each attribute to be ranked. Participation was voluntary, and each assessor was well-informed of the study objective before their consent for assessment.

2.11 Storage experiment

The samples were kept into airtight PET jars and stored under dark conditions to prevent light exposure at room temperature (27 ± 1 °C, relative humidity 61–70 %) for a period of 30 days. Assessments were performed on days 0, 10, 20, and 30 to measure parameters including moisture content, pH, water activity, free fatty acids, peroxide value, and antioxidant capacity.

2.12 Microbiological examination

The spread-plate and pour-plate techniques were employed to quantify yeast and molds at 25 °C for a period of 5 days and total aerobic bacteria (incubation at 30 °C for a period of 3 days) on PDA and PCA media, respectively. Chloramphenicol was included in PDA at a concentration of 100 mg/L for selection of yeast and mold growth from a mixed culture, and serial dilutions of 1:10 and 1:100 were applied (each dilution was plated in triplicate). The number of microbe colonies was given as colony-forming units (CFU/g mayonnaise) [29].

2.13 Statistical analysis

Statistical analysis was carried out using the general linear model in SPSS software (Version 16.0, 2007) to perform ANOVA. When significant differences were detected, means were compared using Duncan’s multiple range test (DMRT). A significance level of p ≤ 0.05 was considered for all analyses. All measurements were conducted in triplicate. Duncan’s multiple range test (DMRT)

3 Results and discussion

3.1 Physical parameters of WM-SO

The physicochemical profile of watermelon seed oil (WM-SO) (Table 1) demonstrates high quality and oxidative stability. The measured free fatty acid (FFA) level of 1.21 ± 0.03 % (expressed as oleic acid) reflects a low degree of hydrolysis, suggesting that the oil maintains its quality and freshness. Such a low FFA concentration implies limited microbial or enzymatic action, which is beneficial for extending the oil’s storage life and preserving its sensory attributes. Also according to the presented data, the determined acid value was 2.19 ± 0.03 mg KOH/g which supports the FFA findings, indicating the oil is chemically stable and appropriate for use in food systems. This metric is a standard indicator of oil quality, and readings below 4 mg KOH/g are generally acceptable for non-refined edible oils [30]. These findings are in agreement with those reported by Ojukwu, Ugwu [31]. In addition, the peroxide value was found to be 0.98 ± 0.05 meq O2/kg, reflecting a low level of initial lipid oxidation. This suggests limited oxidative damage and confirms that the oil remains in a stable condition. The peroxide value lies well below the Codex limit of 15 meq O2/kg for virgin oils [13] and undercuts roasted-seed values of 1.57–3.00 meq O2/kg [32], underscoring the protective effect of minimal heat. Maintaining a low peroxide level is essential for preserving the oil’s nutritional properties, flavor, and storage potential.

Table 1:

Some chemical and physical parameters of watermelon seeds oil (WM-SO).

Parameters Values
Free fatty acid (% as oleic acid) 1.21 ± 0.03
Acid value (mg KOH/g oil) 2.19 ± 0.03
Peroxide value (meq.O2/kg oil) 0.98 ± 0.05
Saponification value (mg KOH/g oil) 187.65 ± 0.02
Iodine value (g I2/100 g oil) 85.78 ± 0.04
Carotenoid (mg β-carotene equivalent/g oil) 1.29 ± 0.06
α- tocopherol (mg/Kg oil) 193.27 ± 0.03
γ- tocopherol (mg/Kg oil) 11.40 ± 0.07
Total tocopherol (mg/Kg oil) 204.76 ± 0.06
Total phenols content (mg EGA/100 g oil) 81.4 ± 0.04
pH 3.86 ± 0.06
Refractive index 1.456 ± 0.01
Color
Yellow 33.60 ± 1.20
Red 2.20 ± 0.30

  1. Mean ± Standard deviation of three values.

The saponification (187.65 ± 0.02 mg KOH/g) and iodine values (85.78 ± 0.04 g I2/100 g) characterize WM-SO as rich in long-chain triglycerides with moderate unsaturation mirroring the predominance of linoleic and oleic acids (>50 %) reported by Azeem et al. [33] thus balancing nutritional benefits and oxidative resistance. Its robust antioxidant complement – 1.29 ± 0.06 mg β-carotene eq./g, 204.76 ± 0.06 mg/kg total tocopherols (α 193.27 mg/kg; γ 11.40 mg/kg), and 81.4 ± 0.04 mg GAE/100 g phenols agrees with supercritical-extracted oils [34] and underlies the low peroxide level and potential health benefits. Finally, the refractive index (1.456 ± 0.01) and color values (Yellow 33.60 ± 1.20; Red 2.20 ± 0.30) confirm purity and a pale-yellow hue favored in foods [35], while the pH (3.86 ± 0.06) suggests trace acidic constituents and minor polar compounds (e.g., phospholipids, free fatty acids, organic acids) co-extracted during cold pressing [36]. Similar values have been reported in other seed oils: for instance, cold-pressed pumpkin seed oil exhibited a pH of 3.9 ± 0.1, attributed to residual phosphatidic acids and mono-/diglycerides [37]. Moreover, low pH values in seed oils often correlate with higher levels of phenolic acids and other hydrophilic antioxidants, which partition at the oil–water interface and enhance radical-scavenging activity [38]. Together, these attributes validate WM-SO as high-quality edible oil for culinary, nutraceutical, and cosmetic applications.

3.2 Fatty acids composition of WM-SO

The detailed fatty acids composition of WM-SO (Table 2) shows a pronounced dominance of unsaturated species, with polyunsaturated fatty acids (PUFAs) comprising 63.85 % of total lipids – almost entirely linoleic acid (C18:2, 63.70 %) and monounsaturated fatty acids (MUFAs) accounting for 17.23 %, principally oleic acid (C18:1, 16.87 %). Saturated fatty acids (SFAs) only proportion to 18.63 %, of which palmitic (C16:0, 11.24 %) and stearic (C18:0, 6.49 %) dominate. This indicated that it has a high unsaturation/saturation (U/S) ratio of 4.35 and total unsaturation of fatty acids (USFAs) of 81.08 %, which indicates WM SO’s good nutritional quality. The obtained data about fatty acids profile of WM-SO is also in line with what Brahmi et al. [39] found which was that watermelon seed oil 66.84 % linoleic acid and 16.11 % oleic acid, with palmitic acid levels (9.60 %) and a high U/S ratio that were similar to our findings. The high PUFA content confers health benefits particularly essential fatty acid intake and favorable effects on cardiovascular risk while the moderate MUFA level enhances oxidative stability compared to oils with even higher PUFA proportions [40]. The relatively low SFA fraction further positions WM SO as heart healthy, since diets low in SFAs correlate with reduced serum cholesterol [41]. Also, WM-SO contained a very low level of α-linolenic acid (0.15 %) compared with linoleic acid (63.70 %), resulting in a low α-linolenic:linoleic ratio. Although not nutritionally favorable, this observation provides a clearer picture of the oil’s nutritional quality.

Table 2:

Fatty acids composition of watermelon seeds oil (WM-SO).

Carbon chain Fatty acid Value (%)
Saturated fatty acid (SFA)
C14:0 Myristic acid 0.30
C15:0 Pentadecyclic acid 0.11
C16:0 Palmitic acid 11.24
C17:0 Margaric acid 0.11
C18:0 Stearic acid 6.49
C20:0 Arachidic acid 0.25
C22:0 Behenic acid 0.13
Total SFA – 18.63
Monounsaturated fatty acid (MUSFA)
C14:1 Tetradecenoic 0.14
C16:1 Palmitoleic acid 0.09
C18:1 Octadecenoic acid 16.87
C20:1 Ecosenoic acid 0.13
Total MUSFA – 17.23
Polyunsaturated fatty acid (PUSFA)
C18:2 Linoleic acid 63.70
C18:3 Linolenic acid 0.15
Total PUSFA – 63.85
Total USFA – 81.08
Unknown – 0.29
U/S ratio – 4.35

Consequently, WM SO’s fatty acid composition characterized by predominance of linoleic acid, substantial oleic acid, and minimal saturated lipids supports its use as functional edible oil and as an ingredient in nutraceutical formulations where both oxidative stability and health-promoting lipid profiles are desired.

3.3 Physicochemical WMSO incorporated mayonnaise

The replacement of soybean oil with WM-SO into both egg-based and eggless mayonnaise formulations significantly influenced their physicochemical and nutritional characteristics, as detailed in Table 3.

Table 3:

Physicochemical and bioactive compounds of watermelon seeds oil (WM-SO) incorporated mayonnaise.

Parameters Mayonnaise type
Control egg-based WM-SO egg-based Control eggless WM-SO eggless
Physicochemical properties
pH 4.13 ± 0.04a 4.16 ± 0.06a 3.74 ± 0.05b 3.79 ± 0.04b
Water activity 0.87 ± 0.01a 0.84 ± 0.01b 0.83 ± 0.01b 0.80 ± 0.01c
Viscosity (cP) 1,486 ± 18.5a 1,131 ± 16.8c 1,169 ± 15.9b 1,045 ± 17.2d
Moisture (%) 24.92 ± 0.97a 24.51 ± 0.88a 22.70 ± 0.95b 22.30 ± 0.84b
Fat (%) 29.17 ± 1.01a 28.04 ± 1.00b 27.85 ± 0.97c 26.23 ± 0.95d
Ash (%) 1.74 ± 0.04c 2.15 ± 0.08b 2.18 ± 0.07b 2.37 ± 0.10a
Free fatty acids (% as oleic acid) 0.19 ± 0.01b 0.18 ± 0.01b 0.22 ± 0.02a 0.20 ± 0.02a
Titratable acidity (mg KOH/g) 0.48 ± 0.03b 0.44 ± 0.02b 0.55 ± 0.06a 0.51 ± 0.04a
Peroxide value (meq.O2/kg) 0.71 ± 0.04a 0.67 ± 0.04b 0.73 ± 0.04a 0.70 ± 0.05ab
Bioactive compounds
Total phenols (mg GAE/g) 99.13 ± 2.13b 109.76 ± 2.90a 85.34 ± 1.28c 97.42 ± 1.65b
Total carotenoids (mg/g) 4.25 ± 0.30b 4.63 ± 0.38a 3.81 ± 0.29c 4.29 ± 0.41b
Antioxidant activity (% RSA) 32.59 ± 3.30d 49.11 ± 3.92b 45.27 ± 3.60c 62.88 ± 5.12a

  1. Mean ± Standard deviation of three values. RSA means radical scavenging activity. In a row, means have the same small superscript letter are not significantly different by Dunken’s test at p ≤ 0.05.

To begin with, pH values remained relatively stable within each formulation. Egg-based samples maintained higher pH (4.13–4.16) compared to eggless ones (3.74–3.79), with no significant change (p ≤ 0.05) upon substituting soybean oil with WM SO. The obtained results aligned with Nishat et al. [42], who found that musk melon seed oil did not significantly affect mayonnaise acidity, which is critical for microbial safety and sensory balance. Furthermore, water activity decreased significantly (p ≤ 0.05) in WM-SO samples, especially in eggless mayonnaise (from 0.83 to 0.80). This reduction likely result from the oil’s hydrophobic nature which binds less free water, ultimately lowering the water activity and thereby enhancing microbial stability and shelf-life [22].

In addition to pH and water activity, viscosity was another parameter notably affected by using WM-SO instead of soybean in mayonnaise formulations. Both egg-based and eggless formulations showed a marked reduction in viscosity, dropping from 1,486 to 1,131 cP and 1,169 to 1,045 cP, respectively. This trend may be attributed to the high degree of unsaturation in WM-SO, which produces smaller lipid droplets and reduces internal friction within the emulsion, leading to a thinner consistency [43]. Likewise, the fat content decreased slightly but significantly (p ≤ 0.05) in WM-SO samples. The fat level dropped from 29.17 % to 28.04 % in egg-based and from 27.85 % to 26.23 % in eggless mayonnaise. This reduction may be linked to improved emulsion stability or lower fat retention capacity in the WM-SO systems, contributing to a lighter texture [21]. Meanwhile, moisture content remained unchanged, confirming preserved water-binding capacity. The obtained data were in the same line with those found by Rezig et al. [44].

Moreover, the ash content increased significantly (p ≤ 0.05) with the using of WM-SO instead of soybean oil, particularly in the eggless mayonnaise (rising from 2.18 % to 2.37 %). This enhancement suggests the contribution of naturally occurring minerals in WM-SO, which are retained during cold pressing. Similar mineral enrichments have been reported in watermelon seed oils with high nutritional value [45].

Lastly, lipid quality indicators namely free fatty acids and titratable acidity remained stable following WM-SO incorporation. This stability indicates that no hydrolytic degradation occurred, confirming the chemical integrity of the oil throughout the formulation process. Notably, peroxide values declined slightly in WM-SO samples across both categories (e.g., from 0.71 to 0.67 meq O2/kg in egg-based mayonnaise and from 0.70 to 0.73 in eggless mayonnaise). This reduction points to enhanced oxidative stability, likely due to WM-SO’s rich antioxidant profile, which includes tocopherols, phenolic compounds, and carotenoids [42]. The obtained data were in the same trend with those found by Sudjatinah [46].

3.4 Bioactive compounds of WMSO incorporated mayonnaise

Replacing soybean oil with WM-SO significantly enhanced the bioactive profile and antioxidant capacity of both egg-based and eggless mayonnaise (Table 3). Total phenolic content rose from 99.13 ± 2.13 to 109.76 ± 2.90 mg GAE/g in egg-based and from 85.34 ± 1.28 to 97.42 ± 1.65 mg GAE/g in eggless formulations (p ≤ 0.05), reflecting an ∼11–14 % increase likely due to the high levels of phenolic acids (e.g., gallic, caffeic, sinapic) naturally found in WM-SO [13]. Total carotenoids also increased significantly from 4.25 ± 0.30 to 4.63 ± 0.38 mg/g in egg-based and from 3.81 ± 0.29 to 4.29 ± 0.41 mg/g in eggless samples consistent with WM-SO’s known β-carotene content [47]. Moreover, radical scavenging activity (%RSA) improved markedly, rising from 32.59 ± 3.30 to 49.11 ± 3.92 % in egg-based (∼51 % increase) and from 45.27 ± 3.60 to 62.88 ± 5.12 % in eggless (∼39 % increase), indicating a synergistic antioxidant effect from phenolics, carotenoids, tocopherols, and unsaturated fatty acids [12], 13]. The superior RSA observed in eggless formulations may be attributed to the unobstructed dispersion of bioactives in a protein-free matrix, enhancing their availability and potency. Similar results were obtained by Abed, Khairy [48].

3.5 Colour characteristics

Replacing soybean oil with WM-SO affected the lightness (L*) of both mayonnaise types. In egg-based samples, L* decreased significantly from 64.21 ± 1.82 to 62.09 ± 1.73 (p ≤ 0.05), indicating a slightly darker appearance (Table 4). This darkening is likely linked to the inherent pigments in WM-SO, including carotenoids and residual chlorophylls, which impart a deeper hue [39], 47]. In contrast, eggless samples showed a significant increase in L* from 66.18 ± 1.90 to 69.08 ± 1.66, suggesting a brighter appearance. This could be due to a more uniform dispersion of these pigments in the absence of egg proteins, enhancing light scattering and perceived brightness [49].

Both mayonnaise types showed shifts in chromaticity values: redness (a*) increased from 0.73 ± 0.01 to 0.80 ± 0.01 in egg-based and from 0.35 ± 0.01 to 0.47 ± 0.01 in eggless, reflecting the warm tint contributed by WM-SO’s carotenoid and phenolic pigments [47]. Yellowness (b*) saw a pronounced increase in egg-based samples, from 9.82 ± 0.90 to 11.79 ± 0.88, driven by the high levels of β-carotene and lutein in WM-SO, which compounded the natural egg yolk pigmentation [39], 47]. In eggless mayonnaise, the increase was modest, from 6.09 ± 0.65 to 6.51 ± 0.74 (both c), due to the absence of synergistic pigmentation from egg proteins. Overall, these color changes underscore the impact of WM-SO’s natural pigment profile and highlight the influence of emulsion matrix composition on color expression [39], 49] (Table 4).

Table 4:

Colour attributes of watermelon seeds oil (WM-SO) incorporated mayonnaise.

Parameters Mayonnaise type
Control egg-based WM-SO egg-based Control eggless WM-SO eggless
Colour attributes
L* 64.21 ± 1.82c 62.09 ± 1.73d 66.18 ± 1.90b 69.08 ± 1.66a
a* 0.73 ± 0.01b 0.80 ± 0.01a 0.35 ± 0.01d 0.47 ± 0.01c
b* 9.82 ± 0.90b 11.79 ± 0.88a 6.09 ± 0.65c 6.51 ± 0.74c

  1. Mean ± Standard deviation of three values. In a row, means have the same small superscript letter are not significantly different by Dunken’s test at p ≤ 0.05.

3.6 Particle size distribution

Substituting soybean oil with WM-SO had notable effects on the particle size distribution and specific surface area (SSA) of both egg-based and eggless mayonnaise (Table 5). In egg-based mayonnaise, D50 decreased significantly from 41.92 ± 1.12 µm to 39.74 ± 1.18 µm (p ≤ 0.05), while D90 plummeted from 143.26 ± 4.91 µm to 12.50 ± 0.89 µm, resulting in a reduced mean droplet size (52.61 ± 1.00 µm to 46.92 ± 1.04 µm) and a dramatic increase in SSA (4,362.6 ± 5.7 to 49,598.3 ± 6.1 cm2/cm3). Meanwhile, D10 (∼5 µm) remained unchanged, indicating the finest droplets were unaffected. These changes are indicative of enhanced homogenization and interfacial stabilization by WM-SO, comparable to findings in tomato seed oil–enhanced egg-based emulsions where reduced D90 and increased SSA were observed [44]. In eggless mayonnaise, D50 remained stable (∼14.2 µm), D10 increased modestly (from 2.15 ± 0.43 to 2.34 ± 0.47 µm), D90 decreased from 78.62 ± 1.60 to 64.13 ± 1.55 µm, and the mean size dropped from 29.86 ± 0.90 to 25.74 ± 0.90 µm, with SSA showing a slight rise (8,624.5 ± 7.1 to 8,732.7 ± 7.9 cm2/cm3), suggesting WM-SO modestly refined droplet distribution. These uniformly smaller and more numerous droplets enhance emulsion stability and texture, as established in similar oil-in-water systems [50], with the effect more pronounced in egg-based formulas due to synergistic emulsifier activity with egg proteins, whereas eggless variants benefit from subtler WM-SO effects. Overall, WM-SO improves physical stability by generating finer emulsions with larger interfacial surface area, supporting its suitability for stable and consumer-pleasing mayonnaise formulations. The obtained data were in the same line with those found by Thakur et al. [51].

Table 5:

Particle size distribution (Mean ± SD) of watermelon seeds oil (WM-SO) incorporated mayonnaise.

Parameters Mayonnaise type
Control egg-based WM-SO egg-based Control eggless WM-SO eggless
D10 (µm) 5.40 ± 0.85a 5.13 ± 0.72a 2.15 ± 0.43b 2.34 ± 0.47b
D50 (µm) 41.92 ± 1.12a 39.74 ± 1.18b 14.20 ± 0.96c 14.46 ± 0.98c
D90 (µm) 143.26 ± 4.91a 12.50 ± 0.89d 78.62 ± 1.60b 64.13 ± 1.55c
Mean size (μm) 52.61 ± 1.00a 46.92 ± 1.04b 29.86 ± 0.90c 25.74 ± 0.90d
Specific surface area (cm2/cm3) 4,362.59 ± 5.70d 49,598.34 ± 6.11c 8,624.51 ± 7.13b 8,732.65 ± 7.88a

  1. Mean ± Standard deviation of three values. In a row, means have the same small superscript letter are not significantly different by Dunken’s test at p ≤ 0.05.

3.7 Optical micrographs

Optical micrographs (Figure 1) reveal clear structural improvements in both egg-based and eggless mayonnaise formulations upon using WM-SO instead of soybean oil. The control eggless mayonnaise (A) displays large, variably sized droplets, indicating weak emulsion stability. In contrast, the control egg-based mayonnaise (B) exhibits smaller, more uniformly distributed oil droplets, reflecting the stronger emulsifying capacity of egg yolk proteins, consistent with reports that egg proteins promote finer droplet formation and improved stability in oil-in-water emulsions [52], 53].

Figure 1: Optical micrographs of (A) control eggless mayonnaise, (B) control egg-based mayonnaise, (C) WM-SO eggless mayonnaise, (D) WM-SO egg-based mayonnaise.
Figure 1:

Optical micrographs of (A) control eggless mayonnaise, (B) control egg-based mayonnaise, (C) WM-SO eggless mayonnaise, (D) WM-SO egg-based mayonnaise.

Upon using WM-SO, eggless WM-SO mayonnaise (C) demonstrates noticeably smaller and more uniform droplets compared to its control, suggesting that the oil enhances the structural stability even without egg proteins. This aligns with findings that plant-based oils can significantly refine droplet size when paired with suitable stabilizers [54]. The most striking microstructure appears in WM-SO egg-based mayonnaise (D), where droplets are the smallest and most homogeneously packed among all samples. These likely results from synergistic interactions between WM-SO’s unsaturated fatty acids and egg yolk lipoproteins, enhancing interfacial stabilization and homogenization efficiency, an effect similar to that reported in tomato seed oil, enriched egg-based emulsions [51].

Overall, WM-SO improves microstructural characteristics across both formulations: droplet size is reduced and distribution becomes more uniform. These structural changes suggest enhanced emulsion stability, reminiscent of trends observed via microstructural analysis and supported by reduced D90 and increased specific surface area metrics [53]. Such improvements translate into smoother texture and improved shelf-life potential key factors in the development of consumer-pleasing and shelf-stable mayonnaise formulations.

3.8 Sensory evaluation

The sensory evaluation (Figure 2) shows that replacing soybean oil with watermelon seed oil (WM-SO) maintains or slightly enhances sensory quality in both egg-based and eggless mayonnaise. Scores for key sensory attributes (appearance, texture, color, taste, and odor) are nearly identical between control and WM-SO formulations, all ranging between 8.3 and 8.8. Notably, overall acceptability improved in the WM-SO egg-based sample (8.6 vs. 8.4 for control) and remained strong in the WM-SO eggless variant (8.5, matching the control). These results suggest that WM-SO preserves organoleptic integrity while potentially increasing consumer appeal. Similar findings have been observed with other seed oils: pumpkin seed oil–enriched eggless mayonnaise maintained sensory quality [48], and tomato seed oil–substituted mayonnaise was well-liked without off-flavors [55]. The absence of bitterness and consistent texture indicate that WM-SO’s mild flavor and unsaturated fat profile integrate seamlessly, making it a promising functional alternative in mayonnaise formulations.

Figure 2: Effect of using watermelon seeds oil WM-SO instead of soybean oil on sensory evaluation score of eggless mayonnaise (A), and egg-based mayonnaise (B).
Figure 2:

Effect of using watermelon seeds oil WM-SO instead of soybean oil on sensory evaluation score of eggless mayonnaise (A), and egg-based mayonnaise (B).

3.9 Changes in physicochemical properties of mayonnaise containing WM-SO during storage experiment

Table 6 presents the physicochemical evolution of mayonnaise samples – both control and watermelon seed oil (WM-SO) enriched – during 30 days of storage at 25 °C. Several quality parameters were monitored to assess the functional impact of WM-SO inclusion in egg-based and eggless formulations.

Table 6:

Changes in physico-chemical properties of WM-SO incorporated mayonnaise during storage at 25 °C for 30 days.

Parameters Storage period (days) Mayonnaise type
WM-SO eggless Control eggless WM-SO egg-based Control egg-based
Moisture (%) Zero 22.30 ± 0.84Cb 22.70 ± 0.95Bb 24.51 ± 0.88Ba 24.92 ± 0.97Ba
10 22.41 ± 0.67Cc 22.95 ± 0.88Bc 24.79 ± 0.82Bb 25.18 ± 0.92Ba
20 22.84 ± 0.91Bd 23.30 ± 0.72Ac 25.12 ± 0.76Ab 25.66 ± 0.95Aa
30 23.17 ± 0.98Ad 23.68 ± 0.76Ac 25.39 ± 0.90Ab 25.98 ± 0.81Aa
pH value Zero 3.79 ± 0.04Ab 3.74 ± 0.05Ab 4.16 ± 0.06Aa 4.13 ± 0.04Aa
10 3.61 ± 0.03Ab 3.55 ± 0.04Bb 3.96 ± 0.04Ba 3.90 ± 0.04Ba
20 3.48 ± 0.02Bb 3.42 ± 0.02Cb 3.72 ± 0.005Ca 3.63 ± 0.03Ca
30 3.35 ± 0.03Cb 3.28 ± 0.03Db 3.65 ± 0.05Ca 3.54 ± 0.02Ca
Water activity Zero 0.80 ± 0.01Dd 0.83 ± 0.01Dc 0.84 ± 0.01Db 0.87 ± 0.01Ca
10 0.82 ± 0.01Cd 0.84 ± 0.01Cc 0.85 ± 0.01Cb 0.88 ± 0.01Ba
20 0.83 ± 0.01Bd 0.85 ± 0.01Bc 0.86 ± 0.01Bb 0.89 ± 0.01Aa
30 0.85 ± 0.01Ad 0.86 ± 0.01Ac 0.87 ± 0.01Ab 0.89 ± 0.01Aa
Free fatty acid (% oleic acid) Zero 0.20 ± 0.02Da 0.22 ± 0.02Da 0.18 ± 0.01Db 0.19 ± 0.01Db
10 0.25 ± 0.01Cb 0.28 ± 0.02Ca 0.23 ± 0.02Cb 0.24 ± 0.02Cb
20 0.33 ± 0.02Bb 0.36 ± 0.01Ba 0.28 ± 0.03Bd 0.31 ± 0.01Bc
30 0.39 ± 0.02Ab 0.41 ± 0.03Aa 0.34 ± 0.01Ad 0.37 ± 0.01Ac
Peroxide value (meq.O2/Kg) Zero 0.70 ± 0.05Dab 0.73 ± 0.04Da 0.67 ± 0.04Db 0.71 ± 0.04Da
10 0.88 ± 0.03Cb 0.92 ± 0.03Ca 0.83 ± 0.02Cc 0.90 ± 0.05Ca
20 0.92 ± 0.06Bc 1.08 ± 0.06Bb 0.90 ± 0.04Bc 1.00 ± 0.07Ba
30 1.12 ± 0.05Ac 1.23 ± 0.07Aa 1.05 ± 0.05Ad 1.18 ± 0.06Ab
Antioxidant activity (%RSA) Zero 62.88 ± 5.12Aa 45.27 ± 3.60Ac 49.11 ± 3.92Ab 32.59 ± 3.30Ad
10 45.11 ± 4.33Ba 34.92 ± 3.11Bc 36.82 ± 2.75Bb 27.05 ± 3.07Bd
20 36.29 ± 4.70Ca 23.15 ± 2.09Cc 27.29 ± 1.80Cb 19.80 ± 1.21Cd
30 22.17 ± 2.17Da 16.30 ± 1.80Dc 19.21 ± 1.95Db 12.44 ± 1.82Dd

  1. Mean ± Standard deviation of three values. RSA means radical scavenging activity. In a row, means have the same small superscript letter are not significantly different by Dunken’s test at p ≤ 0.05. In a column, means have the same capital superscript letter are not significantly different by Dunken’s test at p ≤ 0.05.

3.9.1 Moisture content

During the 30-day storage at 25 °C, all mayonnaise samples exhibited a statistically significant increase in moisture content (p ≤ 0.05) (Table 6). However, formulations contained WM-SO instead of soybean oil consistently displayed lower moisture uptake compared to their control counterparts. In the egg-based group, WM-SO mayonnaise absorbed 0.88 percentage points less moisture over time (increased from 24.51 ± 0.88 to 25.39 ± 0.90 %) than the soybean-oil control (increased from 24.92 ± 0.97 to 25.98 ± 0.81 %). Similarly, in the eggless variants, WM-SO samples increased from 22.30 ± 0.84  to 23.17 ± 0.98 %, while controls rose from 22.70 ± 0.95  to 23.68 ± 0.76 %.

This variance suggests that WM-SO subtly alters the emulsion structure – possibly by enhancing droplet packing density or modifying interfacial layer composition – thereby reducing the system’s capacity to bind free water. Such structural adjustments can be explained by WM-SO introducing hydrophobic components with higher unsaturated fatty acid content, which increase oil droplet cohesion and reduce available water-binding sites. This is supported by evidence in seed oil-enriched emulsions; Siol et al. [13] observed that increased oil presence resulted in tighter droplet networks and reduced moisture retention, emphasizing the link between droplet structuring and water dynamics. Moreover, enhanced moisture restriction in WM-SO formulations can improve textural attributes and limit microbial growth, since excessive moisture in emulsions is known to promote texture degradation and spoilage [48].

3.9.2 pH value

All formulations (both control and prepared with WM-SO) underwent a statistically significant (p ≤ 0.05) pH decline over 30 days at 25 °C, underscoring a classic acidification trend in oil-in-water emulsions (Table 6). Egg-based samples fell from an initial pH of ∼4.13 to ∼3.65, and eggless samples from ∼3.79 to ∼3.35, driven primarily by lipid hydrolysis releasing free fatty acids, which matches observations in long-term stored mayonnaise [56]. Notably, the trajectory of pH reduction did not differ significantly between WM-SO and control samples, indicating that WM-SO neither contributes additional acidifying substances nor disrupts the system’s buffering capacity. Instead, its rich phenolic content may gently inhibit hydrolysis, slowing acid accumulation – similar to stabilizing effects seen in other phenolic-rich emulsions. Egg-based samples began with slightly higher pH values, likely due to the buffering action of egg yolk proteins; yet by day 30 both egg-based and eggless samples converged to similar pH levels. As the final pH approached the egg yolk isoelectric point (pH 3.5–3.9), one would anticipate destabilization – given that protein charge is neutralized, reducing structural integrity – but no physical breakdown was observed. This suggests that WM-SO’s antioxidant profile (tocopherols, phenolics, carotenoids) may preserve emulsion structure even as the system acidifies. From a safety perspective, the maintained pH range (3.3–3.7) across treatments remains well below the threshold (∼4.1) that inhibits Salmonella survival in mayonnaise [1], 56]. Consequently, WM-SO fortification does not compromise safety or regulatory compliance; rather, it enhances pH resilience, supporting its use as a functional, stable ingredient in mayonnaise formulations.

3.9.3 Water activity

Water activity, the measure of unbound water that can support microbial growth, increased slightly in all mayonnaise samples during storage at 25 °C (Table 6). However, formulations contained WM-SO instead of soybean oil consistently retained significantly lower water activity values than their soybean oil controls (p ≤ 0.05). In egg-based emulsions, WM-SO samples rose from 0.84 to 0.87 over 30 days, compared to controls that increased from 0.87 to 0.89. Similarly, eggless WM-SO variants climbed from 0.80 to 0.85, while controls shifted from 0.83 to 0.86. This pattern suggests that the hydrophobic nature of WM-SO oil droplets helps retain water within the oil phase, reducing the availability of free water in the aqueous phase – a finding consistent with studies in seed-oil-enriched emulsions [46]. Lower water activity directly correlates with better microbial stability, and maintaining below 0.90 is critical, as higher values increase the risk of spoilage by yeasts and bacteria [57]. In this context, WM-SO not only supports product stability from a chemical standpoint – as evidenced by favorable moisture and oxidation metrics – but also enhances microbial safety, affirming its suitability for shelf-stable, low-pH, oil-in-water emulsions like mayonnaise.

3.9.4 Free fatty acids

Free fatty acids are critical markers of lipid hydrolysis in oil-in-water emulsions, and all mayonnaise samples – including those prepared by substituting WM-SO with soybean oil – exhibited a statistically significant increase in free fatty acids percentage during 30 days of storage at 25 °C (p ≤ 0.05) (Table 6). Notably, WM-SO enrichments consistently limited free fatty acids accumulation compared to their soybean-oil controls. In egg-based formulations, control samples increased from 0.19 ± 0.01 to 0.37 ± 0.01 %, while WM-SO samples rose from 0.18 ± 0.01 to 0.34 ± 0.01 %, representing a ∼9 % reduction by day 30. Eggless variants showed similar trends, with free fatty acids percentage rising from 0.22 ± 0.02 to 0.41 ± 0.03 % in controls versus 0.20 ± 0.02 to 0.39 ± 0.02 % in WM-SO samples – about a 5 % decrease. This attenuation suggests that WM-SO’s natural antioxidants (e.g., phenolics, tocopherols, flavonoids) stabilize the oil–water interface, effectively delaying lipolytic reactions. Similar findings were reported in tomato seed oil–fortified mayonnaise, where free fatty acids in WM-SO samples increased to 0.23 %, compared to 0.27 % in controls – a ∼15 % reduction in free fatty acids accumulation [51]. Mechanistically, WM-SO’s antioxidant compounds likely shield triglycerides at the droplet interface, reducing enzyme accessibility and hydrolytic activity, supported by its high unsaturated fatty acid content [52].

From a quality standpoint, lower free fatty acids levels in WM-SO formulations help prevent bitterness and maintain emulsion viscosity, supporting better texture and flavor retention during shelf life. Additionally, reduced free fatty acids accumulation aligns with consumer safety and regulatory standards – like those set by the FDA and EMA – which typically accept free fatty acids levels below 0.5 % for edible oils and emulsion-based foods like mayonnaise [58]. Overall, the integration of WM-SO into both egg-based and eggless mayonnaise effectively decelerates hydrolytic degradation, underscoring its role as a functional ingredient that enhances product stability and sensory quality.

3.9.5 Peroxide value

Peroxide value, a primary marker of lipid oxidation and rancidity in emulsions, increased significantly (p ≤ 0.05) in all mayonnaise samples stored at 25 °C for 30 days (Table 6); however, those prepared by using WM-SO instead of soybean oil displayed a consistently slower peroxide value rise, indicating enhanced oxidative stability. In egg-based samples, control peroxide value climbed from 0.71 ± 0.04 to 1.18 ± 0.06 meq O2/kg, whereas WM-SO formulations rose from 0.67 ± 0.04 to 1.05 ± 0.05 meq O2/kg – an approximate 11 % reduction. Eggless variants followed suit: control PV reached 1.23 ± 0.07, compared to 1.12 ± 0.05 meq O2/kg in WM-SO samples – a nearly 9 % decrease. The slower PV rise can be attributed to WM-SO’s high antioxidant content – such as tocopherols, phenolics, and carotenoids – that inhibit hydroperoxide formation, alongside its unsaturated fatty acid composition and enhanced emulsion structure, which limit pro-oxidant penetration [59]. Comparable findings in tomato seed oil–fortified mayonnaise further support this stabilizing effect, where peroxides accumulated more slowly compared to soybean oil controls [42]. Mechanistically, WM-SO’s antioxidants likely quench free radicals at the oil–water interface and reinforce interfacial stability, while its unsaturated fatty acid composition, balanced by natural antioxidants, ensures comprehensive protection. Additionally, tighter droplet packing may limit oxygen access, slowing oxidation. Practically, the WM-SO samples maintained peroxide value well below both sensory thresholds for rancidity (∼20–30 meq O2/kg) and Codex Alimentarius regulatory limits (10–15 meq O2/kg) [60]. These results confirm that WM-SO effectively boosts oxidative resilience, preserving flavor, texture, and regulatory compliance in mayonnaise formulations.

3.9.6 Antioxidant activity

Over a 30-day storage at 25 °C as shown in Table 6, mayonnaise prepared by using WM-SO instead of soybean oil retained significantly higher radical scavenging activity (RSA%) compared to soybean oil controls (p ≤ 0.05), underscoring enhanced antioxidant capacity. In egg-based WM-SO formulations, RSA% declined from 49.1 ± 3.9 to 19.2 ± 2.0 %, whereas control samples dropped from 32.6 ± 3.3 % to 12.4 ± 1.8 %, preserving approximately 39 % more activity by day 30. Similarly, eggless WM-SO variants fell from 62.9 ± 5.1 to 22.2 ± 2.2 %, while controls decreased from 45.3 ± 3.6 to 16.3 ± 1.8 %, maintaining around 36 % of the initial antioxidant reserve. This sustained activity reflects WM-SO’s richness in phenolics, flavonoids, and tocopherols, which intercept free radicals at the oil–water interface, effectively delaying oxidative degradation. Comparable protective effects were observed in grape seed extract–enriched mayonnaise, where grape seeds extract addition notably preserved RSA % and limited hydroperoxide build up over 8 weeks at ambient temperature [61]. Likewise, Rosa canina fruit extract notably enhanced oxidative resistance in mayonnaise, attributed to its high phenolic content [62].

3.10 Microbial count in mayonnaise

Table 7 presents the evolution of the microbial population (log CFU/g) in mayonnaise formulations enriched with watermelon seed oil (WM-SO) during 30 days of storage at 25 °C. Yeasts and moulds were absent in all samples up to the 20th day, reflecting both the microbiological quality of the formulations and adequate hygienic handling during preparation. After 30 days, fungal growth was observed only at low levels, with the control egg-based sample showing the highest count (1.27 ± 0.05 log CFU/g) and the WM-SO eggless sample exhibiting the lowest (0.62 ± 0.02 log CFU/g). These differences were statistically significant (p ≤ 0.05), suggesting that WM-SO incorporation effectively suppressed fungal proliferation throughout storage.

Table 7:

Changes in microbial count (Log CFU/g mayonnaise) of WM-SO incorporated mayonnaise during storage at 25 °C for 30 days.

Microbial count (log CFU/g) Storage period (days) Mayonnaise type
WM-SO eggless Control eggless WM-SO egg-based Control egg-based
Yeast and moulds Zero ND ND ND ND
10 ND ND ND ND
20 ND ND ND ND
30 0.62 ± 0.02d 1.01 ± 0.04b 0.85 ± 0.03c 1.27 ± 0.05a
Total plate count Zero ND ND ND ND
10 ND ND ND ND
20 ND ND ND 1.13 ± 0.06B
30 ND ND ND 1.45 ± 0.04A

  1. ND means not detected. In a row, means have the same small superscript letter are not significantly different by Dunken’s test at p ≤ 0.05. In a column, means have the same capital superscript letter are not significantly different by Dunken’s test at p ≤ 0.05.

For total plate counts, bacterial growth remained undetectable in all formulations until day 20, except for the control egg-based mayonnaise (1.13 ± 0.06 log CFU/g). By day 30, this sample exhibited a modest increase to 1.45 ± 0.04 log CFU/g, whereas no detectable bacterial colonies were found in the other samples. The inhibition of microbial development in WM-SO–based mayonnaise can be attributed to the presence of phenolic compounds and natural antioxidants inherent in watermelon seed oil, which exhibit known antimicrobial properties.

Collectively, these findings demonstrate that substituting conventional oil with WM-SO markedly enhances the microbiological stability of mayonnaise, particularly in eggless formulations. The lower yeast and mould counts, together with the absence of bacterial growth up to 30 days, highlight WM-SO’s capacity to extend product shelf life. This stability may also be reinforced by the combined effects of its acidic pH and relatively low water activity (0.80–0.89), both of which hinder bacterial proliferation while permitting only minimal fungal survival. Overall, the integration of WM-SO contributes to a safer, more stable, and functionally superior mayonnaise, with the statistical validation (mean ± SD; Duncan’s test, p ≤ 0.05) confirming the robustness and reproducibility of these results.

Generally, all findings of our study are consistent with recent literature that highlights the promising functionalities of plant-based emulsions and lipid alternatives in the reformulation of innovative plant-based foods. Recently, the literature indicates that replacing conventional oils with seed-derived or plant-based oils increases oxidative stability, bioactive retention, and microstructural integrity of emulsion-based foods [44], 51], 54]. Similar patterns of improved oxidative stability of microencapsulated or emulsion systems containing chia seed oil and other formulations with pumpkin seed oil in structured emulsions and fat-replacements [44], 48] have been documented. Thakur et al. [51] characterized tomato seed oil and described its functional lipid profile and potential as an alternative oil in food applications. The consistent increase in antioxidant capacity as well as emulsion quality confirms the potential of watermelon seed oil as a nutritionally improved and sustainable substitute to conventional edible oils in mayonnaise systems. This encourages the manufacturing of clean-label, plant-based emulsions that are aligned with the health functionality and environmental sustainability.

4 Conclusions

This study revealed that watermelon seed oil (WM-SO) could be used as a superior, nutritious alternative to soybean oil in both egg-based and eggless mayonnaise. Incorporation of WM-SO markedly improved oxidative stability, decreased the formation of free fatty acids (∼9 % less in egg-based), and decreased peroxide values (∼11 % reduced). Using WM-SO in the mayonnaise formula also improved antioxidant activity, with retention of as much as 39 % more RSA, and microbial stability during storage of 30 days. Using WM-SO in mayonnaise preparation also gave a smaller droplet size, enhanced microstructure, and superior sensorial properties without altering acceptability. These results make WM-SO a potential functional ingredient to produce healthier, more stable, and consumer-friendly mayonnaise products. Further investigations should be carried out to determine the functional and nutritional impact of WM-SO mayonnaise, such as bioactive component bioavailability, consumer acceptability, and the sustainability and economic viability of using it as a functional, environmentally friendly alternative to oils.

Corresponding author: Essam Mohamed Elsebaie, Food Technology Department, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Shaikh, Egypt, E-mail:

  1. Funding information: No sources of funding for this study

  2. Author contributions: Conceptualisation: E. M. E. S., R.A.S and H. A. Y. S.; methodology: E. M. E. S., S. A. H., H. A. Y. S and S. S. I.; software: A. S. M. F. and S. S. I.; validation: E. M. E., D. M. E and R. A. S.; formal analysis: E. M. E. S. and M. F. A. E.; investigation: E. M. E. S., E. M. A., M. F. A. E., A. S. M. F and H. A. Y. S.; resources: E. M. A.; data curation: E. M. E. S. and H. A. Y. S.; writing – original draft preparation: E. M. E. S., E. M. E. and D. M. E.; writing – review and editing: E. M. E.; visualisation: S. A. H.; funding acquisition: E. M. A. All the authors agreed on the final version of the manuscript.

  3. Conflict of interest: Authors state no conflicts of interest.

  4. Ethical approval: No experiments were conducted on animales or humans.

  5. Data availability stament: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Received: 2025-08-08
Accepted: 2025-11-04
Published Online: 2025-11-27

© 2025 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/chem-2025-0215
Keywords for this article
agro-waste; antioxidants; DPPH; fatty acids; oil; quality
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BY 4.0

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