Evaluation of the Quality and Composition of the Lipid Fraction Obtained from Acorns

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Acorns from native (Q. robur) and invasive (Q. rubra) oaks can be used as alternative sources of plant oils. The current study offers a screening method that combines traditional quality measurements, GC-based fatty acid analysis, and PDSC-based oxidative stability assessment. It helps in choosing suitable species and extraction techniques for food, cosmetic, or technical uses, promoting the sustainable utilization of forest biomass.

Abstract

Acorns represent an underutilized source of forest biomass with potential for producing edible oils and bioactive compounds. This research compared lipid fractions from pedunculate oak (Quercus robur L.) and northern red oak (Quercus rubra L.) collected in Poland, examining how different extraction methods influence oil yield and quality. Oils were extracted using Soxhlet with hexane, cold hexane extraction for both species, and mechanical pressing for Q. rubra. Fatty acid profiles analyzed by GC-FID facilitated calculation of lipid quality indices. Oxidative stability was assessed through isothermal PDSC, and total phenolics, flavonoids, and antioxidant activity (DPPH, ABTS) were measured in acorn extracts. Q. rubra produced more oil than Q. robur regardless of extraction method, but Q. robur oils exhibited significantly higher PDSC oxidation times (τ_on, τ_max). Pressed Q. rubra oil showed higher acid and peroxide values compared to solvent-extracted oils. Fatty acid composition was predominantly influenced by species rather than by extraction method, as confirmed by multivariate analysis, which indicated species as the main driver of variability. Overall, these results highlight a trade-off between oil yield and oxidative stability, suggesting acorns as a promising, species-dependent oil resource.

1. Introduction

Forests are essential for maintaining environmental stability, regulating biogeochemical cycles, and conserving biodiversity. Compared to many European countries, Poland is relatively well forested. Forests cover over 9.3 million hectares, which is about 29.6% of the country’s total area, and this percentage has been steadily rising in recent decades [1]. The growth of forested areas benefits ecosystem functions, climate regulation, and biological diversity. Among Poland’s main tree species that form forests, oaks (Quercus spp.) hold a particularly significant ecological, economic, and cultural role.

Among broadleaved tree taxa, oaks (Quercus spp., Fagaceae) have a prominent ecological and socio-economic importance across the temperate zone of the Northern Hemisphere. Oaks comprise approximately 400–500 species and represent a taxonomically complex group with extensive interspecific variation and frequent hybridization [2]. In Poland, three oak species grow naturally, i.e., pedunculate oak (Q. robur L.), the most common species, mainly found in northern areas, sessile oak (Q. petraea (Matt.) Liebl.), mostly present in southern Poland, and downy oak (Q. pubescens Willd.), which is limited to specific locations like the Bielinek Nature Reserve along the Oder River [3]. Additionally, northern red oak (Q. rubra L.), a species native to North America, has been increasingly spotted in Polish forests [4]. Because of its high invasiveness and ability to replace native species, Q. rubra is regarded as an invasive species in Poland, and forest management plans recommend restricting its planting and monitoring existing stands [5].

The fruit of oak trees is the acorn, which shows significant variation among species in its shape, development, and are of significant scientific interest because of their diverse and rich chemical composition. On a dry-matter basis, carbohydrates, often labeled as nitrogen-free extract, typically constitute the largest part, reaching approximately 89.7% in some taxa [6]. The primary carbohydrate is starch, which can make up about 55% of acorn dry weight [7]. In addition to carbohydrates, acorns contain proteins, lipids, water, vitamins, and essential minerals [7,8]. Protein levels vary, with crude protein ranging from 2.75% to 8.44% among 20 Quercus taxa from Turkey [9], and around 8.77% in Q. robur seeds [10]. Lipid content varies considerably among species and sources, generally from about 1.1% to 31.3% [6], with many acorns containing 3–10% lipids [11]. Notably, Q. robur seeds had roughly 4.55% lipids in one study, similar to maize kernels at approximately 4.4% [10]. Acorn oils are rich in unsaturated fatty acids, often representing 75–90% of total fatty acids, with oleic acid as a major component, plus linoleic (n-6) and smaller amounts of α-linolenic (n-3) acids [12,13]. Historically, acorn oil has been used for cooking in Algeria and Morocco and is noted for having physicochemical properties similar to those of olive oil, indicating its potential as an alternative edible oil source [12,14].

Beyond macronutrients, acorns contain many bioactive compounds, with polyphenols being the best documented group, especially tannins, phenolic acids, and flavonoids [15]. Phenolic compounds mainly cause the high antioxidant activity of acorns, while tannins add to their astringent properties and protective effects against oxidative stress [16]. Depending on species and processing, extracts and fractions from acorns (and more broadly from Quercus tissues) have been reported to show bioactivities such as anti-inflammatory, antimicrobial, and anticancer effects, mainly in in vitro and/or preclinical models [15].

In recent years, attention has increasingly focused on acorns as a more sustainable, underutilized food resource. Historically, acorns have been consumed for thousands of years and served as a staple food in many regions, including East Asia, the Mediterranean basin, and North America, especially among Indigenous communities in California [17]. Today, acorns are used to produce flour, coffee-like roasted beverages, and edible oils, with studies also characterizing the polyphenols and mineral composition of acorn-based brews and other products [15].

In addition to nutritional uses, oak-derived materials have long been used in traditional medicine, largely linked to astringent tannins and related phytochemicals [18]. Extracts from oak leaves and acorns have demonstrated effectiveness against various bacterial strains, including Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae [19]. Ecologically, acorns play a crucial role as a food source for numerous invertebrate and vertebrate species, contributing significantly to forest food webs and natural regeneration processes [20].

This study aimed to conduct a detailed comparative analysis of acorns from two oak species found in Poland, i.e., pedunculate oak (Q. robur L.) and northern red oak (Q. rubra L.), focusing on their potential as raw materials for oil production. The lipid fraction was extracted from the acorns using three methods: Soxhlet extraction with hexane, cold hexane extraction, and mechanical pressing. The quality of the oils was assessed by measuring acid and peroxide values, and the fatty acid profile was analyzed with gas chromatography (GC). Additionally, the oxidative stability of the acorn lipid extracts was evaluated through isothermal oxidation using high-pressure differential scanning calorimetry (PDSC).

In addition, this study evaluates both the quality and oxidative stability of lipid fractions from acorns of pedunculate oak (Q. robur) and northern red oak (Q. rubra), using traditional quality metrics, detailed fatty acid analysis, and advanced thermal techniques. Notably, applying PDSC to gauge the oxidative stability of acorn oils is rarely reported in existing research. Additionally, comparing a native oak species (Q. robur) with an invasive species (Q. rubra) offers new insights into the potential of acorns as alternative plant oil sources, contributing to the broader discussion on the sustainable use of forest biomass.

2. Materials and Methods

2.1. Materials

Acorns of two oak species, Q. rubra L. (northern red oak) and Q. robur L. (pedunculate oak), were used in this study (Figure 1). Sampling was conducted during the main acorn-fall season (September–October 2024) near Krotoszyn, Greater Poland Voivodeship, Poland (51.662° N, 17.461° E). The acorns were collected at physiological maturity, i.e., immediately after natural fall, and subsequently stored at room temperature for one month prior to analysis.

Prior to analysis, the acorns were manually cracked to remove the shells, and the kernels were ground into small particles using a coffee grinder (Esperanza, Ożarów Mazowiecki, Poland). The milled material was transferred to sealed, light-protected containers and kept at room temperature until analysis.

All chemicals and reagents used in the study were of analytical grade (unless stated otherwise) and were purchased from Merck Life Science sp. z o.o. (Poznań, Poland) and Avantor Performance Materials Poland S.A. (Gliwice, Poland). Key chemicals included: n-hexane (≥95%), ethanol (96%, v/v), toluene (≥99.5%), chloroform (≥99%), glacial acetic acid (≥99.8%), anhydrous magnesium sulfate (MgSO₄; ≥99%), sodium thiosulfate (Na₂S₂O₃·5H₂O; ≥99%), Folin–Ciocalteu reagent, sodium carbonate (Na₂CO₃; ≥99.5%), aluminum chloride (AlCl₃·6H₂O; ≥99%), sodium acetate (≥99%), DPPH (≥95%), ABTS (≥98%), Trolox (≥97%), and ammonium persulfate (≥98%).

2.2. Oil Extraction from Acorns

Three methods were used to extract oil from acorns: Soxhlet extraction, cold solvent extraction by shaking, and mechanical pressing. Acorns of northern red oak (Q. rubra L.) were subjected to all three methods, while acorns of pedunculate oak (Q. robur L.) were only processed with solvent-based techniques due to their low lipid content, which made mechanical pressing ineffective.

2.2.1. Soxhlet Extraction

Soxhlet extraction was used to recover oil from both northern red oak and pedunculate oak acorns. A total of 20 g of ground acorn material was extracted with 150 mL of hexane as the solvent. The extraction was performed for 2 h at the boiling point of hexane. After the extraction, the hexane–oil solution was dried with anhydrous magnesium sulfate (MgSO₄). After 10 min of drying, the solution was filtered to remove the drying agent. The solvent was then evaporated under reduced pressure using a rotary evaporator (Büchi Rotavapor R-200, Büchi AG, Flawil, Switzerland), yielding crude oil. The collected oil was weighed to determine the oil yield.

2.2.2. Cold Solvent Extraction

Cold solvent extraction was conducted for both northern red oak and pedunculate oak acorns. In each case, 25 g of ground acorns was placed in a 500 mL flask and mixed with 220 mL of hexane. The extraction was performed at room temperature for 3 h using a laboratory shaker (IKA KS 4000 ic control, IKA, Königswinter, Germany). Following extraction, the solid material was separated by filtration. Anhydrous magnesium sulfate (MgSO₄) was added to the filtrate to remove residual moisture. After a short drying period, the solution was filtered again, and hexane was removed using a rotary evaporator. The obtained oil was collected and weighed for further analyses.

2.2.3. Mechanical Pressing

Mechanical pressing was carried out solely on northern red oak acorns. Initial attempts to press pedunculate oak acorns failed due to their low oil content. For pressing, 150 g of ground acorn material was used. The ground material was placed into a mechanical oil press (YODA YD-ZY-02A, Yoda Polska, Warsaw, Poland). During pressing, the oil separated from the solid residue, and the remaining press cake was expelled from the chamber. After pressing, the crude oil was collected and simply clarified to remove mechanical impurities. The clarified oil was then stored and prepared for further analysis.

2.3. Determination of Oil Yield

Oil yield was measured gravimetrically following either solvent extraction or mechanical pressing. It was calculated by dividing the mass of extracted oil by the dry acorn material’s mass, then expressed as a percentage. The calculation was based on Equation (1).

$$\mathrm{Oil} \mathrm{yield} \left(\%\right)={\displaystyle \frac{{\mathrm{m}}_{\mathrm{oil}}}{{\mathrm{m}}_{\mathrm{sample}}}} \times 100$$

(1)

where m_oil is the mass of the extracted oil (g) and m_sample is the mass of dry acorn material used for extraction (g).

2.4. Determination of Acid Value

The acid value (AV) was determined to evaluate hydrolytic degradation of the oil in accordance with the AOCS Official Method Te 1a-64 [21] using an automatic titrator (TitraLab AT1000 Series, HACH LANGE, Wrocław, Poland). Briefly, 2 g of oil was transferred to a titration vessel and dissolved in 70 mL of a toluene–ethanol mixture (1:1, v/v). The sample was titrated with 0.1 M potassium hydroxide (KOH), and the results were expressed as mg KOH/g of oil.

2.5. Determination of Peroxide Value

Peroxide value (PV) was quantified according to the AOCS Official Method Cd 8b-90 [22] using the automated titration system described above. In brief, 2.0 g of oil was dissolved in 25 mL of a chloroform/acetic acid mixture (2:3, v/v) and treated with 1 mL of saturated potassium iodide. The solution was mixed for 30 s and then left in the dark for 5 min. Next, 75 mL of distilled water was added, and the liberated iodine was titrated with 0.002 M sodium thiosulfate. PV was expressed as mEq O₂/kg of oil.

2.6. Fatty Acid Methyl Ester Analysis by Gas Chromatography

Fatty acid composition was determined by GC-FID using a YL6100 GC Clarity system (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). Fatty acid methyl esters (FAMEs) were prepared in accordance with EN ISO 5509:2001 [23]. Nitrogen served as the carrier gas at a steady flow rate of 1.0 mL/min under constant-flow conditions. Samples were injected in split mode with a split ratio of 1:50. The oven temperature program was set as follows: 70 °C for 0.5 min, then increased at 15 °C/min to 160 °C, followed by a rise at 1.1 °C/min to 200 °C with a 12 min isothermal hold, and finally increased at 30 °C/min to 225 °C with a 1 min hold. The injector and detector temperatures were maintained at 225 °C and 250 °C, respectively. Fatty acids were assigned by matching retention times with those of a certified reference standard mixture (Supelco 37 Component FAME Mix, Sigma-Aldrich, Bellefonte, PA, USA). The composition was reported as the relative percentage of each fatty acid in the total fatty acid pool [24].

Additionally, the fatty acid profiles were employed to determine the health-related lipid indices of the tested oils. The atherogenic index (AI), thrombogenic index (TI), and the hypocholesterolaemic/hypercholesterolaemic ratio (h/H) were computed using Equations (2)–(4) [25].

$$\mathrm{AI}={\displaystyle \frac{\mathrm{C}12:0+\left(4 \times \mathrm{C}14:0\right)+\mathrm{C}16:0}{\sum \mathrm{MUFA}+\sum n–6 \mathrm{PUFA}+\sum n–3 \mathrm{PUFA}}}$$

(2)

$$\mathrm{TI}={\displaystyle \frac{\mathrm{C}14:0+\mathrm{C}16:0+\mathrm{C}18:0}{0.5 \times \sum \mathrm{MUFA}+\left(0.5 \times \sum n–6 \mathrm{PUFA}\right)+\left(3 \times \sum n–3 \mathrm{PUFA}\right)+\frac{\sum n–3 \mathrm{PUFA}}{\sum n–6 \mathrm{P}\mathrm{UFA}}}}$$

(3)

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

(4)

2.7. Oxidative Stability Analysis by Isothermal Pressure Differential Scanning Calorimetry

Oxidative stability was evaluated by pressure differential scanning calorimetry (PDSC) using a DSC Q20P instrument (TA Instruments, New Castle, DE, USA). The calorimeter was calibrated for baseline with an empty oven and for temperature with high-purity indium as a standard. An empty open aluminum pan served as the reference for all measurements. For each test, 3–4 mg of oil was placed in an open aluminum pan and subjected to oxidative conditions at a constant temperature of 120 °C under an oxygen pressure of 1350–1400 kPa. During the analysis, the time corresponding to the oxidation onset time (τ_on) and the maximum heat flow rate (τ_max) were recorded and used as the indicators of oxidative stability.

2.8. Determination of Bioactive Compounds

Crushed acorn kernels were extracted using 80% (v/v) ethanol at a solid-to-solvent ratio of 1:10 (w/v). The mixtures were vortexed vigorously for 2 min and then incubated in a water bath at 40 °C for 30 min. After extraction, the samples were centrifuged at 8000 rpm for 10 min (MPW-352, MPW Med. Instruments, Warsaw, Poland). The supernatants were collected and used to determine total phenolic, total flavonoid, and antioxidant activity.

2.8.1. Determination of Total Phenolic Content (TPC)

All absorbance readings were recorded with a UV–Vis spectrophotometer (Rayleigh UV1601, Beijing Beifen-Ruili Analytical Instrument (Group) Co., Ltd., Beijing, China) using 1 cm pathlength cuvettes. Total phenolic content (TPC) was estimated using the Folin–Ciocalteu reaction as an indicator of phenolic reducing capacity [26]. For the assay, 0.18 mL of ethanolic extract was diluted with 4.92 mL of distilled water and combined with 0.30 mL of Folin–Ciocalteu reagent. After 3 min, 0.60 mL of 17.7% (w/v) Na₂CO₃ solution was added. The mixture was vortexed and incubated in the dark for 60 min. Absorbance was then measured at 750 nm against ethanol. Results were calculated from a chlorogenic acid calibration curve and expressed as mg CGA/g DW.

2.8.2. Determination of Total Flavonoid Content (TFC)

Total flavonoid content (TFC) was measured based on complex formation with aluminum chloride, following the method described by Aryal et al. [27]. For each measurement, 1.00 mL of ethanolic extract was mixed with 0.20 mL of 0.3 mM aqueous aluminum chloride (AlCl₃), 0.20 mL of 1 M sodium acetate, and 5.60 mL of distilled water. After thorough mixing, the samples were incubated in the dark for 30 min. Absorbance was recorded at 430 nm (ethanol blank). The flavonoid content was calculated from the quercetin calibration curve and expressed as mg QE/g DW.

2.8.3. DPPH Radical Scavenging Activity Assay

DPPH radical scavenging activity was determined following the procedure described in [28]. A diluted ethanolic extract (0.30 mL) was added to 2.70 mL of 0.004% (w/v) DPPH solution prepared in methanol. After 30 min incubation in the dark, absorbance was measured at 517 nm relative to ethanol. Antioxidant activity was determined from the Trolox calibration curve and expressed as mmol Trolox equivalents per gram of dry weight (mmol TE/g DW).

2.8.4. ABTS Radical Cation Scavenging Activity Assay

ABTS radical cation scavenging capacity was assessed based on the method of Moreira [29] with minor adjustments. A 7 mM ABTS solution was prepared in distilled water and activated with ammonium persulfate to a final concentration of 2.45 mM. The mixture was kept in the dark at room temperature for 16 h to generate ABTS⁺. Before analysis, the working solution was diluted with distilled water to an absorbance of 0.700 ± 0.020 at 734 nm. For the assay, 40 µL of ethanolic extract was mixed with 4.0 mL of ABTS·⁺ solution. After 6 min, absorbance was read at 734 nm against distilled water. Antioxidant activity was calculated using a Trolox calibration curve and expressed as mmol TE/g DW.

2.9. Statistical Analysis

All statistical computations were performed using Statistica 13.3 (TIBCO Software Inc., Palo Alto, CA, USA). Results are shown as mean ± standard deviation (SD). The normality of the data distribution was tested with the Shapiro–Wilk test, and the homogeneity of variances was assessed using Levene’s test. When these assumptions were satisfied, one-way analysis of variance (ANOVA) was used, followed by Tukey’s honestly significant difference (HSD) post hoc test at a significance level of α = 0.05. Additionally, multivariate analyses were used to explore relationships among variables and to characterize the extracts. These included principal component analysis (PCA), while hierarchical clustering and heatmap visualization were performed using the ChiPlot online platform [30], based on standardized data and applying Ward’s linkage method with Euclidean distance.

3. Results and Discussion

The lipid fractions from Q. rubra and Q. robur acorns were thoroughly analyzed to compare species differences and examine how extraction methods affect oil quality. The study systematically assessed variations in oil yield, fatty acid composition, physicochemical quality parameters, oxidative stability, and bioactive compounds. Multivariate statistical techniques were then used to uncover patterns and correlations among the compositional and stability-related variables. To minimize environmental variability and focus on the effects of species and processing, acorns were gathered from a single geographic location and harvest season. Future studies should apply the same analytical methods across different regions and harvest years, and also investigate how the maturity stage and storage time after harvest affect oil yield and quality.

Oil yield showed significant variation between oak species and extraction techniques (

Table 1. Oil content (%) in the dry matter of acorns.

Oak Species	Oil Yield (%)
	Soxhlet Extraction	Cold Hexane Extraction	Mechanical Pressing
Q. rubra	17.56 ± 1.29 Aa	14.87 ± 1.13 Ab	7.28 ± 0.42 c
Q. robur	7.01 ± 0.27 Ba	4.18 ± 0.38 Bb	–

Different lowercase letters (a–c) denote significant differences among extraction methods within the same oak species, while different uppercase letters (A,B) denote significant differences between oak species within the same extraction method (Tukey’s HSD, α = 0.05).

). Acorns of Q. rubra produced considerably more oil than those of Q. robur across all extraction methods. For Q. rubra, Soxhlet extraction yielded the highest amount of oil (17.56%), followed by cold hexane extraction (14.87%). Mechanical pressing resulted in a much lower yield (7.28%). Conversely, oil yield from Q. robur was notably lower, reaching 7.01% for Soxhlet extraction and 4.18% for cold hexane extraction. The low lipid content of Q. robur acorns did not allow effective oil recovery by mechanical pressing, and therefore this method was not applicable for this species. For both species, significant differences were observed among extraction methods, with solvent extraction consistently outperforming mechanical pressing in Q. rubra.

The noticeable oil yield gap between Q. rubra and Q. robur aligns with the significant, species-specific differences in acorn lipid content documented in the literature. For example, Górnaś et al. [31] reported significantly higher oil yields for Q. rubra (21.8%) compared to Q. robur (5.3%) when oil was extracted using an ultrasound-assisted n-hexane method. The higher oil content in Q. rubra helps explain why mechanical pressing was only successful for Q. rubra in this study, while the lower-lipid Q. robur material did not allow for efficient oil recovery through pressing.

The significant impact of the extraction method observed here, i.e., Soxhlet > cold hexane > pressing, aligns with previous research showing that solvent contact time, energy input, and mass transfer enhancement affect oil recovery. In Algerian acorns, Makhlouf et al. [32] employed Soxhlet extraction with hexane to extract oils from Q. ilex, Q. suber, and Q. coccifera, reporting yields of 7.05–8.40%. Similarly, for Q. brantii, Niari et al. [33] used n-hexane Soxhlet extraction as the standard method and also tested assisted techniques. They found average yields of around 5.67% with ultrasound-assisted extraction (UAE) and 5.24% with microwave-assisted extraction (MAE), with UAE reaching a maximum of 8.27% under optimized conditions. These yields are comparable to the Soxhlet yield obtained here for Q. robur, suggesting that many Quercus species, particularly those with lower natural lipid content, tend to produce moderate to low oil yields even with exhaustive solvent extraction.

Regarding production potential, the yields achieved here (up to 17.56% of dry matter for Q. rubra via Soxhlet extraction) suggest that high-lipid acorn species can yield oil levels similar to some traditional oilseeds. Still, industrial use will depend on raw material availability, collection logistics, and annual crop variations. Thus, acorn oil might be best suited as a seasonal or niche product or as a co-product in forestry or agroforestry systems, with storage and processing methods to manage supply fluctuations.

Maximizing oil recovery is essential, but extraction conditions can affect oil quality. Factors such as temperature, contact time with solvents, and mechanical disruption may cause hydrolysis of triacylglycerols, increasing free fatty acids, and promote oxidation processes. After comparing yields, the key physicochemical quality indices were assessed. The acid value (AV) showed considerable variation across different species and extraction methods (

Table 2. Acid value of oils obtained by different extraction methods.

Oak Species	Acid Value (mg KOH/g Oil)
	Soxhlet Extraction	Cold Hexane Extraction	Mechanical Pressing
Q. rubra	0.57 ± 0.04 Bc	0.43 ± 0.07 Bb	0.77 ± 0.09 a
Q. robur	3.77 ± 0.22 Aa	3.55 ± 0.31 Aa	–

Different lowercase letters (a–c) denote significant differences among extraction methods within the same oak species, while different uppercase letters (A,B) denote significant differences between oak species within the same extraction method (Tukey’s HSD, α = 0.05).

). Oils from Q. robur exhibited notably higher AV levels (3.55–3.77 mg KOH/g oil) than those from Q. rubra (0.43–0.77 mg KOH/g oil). Within Q. rubra, mechanical pressing produced the highest AV, whereas cold hexane extraction resulted in the lowest. No significant difference was observed between Soxhlet and cold hexane extraction for Q. robur.

The significantly higher AV in Q. robur suggests either a greater extent of pre-extraction hydrolysis in the acorn matrix or a higher susceptibility to hydrolysis under the applied processing conditions. Similar free fatty acid levels have been reported for Q. brantii oils extracted by conventional and assisted methods, supporting the idea that FFA around 1.8% can occur in acorn oils depending on raw material and processing [33]. In contrast, Makhlouf et al. [32] reported lower free acidity (0.92–1.13 g/100 g oil) for Soxhlet-hexane extracted oils. Within Q. rubra, the higher AV after mechanical pressing is mechanistically plausible, i.e., pressing enhances cell disruption and promotes contact between residual moisture, endogenous enzymes, and the lipid phase, while also increasing local temperature. Notably, even under “cold-press” conditions, oil temperatures can reach approximately ~55 °C due to friction and compression, which may facilitate hydrolysis and explain why pressed oils can have higher acidity than solvent-extracted oils under similar raw material conditions [34]. This supports the broader observation that harsher, more energy-intensive extraction methods can negatively impact acidity/FFA levels, as seen with MAE increasing FFA compared to UAE in acorn oil extraction [33].

The peroxide value (PV) varied depending on the species and extraction method, as shown in

Table 3. Peroxide value of oils obtained by different extraction methods.

Oak Species	Peroxide Value (mEq O2/kg Oil)
	Soxhlet Extraction	Cold Hexane Extraction	Mechanical Pressing
Q. rubra	1.10 ± 0.08 Bb	1.23 ± 0.10 Bb	2.89 ± 0.10 a
Q. robur	2.78 ± 0.12 Aa	2.37 ± 0.08 Ab	–

Different lowercase letters (a,b) denote significant differences among extraction methods within the same oak species, while different uppercase letters (A,B) denote significant differences between oak species within the same extraction method (Tukey’s HSD, α = 0.05).

. For oils extracted with solvents, Q. rubra exhibited lower PVs (ranging from 1.10 to 1.23 mEq O₂/kg oil), whereas Q. robur had higher values (2.37 to 2.78 mEq O₂/kg oil). In Q. rubra, mechanical pressing significantly increased PV to 2.89 mEq O₂/kg oil, compared to solvent extraction. For Q. robur, Soxhlet extraction resulted in slightly higher PV than cold hexane extraction.

PV remained relatively low across all samples (1.10–2.89 mEq O₂/kg oil), well below edible oil limits (i.e., 10 mEq O₂/kg) [35]. The higher PV in pressed oil correlates with increased oxidative stress during pressing, due to greater oxygen exposure and frictional heating, even in cold-press processes [34]. This localized heating and significant matrix disruption accelerate hydroperoxide formation, possibly explaining the higher PV in pressed Q. rubra compared to solvent extraction. Similar PV levels have been reported for conventional acorn oils: in Q. brantii, the conventional method yielded PV around 2.89 mEq O₂/kg, while assisted methods such as UAE/MAE produced slightly lower PVs (2.48–2.65 mEq O₂/kg). This suggests shorter processing times and reduced thermal/oxidative exposure can help limit primary oxidation [33].

The PV levels observed here were below the commonly cited range for fresh edible oils and below Codex maximum limits (≤10 mEq O₂/kg for refined oils; ≤15 mEq O₂/kg for cold-pressed/virgin oils), indicating minimal primary oxidation at the time of analysis [36]. The low PV can be due to prompt harvesting at physiological maturity, limited oxygen and light exposure during extraction, and high oleic acid content, especially in Q. rubra, combined with natural lipophilic antioxidants that transfer into the oil. Since PV only measures primary oxidation, a full quality assessment should also include secondary oxidation markers like p-anisidine value, volatile products, and shelf-life studies under realistic conditions.

Storage studies show that acorn oils maintain relatively low PV over time: after 180 days, PV ranged from 1.46 to 1.94 mEq O₂/kg, with no significant differences among species, though they increased slightly from initial levels [37]. This stability is often attributed to natural antioxidants, such as phenolics and tocopherols, which help slow peroxide buildup. Extracts from acorns have demonstrated their ability to reduce peroxide levels in other lipid systems [35,37].

The fatty acid profiles showed significant differences between species, with the extraction method having a relatively minor impact (

Table 4. Fatty acid composition (%) of oils obtained by different extraction methods.

Fatty Acid	Q. rubra Mechanical Pressing	Q. rubra Cold Hexane Extraction	Q. rubra Soxhlet Extraction	Q. robur Cold Hexane Extraction	Q. robur Soxhlet Extraction
C14:0	0.11 ± 0.03 a*	0.12 ± 0.01 a	0.11 ± 0.02 a	0.08 ± 0.01 a	0.10 ± 0.03 a
C16:0	9.05 ± 0.24 c	10.06 ± 0.95 b	9.85 ± 0.25 bc	13.36 ± 0.18 a	13.09 ± 0.35 a
C16:1	0.20 ± 0.01 b	0.23 ± 0.04 b	0.21 ± 0.01 b	0.27 ± 0.03 a	0.24 ± 0.01 ab
C18:0	2.37 ± 0.06 a	2.16 ± 0.10 b	2.12 ± 0.01 b	1.78 ± 0.08 c	1.89 ± 0.01 c
C18:1	64.59 ± 1.44 a	64.02 ± 0.78 a	64.22 ± 0.18 a	49.48 ± 0.93 c	52.08 ± 0.04 b
C18:2 n-6	22.26 ± 1.22 c	22.13 ± 0.05 c	22.19 ± 0.06 c	32.11 ± 0.89 a	30.26 ± 0.35 b
C18:3 n-3	0.39 ± 0.01 c	0.38 ± 0.01 c	0.38 ± 0.01 c	1.93 ± 0.06 a	1.34 ± 0.01 b
C20:0	0.51 ± 0.01 a	0.42 ± 0.08 b	0.43 ± 0.01 b	0.42 ± 0.03 b	0.52 ± 0.01 a
C20:1	0.56 ± 0.01 a	0.51 ± 0.09 a	0.52 ± 0.01 a	0.61 ± 0.10 a	0.52 ± 0.01 a
SFA	12.04 ± 0.23 b	12.75 ± 0.78 b	12.50 ± 0.27 b	15.64 ± 0.11 a	15.60 ± 0.33 a
MUFA	65.35 ± 1.41 a	64.75 ± 0.83 a	64.95 ± 0.19 a	50.36 ± 1.00 c	52.84 ± 0.03 b
PUFA	22.65 ± 1.23 c	22.51 ± 0.04 c	22.56 ± 0.06 c	34.04 ± 0.83 a	31.60 ± 0.34 b
PUFA/SFA	1.88 ± 0.07 c	1.77 ± 0.11 c	1.81 ± 0.04 c	2.18 ± 0.04 a	2.03 ± 0.06 b
n-6/n-3	57.06 ± 1.05 a	58.27 ± 2.30 a	59.17 ± 1.29 a	16.65 ± 0.95 c	22.58 ± 0.50 b
AI	0.11 ± 0.01 c	0.12 ± 0.01 b	0.12 ± 0.01 bc	0.16 ± 0.01 a	0.16 ± 0.01 a
TI	0.26 ± 0.01 b	0.28 ± 0.02 b	0.27 ± 0.01 b	0.32 ± 0.01 a	0.33 ± 0.01 a
h/H	9.53 ± 0.30 a	8.54 ± 0.97 b	8.72 ± 0.27 ab	6.21 ± 0.09 c	6.35 ± 0.19 c

* Different lowercase letters (a–c) denote significant differences among treatments (Tukey’s HSD, α = 0.05). Abbreviations: PUFA—polyunsaturated fatty acids; MUFA—monounsaturated fatty acids; SFA—saturated fatty acids; AI—atherogenicity index; TI—thrombogenicity index.

). Overall, species identity influenced fatty acid composition more than the extraction technique. Oleic acid (C18:1) was the predominant fatty acid across all samples. Oils from Q. rubra contained notably higher oleic acid levels (64.02–64.59%) compared to Q. robur (49.48–52.08%). Conversely, Q. robur oils had higher amounts of linoleic acid (C18:2 n-6; 30.26–32.11%) and α-linolenic acid (C18:3 n-3; 1.34–1.93%) than those from Q. rubra (22.13–22.26% and 0.38–0.39%, respectively). Total monounsaturated fatty acids (MUFA) were significantly greater in Q. rubra (64.75–65.35%) than in Q. robur (50.36–52.84%), while total polyunsaturated fatty acids (PUFA) were higher in Q. robur (31.60–34.04%) compared to Q. rubra (22.51–22.65%). Saturated fatty acids (SFA) were also more prevalent in Q. robur (15.60–15.64%) than in Q. rubra (12.04–12.75%). Consequently, the PUFA/SFA ratio was significantly higher in Q. robur, and the n-6/n-3 ratio was notably higher in Q. rubra.

Lipid quality indices reflected these species differences, i.e., oils from Q. robur showed significantly higher atherogenicity (AI = 0.16) and thrombogenicity (TI = 0.32–0.33) indices than those from Q. rubra (AI = 0.11–0.12 and TI = 0.26–0.28). Conversely, the hypocholesterolemic/hypercholesterolemic ratio (h/H) was considerably higher in Q. rubra (8.54–9.53) than in Q. robur (6.21–6.35). Within each species, the extraction method had minimal impact on these indices, with statistically significant but relatively small differences mainly observed in Q. rubra.

In Quercus oils, oleic acid (C18:1) is typically the predominant fatty acid, with palmitic (C16:0) and linoleic (C18:2) constituting the remaining composition. This aligns with current study findings, where oleic acid was prevalent in both species, and the combined levels of C16:0, C18:1, and C18:2 represented most of the total fatty acids. Similar patterns have been observed in Q. rubra and Q. robur, where oleic, linoleic, and palmitic acids together made up approximately 94–98% of detected fatty acids [31]. The observed species effect (higher C18:1 in Q. rubra and higher C18:2 and C18:3 in Q. robur) agrees well with published comparative datasets. Górnaś et al. [31] reported that Q. rubra oils were richer in oleic acid (C18:1 range 55.7–65.7%) and poorer in linoleic acid (C18:2 range 24.1–34.2%) and palmitic acid (C16:0 range 7.5–8.5%) than Q. robur, while Q. robur contained distinctly more α-linolenic acid (C18:3 range 1.2–3.0%) than Q. rubra (0.3–0.4%).

Comparisons with other Quercus species reveal that acorn oils range from oleic-rich to more linoleic-rich profiles, with the main fatty acids remaining quite similar. For instance, Q. brantii oil has been reported to contain approximately 63–64% oleic acid, about 17–18% linoleic acid, around 15–16% palmitic acid, and roughly 2–2.5% stearic acid [33]. Similarly, oils from Mediterranean oaks (Q. ilex, Q. suber, Q. coccifera) were characterized by palmitic acid (~12%), stearic acid (~2.7%), oleic acid (~67.5%), and linoleic acid (~15%) as the predominant fatty acids, indicating a strongly oleic acid–dominated profile [37]. Additionally, these studies indicate that fatty acid proportions can vary within Quercus due to geographic origin, local growing conditions, genetic/genotypic variability, and the time of sampling (year and harvest date), alongside species identity [31,32,33,34,35,36,37].

Oxidative stability parameters measured by PDSC varied considerably between different oak species and extraction methods (

Table 5. PDSC oxidation parameters of oils obtained by different extraction methods.

Oak Species	Onset Oxidation Time (τon, Min)
	Soxhlet Extraction	Cold Hexane Extraction	Mechanical Pressing
Q. rubra	126.11 ± 1.22 Ba	106.29 ± 1.11 Bb	99.78 ± 0.67 c
Q. robur	212.95 ± 2.56 Aa	149.41 ± 1.25 Ab	–
Oak species	Maximum oxidation time (τmax, min)
	Soxhlet Extraction	Cold Hexane Extraction	Mechanical Pressing
Q. rubra	136.38 ± 1.43 Ba	117.12 ± 1.16 Bb	110.44 ± 0.87 c
Q. robur	227.92 ± 2.43 Aa	156.04 ± 0.86 Ab	–

Different lowercase letters (a–c) denote significant differences among extraction methods within the same oak species, while different uppercase letters (A,B) denote significant differences between oak species within the same extraction method (Tukey’s HSD, α = 0.05).

). For both solvent-based techniques, oils from Q. robur exhibited significantly higher τ_on and τ_max values than those from Q. rubra, indicating a strong species effect. Specifically, in Soxhlet extraction, τ_on and τ_max for Q. robur reached 212.95 and 227.92 min, while Q. rubra registered 126.11 and 136.38 min for these parameters. A similar pattern was observed with cold hexane extraction, with τ_on and τ_max measuring 149.41 and 156.04 min for Q. robur, versus 106.29 and 117.12 min for Q. rubra.

Within Q. rubra, the extraction method significantly influenced oxidative stability. Soxhlet extraction resulted in the highest τ_on and τ_max (126.11 and 136.38 min), followed by cold hexane extraction (106.29 and 117.12 min). Mechanical pressing yielded the lowest stability markers (τ_on = 99.78 min and τ_max = 110.44 min). For Q. robur, Soxhlet extraction also provided notably higher oxidative stability than cold hexane extraction, increasing τ_on from 149.41 to 212.95 min and τ_max from 156.04 to 227.92 min.

The notable species effect seen in the PDSC measurements, i.e., higher τ_on and τ_max in Q. robur oils from both solvent-based extractions, suggests that oxidative resistance in the lipid phase depends largely on species-specific compositional features beyond simple bulk indices. Crucially, the extraction method also influenced stability within each species, with Soxhlet extraction being more effective than cold hexane or pressing in Q. rubra. This aligns with the idea that more thorough solvent extraction can shift the balance of oxidation-promoting and protecting minor constituents in the oil. Findings from assisted extraction studies on Q. brantii reinforce that extraction conditions can modify the recovery of phenolics and markers associated with oxidative stability, highlighting that stability depends not only on the native matrix but also on how the oil is released from it [33].

To better understand the factors contributing to the observed differences in PDSC-derived oxidative stability between species, measurements of bioactive compounds and antioxidant activity were conducted on whole acorns (

Table 6. Bioactive compounds and antioxidant activity of acorn kernels.

Oak Species	TPC (mg CGA/g DW)	TFC (mg QE/g DW)	DPPH (mmol TE/g DW)	ABTS (mmol TE/g DW)
Q. rubra	79.43 ± 2.09 a	0.69 ± 0.09 a	0.34 ± 0.01 a	0.53 ± 0.02 a
Q. robur	49.28 ± 0.38 b	0.41 ± 0.05 b	0.21 ± 0.03 b	0.37 ± 0.03 b

Different lowercase letters (a–b) denote significant differences between oak species (Tukey’s HSD, α = 0.05). Abbreviations: TPC—total phenolic content; TFC—total flavonoid content; DPPH—2,2-diphenyl-1-picrylhydrazyl radical scavenging activity; ABTS—2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation scavenging activity; CGA—chlorogenic acid equivalents; QE—quercetin equivalents; TE—Trolox equivalents; DW—dry weight.

). There were significant variations in phenolic composition and radical scavenging capacity among the species. Specifically, Q. rubra acorns exhibited a higher total phenolic content (TPC), measuring 79.43 mg CGA/g DW, compared to 49.28 mg CGA/g DW in Q. robur. Additionally, Q. rubra showed a substantially higher total flavonoid content (TFC) of 0.69 mg QE/g DW, versus 0.41 mg QE/g DW in Q. robur.

Consistent with the phenolic component differences, antioxidant activity measured by both assays was notably higher in Q. rubra extracts than in Q. robur. The DPPH radical scavenging activity was 0.34 mmol TE/g DW for Q. rubra, compared to 0.21 mmol TE/g DW for Q. robur. A similar trend appeared in the ABTS assay, with values of 0.53 mmol TE/g DW for Q. rubra and 0.37 mmol TE/g DW for Q. robur (Table 6). Overall, all tested bioactive and antioxidant parameters clearly differentiated the two oak species.

The link between antioxidant metrics and actual oxidative stability is not always clear. In this research, Q. rubra kernel extracts had higher TPC/TFC and better radical scavenging activity (DPPH, ABTS) than Q. robur, yet Q. robur oils were notably more stable in PDSC tests. Similar caution applies to stored acorn oils, where antioxidant activity tests gave different results: DPPH more reliably aligned with TPC, whereas ABTS occasionally diverged from TPC trends. This shows that different assays can highlight different antioxidant chemistries and may not match lipid oxidation behaviors equally [37].

Research on Quercus oils indicates that the crucial stabilizing antioxidants are those actually present in the oil fraction, rather than necessarily being the most abundant in the entire acorn extract. In Algerian Quercus oils, the phenolic component mainly consisted of hydrolysable tannin derivatives. Both antioxidant activity (measured by DPPH and ABTS assays) and pigment profiles, characterized by low chlorophyll levels and relatively high carotenoids, were discussed as factors influencing oxidation resistance under relevant conditions [32]. In Q. ilex oil, a high total phenolic content was clearly associated with enhanced oxidative stability, as demonstrated by the Rancimat oil stability index alongside TPC and DPPH results. This reinforces the idea that phenolics within the oil can play a significant role when they partition into the lipid phase [38].

TPC/TFC and radical-scavenging assays were conducted on hydroalcoholic kernel extracts, which primarily contain polar constituents like tannin-related phenolics [15]. These compounds are likely mainly found in polar fractions and only partially in non-polar oil fractions. However, phenolic compounds identified in Quercus oils include hydrolysable tannin derivatives, suggesting that small amounts can be present in the oil and may affect its properties [32].

Meanwhile, the greater PDSC stability of Q. robur oils, despite having lower total phenolics (TPC/TFC) and DPPH/ABTS values in the whole acorn, can likely be attributed to species-specific differences in lipophilic antioxidant systems that are not captured by bulk phenolic measurements. Notably, Q. robur acorn oils have been reported to contain much higher total tocopherol levels than Q. rubra oils, about 6 times on average, with Q. robur mainly containing γ-tocopherol, a strong chain-breaking antioxidant during lipid oxidation. In contrast, Q. rubra exhibits significantly lower total tocopherols and an unusual dominance of β-tocopherol [31]. This species-dependent tocopherol advantage in Q. robur may compensate for, or even outweigh, the lower antioxidant capacity observed in polar extracts, resulting in higher τ_on and τ_max in PDSC despite the lower whole-acorn TPC/TFC. In summary, the overall pattern indicates that PDSC stability depends on the balance of antioxidants and pro-oxidants within the oil itself, and increasing extraction severity can enhance PDSC performance by either facilitating the transfer of oil-relevant antioxidants or decreasing co-extraction of destabilizing impurities [31,37].

Previous research indicates that acorn oils are rich in tocopherols, with considerable variation across species. For instance, Q. rubra acorn oil primarily contains β-tocopherol, whereas Q. robur oil generally has higher levels of γ-tocopherol, along with notable differences in total tocopherol content [31,32]. These lipophilic antioxidants, including tocopherols and oil-phase phenolics, can transfer into the extracted oil and are likely to influence oxidation rates and PDSC-based stability.

To integrate these interrelated compositional, quality, and stability variables and visualize overall similarities among samples, multivariate analyses (HCA and PCA) were applied to the standardized dataset. Hierarchical clustering of the standardized data revealed a distinct separation of samples based on oak species (Figure 2). Oils from Q. rubra formed one group, while those from Q. robur constituted a different cluster, highlighting that species was the primary factor influencing differences.

Within the Q. rubra group, oils obtained through mechanical pressing showed more differences from solvent-extracted samples, mainly in peroxide value and oxidative stability parameters. Variables associated with oxidative stability and saturated fatty acids grouped with Q. robur, whereas MUFA, oil yield, and the h/H ratio were linked to Q. rubra samples.

Furthermore, principal component analysis (PCA, Figure 3) corroborated the clustering pattern observed in HCA (Figure 2). The first two principal components accounted for 94.20% of the total variance (PC1 = 83.31%, PC2 = 10.89%). PC1 distinctly separated samples by species: Q. rubra oils were positioned on the positive side of PC1, whereas Q. robur oils were on the negative side. PC1 showed positive correlations with MUFA, n-6/n-3 ratio, h/H, and oil yield, and negative correlations with SFA, PUFA, AV, AI, TI, and parameters related to oxidative stability (τ_on and τ_max). PC2 primarily related to peroxide value and helped distinguish mechanically pressed Q. rubra oil from other Q. rubra samples. Overall, the multivariate analyses consistently indicated that oak species was the main factor affecting oil yield, fatty acid composition, quality indices, and oxidative stability, with the extraction method playing a secondary role.

4. Conclusions

Acorn oils from pedunculate oak (Q. robur L.) and northern red oak (Q. rubra L.) exhibit significant differences, emphasizing a strong influence of species on oil yield, physicochemical properties, fatty acid profile, and oxidative stability. Q. rubra consistently yields more oil than Q. robur across various extraction methods. Conversely, oils from Q. robur show higher PDSC-derived oxidation times (τ_on and τ_max), indicating better oxidative stability. The extraction technique impacts both yield and quality, particularly in Q. rubra, where Soxhlet extraction achieves the highest yield and stability. Mechanical pressing yields less oil and results in higher acid and peroxide values compared to solvent extraction, confirming that processing conditions can affect product quality.

Fatty acid composition and lipid health indices are primarily determined by species, with only minor effects from extraction methods on fatty acid ratios and related metrics. Antioxidant parameters like TPC, TFC, DPPH, and ABTS in acorn kernels do not directly correlate with oil stability, implying that stability depends mainly on the balance between pro-oxidants and antioxidants in the oil phase, transferred during extraction, rather than total polar-extract antioxidant capacity. Overall, acorns serve as a viable, species-specific raw material for oil production. Proper selection of oak species and extraction method is crucial to maximize oil yield while preserving quality and oxidative stability. From an application perspective, industrial feasibility will also depend on raw-material availability and interannual variability in acorn crops, as well as on collection and processing logistics.

This study offers a clear, comparative dataset for two Quercus species under specific laboratory extraction conditions, establishing a baseline for their physicochemical and oxidative stability in acorn oils. Although focused on a single harvest season and region, the observed differences between species and the effects of methods provide a strong basis for expanding research to include additional seasons, locations, and processing scales. Future work could enhance this dataset by directly analyzing minor oil components that influence stability, such as tocopherols and other antioxidants, standard thermal performance measures like smoke point, sensory evaluations, and broader quality assessments under typical storage conditions, including secondary oxidation markers and safety checks relevant to food use. These follow-up studies would improve application-specific guidance and facilitate the translation of the advantageous species-specific traits into scalable practices.