Impact of Sonication Duration on Oil Yield, Phenolic and Flavonoid Contents, Antioxidant Capacity, Fatty Acid Profile, and Phenolic Composition of Bitter and Sweet Lupine (Lupinus albus L.) Seeds

TL;DR

This study examines how sonication time affects oil and nutrient content in bitter and sweet lupine seeds.

Contribution

The study reveals how sonication duration influences phytochemical and lipid profiles in bitter and sweet lupine seeds.

Findings

Sonication increased oil yield in both bitter and sweet lupine seeds.

Bitter lupine seeds had higher phenolic and flavonoid content than sweet ones.

Oleic acid levels in bitter lupine oil increased with longer sonication times.

Abstract

Lupine seeds are not consumed directly because they are bitter. Therefore, differences in the phytochemical, lipidomic, and bioactive properties of bitter and sweetened lupine seeds were revealed using analytical and chromatographic methods. In this study, influence of ultrasound treatment time on extraction efficiency, phenolic and flavonoid contents, antioxidant capacity, fatty acid and phytochemical profile of bitter and sweet lupine (Lupinus spp.) seeds was investigated. The oil quantities of the bitter and sweet lupine seeds were specified to be between 8.60% (control) and 9.90% (5 and 10 min) to 11.0% (25 min) and 13.00% (5 min), respectively. The total phenolic content of bitter lupine seeds ranged from 124.32 mg gallic acid equivalent (GAE)/100 g (15 min) to 142.0 mg GAE/100 g (control), while sweet lupine seeds ranged from 4.21 to 6.61 mg GAE/100 g, respectively. Total flavonoids in bitter seeds varied between 35.00 mg/100 g (control) and 52.30 mg/100 g (15 min), whereas sweet seeds contained between 5.00 and 9.92 mg/100 g. Gallic acid, catechin and kaempferol were detected as the predominant phenolic compounds in sweet and bitter lupine seeds. Oleic acid quantities of bitter and sweet lupine oils were demonstrated to be between 36.78% (25 min) and 52.47% (15 min) to between 55.89% (15 min) and 56.69%, respectively. The phenolic compound amounts of bitter lupine were slightly higher than those of sweet lupine seeds. The highest phenolic compounds detected in both lupine seeds were catechin, kaempferol and rutin. As the sonication time increased, the amount of linoleic acid in bitter lupine oil became higher than in the control sample. In contrast, sweet lupine oil showed a slight decrease in linoleic acid content compared to its control.

Full text

1. Introduction

Lupines are annual legumes belonging to the genus Lupinus within the Fabaceae family. More than 400 species have been identified [1], originating from the Mediterranean region, North Africa, and both North and South America [2,3,4]. Lupines are regarded as soil-improving crops because they can thrive in a wide range of soil and climatic conditions where many other crops fail. Like other legumes, they enhance soil fertility by fixing atmospheric nitrogen through a symbiotic relationship with Rhizobium bacteria, making the fixed nitrogen available to subsequent crops [1]. The fruit of the lupine plant is a pod that splits open along two seams. In Türkiye, the lupine plant is generally called “termiye” [3,4].

Lupine seeds are consumed as snacks, added to salads and side dishes, and processed into flour. Lupine flour is widely used in the production of various food products, including plant-based milk, cheese and meat alternatives, fermented foods, soup, salad, milk, meat and soybean substitutes, mayonnaise, chips, noodles, pasta, and baked goods [3,5]. Due to its high protein content, lupine flour is regarded as an excellent ingredient for enriching a wide range of food products [6,7]. It is frequently used as an egg replacer in baked goods such as cakes, pancakes, biscuits, and brioche [8]. As lupine is naturally gluten free, it is also employed as a functional ingredient in gluten-free formulations [9].

The importance of chemical properties, phenolic acids and flavonoids found in lupine seeds has increased significantly [10,11,12,13,14]. Andean lupine (Lupinus mutabilis) seeds are used as ingredients in the food and pharmaceutical industries due to their high protein and lipid contents, and the protein, lipid and ash contents of Andean lupine grains are 32.0–46.9, 13.6–18.6 and 2.7–4.4 g/100 g (dw), respectively [13]. Higher oil contents have also been reported in the literature. For example, Stone et al. [15] determined a 59.1 to 96.3 g/kg lipid content with an observed average value of 24 g/kg lipid, while certain legume genotypes were found to reach levels as high as 20 g/100 g [16]. Lupine oil is distinguished by its low proportion of saturated fatty acids (10–27%) and its high content of unsaturated fatty acids (78–87%). The predominant fatty acids are oleic acid (25–63%), linoleic acid (13–57%), and linolenic acid (3–11%) [17,18,19,20,21]. The seed hulls are primarily composed of cellulose, hemicellulose, and pectin, whereas oligosaccharides are mainly concentrated in the cotyledons [22]. In comparison with many other legumes, lupine seeds contain relatively low levels of antinutritional factors such as trypsin inhibitors, lectins, and saponins [23]. They also have a low phytic acid content (0.25–0.62 g/100 g in white lupine seeds), which is noteworthy because phytic acid can reduce mineral bioavailability by chelating cations [23,24].

The principal antinutritional components in lupine seeds are quinolizidine alkaloids [25]. These compounds impart a bitter taste and may be toxic; therefore, they must be removed through appropriate processing before consumption. To address this issue, plant breeding efforts have led to the development of varieties with genetically reduced alkaloid content [26,27]. Debittering is typically carried out by extracting alkaloids from whole lupine seeds using water as the solvent. However, this process also affects the seeds’ overall chemical composition. In addition to alkaloids, various water-soluble components—such as minerals, simple carbohydrates, and oligosaccharides—are partially removed. As a result, the relative proportions of lipids and proteins increase, mainly due to the concentration effect caused by the loss of these soluble compounds [28,29].

Although alkaloids obtained from lupines are toxic in human nutrition, they can be used in medical applications due to their antiarrhythmic, hypolipidemic, hypoglycemic, hypotensive, anticarcinogenic, anti-obesity and hypocholesterolemic capacities [30,31,32]. In addition, lupine seeds have phenolic compounds and prebiotic oligosaccharides that support the growth of bifidobacteria [33].

Lupine seeds are widely utilized as a valuable protein source in both animal feed and human diets across many regions of the world. Their popularity stems not only from their high nutritional quality but also from the plant’s ability to thrive in marginal soils and under challenging climatic conditions. In recent years, the consumption of lupin-based foods by humans has shown a marked increase [6]. Beyond their nutritional applications, lupines serve multiple agricultural and environmental purposes. They are used for pasture enhancement, ornamental planting, erosion control, and soil stabilization [34,35]. Additionally, they function as green forage and green manure, while contributing to soil fertility through atmospheric nitrogen fixation [34,35,36].

Ultrasonic-assisted extraction has great importance in the food and herbal product industry due to its practicality, cost-effectiveness, time and energy saving, low temperature extraction and preservation of the quality of the extract. It has been reported that an ultrasonic extraction system increases the extraction efficiency of bioactive compounds to a greater extent than the traditional extraction method [23,24,25]. It has also been stated that an ultrasonic-assisted extraction method increases the oil yield extracted from seeds [26]. Chemical effects produced during acoustic cavitation can be used advantageously to increase the functionality of certain food compounds [27]. The most important components of lupine seeds among the health-beneficial chemical compounds are bioactive compounds. Research has also explored the incorporation of lupine into cereal-based products as a partial substitute for traditional ingredients. In order for lupine seeds to be consumed directly as food, the bitter compounds they contain must first be removed. Therefore, in this study, after the bitterness of bitter lupines was eliminated with tap water, the seeds were roasted for different durations, and the differences in the phytochemical contents of both bitter and sweet lupine seeds needed to be determined. The present study aimed to evaluate the effect of varying sonication durations on oil yield, total phenolic content, total flavonoid content, antioxidant activity, fatty acid composition, and phenolic compound profile in both bitter and sweet lupine seeds.

2. Material and Methods

2.1. Material

The bitter and sweet lupine seeds (Lupinus albus L.) used in this study were supplied by a producer cultivating in Doğanhisar, Konya. Lupine seeds were first separated into two groups. One of these lupine groups was soaked in tap water for 5 days to sweeten it, with the water changed every 6 h. This removed the bitterness and sweetened the lupine seeds, and this group was used as sweet lupine in this study. The lupines were then air-dried and ground to pass through a 0.5 mesh sieve for analysis. Doğanhisar, where the lupine seeds are sourced, is a district in the Konya province. The geographical coordinates of Doğanhisar in the Konya province are as follows: Latitude: 38.145951, Longitude: 31.676744.

As reagents, methanol, n-hexane, NaNO_2_, AlCl_3_, petroleum ether, and NaOH, all produced by Merck (Darmstadt, Germany), were used in this study. Additionally, Folin–Ciocalteu and DPPH, both produced by Sigma company (Milwaukee, WI, USA), were used in this study.

2.2. Methods

2.2.1. Moisture Content

The moisture quantities of lupine seed samples were defined by the KERN & SOHN GmbH (Balingen-Frommern, Germany) infrared moisture analyzer [37]. The moisture contents of bitter and sweet lupine seeds were 8.17 ± 0.59 and 7.43 ± 0.38%, respectively.

2.2.2. Oil Extraction

The powdered lupine seed samples (5 g) were placed on filter paper and combined with 150 mL of petroleum ether in individual flasks. Each flask was subjected to ultrasonication (35 kHz; Bandelin Sonorex, Berlin, Germany) for 5, 15, or 25 min. Following this pre-sonication step, the solvent was removed by evaporating at 50 °C. With the Soxhlet system, only the oil of the control group was obtained with petroleum ether at 50 °C for 5 h [2].

2.2.3. Fatty Acid Composition of Bitter and Sweet Lupine Oils

Fatty acid methyl esters of samples of lupine oil esterified according to the method of Multari et al. [38] were analyzed by gas chromatography (Shimadzu, GC-2010, Kyoto, Japan) equipped with flame-ionization detector (FID) and capillary column (Tecnocroma, Barcelona, Spain TR-CN100, 60 m × 0.25 mm, film thickness: 0.20 µm). The temperature of the injection block and detector was 260 °C. The mobile phase was nitrogen with a 1.51 mL/min flow rate. Total flow rate was 80 mL/min and split rate was also 1/40. Column temperature was programmed at 120 °C for 5 min and increased to 240 °C at 4 °C/min and held for 25 min at 240 °C.

2.2.4. Calculation of Lipid Indices of Bitter and Sweet Lupine Oils

Nutritive value index, calculated oxidizability value (Cox), oleic (ODR) and linoleic desaturation ratio (LDR) values of lupine seed oils were established according to the reports recommended by Chen et al. [39], Fatemi and Hammond [40] and Pleines and Friedt [41].

2.2.5. Extraction of Bioactive Compounds

Lupine seed samples were extracted according to Jakopic et al. [42] with some modifications. Ground samples (3 g) were added to 10 mL of methanol:water (70:30, v/v). Temperature control was implemented during the sonication process, and the extraction was performed at 30 °C. The mixture was kept in an ultrasonic water bath (Bandelin Sonorex 35 Khz) for 0 min (control), 5 min, 15 min, and 25 min, followed by centrifugation at 6000 rpm for 10 min. The supernatants were evaporated at 40 °C. The dried extracts were dissolved in 10 mL of methanol. A 1 g sample of lupine seed oil was combined with 10 mL of methanol:water (70:30, v/v). The mixture was ultrasonically treated in a water bath for 0 (control), 5, 15 and 25 min, and then centrifuged at 6000 rpm (Hermle Z-200A-Wehingen, Germany) for 10 min. The resulting supernatants were washed three times with 5 mL of n-Hexane. Before injection, the extract was filtered [43].

2.2.6. Total Phenolic Content

Total phenolic contents of lupine extracts were characterized using the Folin–Ciocalteu (FC) reagent according to the report suggested by Yoo et al. [44]. FC (1 mL) and Na_2_CO_3_ (10 mL) were added to the extracts and mixed by vortexing. The deionized water was added until the final volume was 25 mL, and kept at dark for 1 h. The absorbance was measured at 750 nm in a spectrophotometer (Shimadzu UV mini 1240, Kyoto, Japan). A calibration curve was prepared with gallic acid (0–200 mg/mL) as the standard. The results are shown as mg gallic acid equivalent (GAE)/100 g.

2.2.7. Total Flavonoid Content

The total flavonoid content of the lupine seeds was determined according to the method described by Hogan et al. [45]. Briefly, 0.3 mL of NaNO_2_, 0.3 mL of AlCl_3_, and 2 mL of NaOH were added to 1 g of the extract, and the reaction mixture was incubated in the dark for 15 min. Subsequently, the absorbance was measured at 510 nm using a spectrophotometer (Shimadzu UV mini 1240, Japan).

2.2.8. DPPH Free Radical Scavenging Activity

The antioxidant activities of lupine seed extracts were characterized using 1,1-diphenyl-2-picrylhydrazyl (DPPH) according to the study proposed by Lee et al. [46]. The extract was mixed with 2 mL methanolic solution of DPPH. After shaking vigorously, it was stored at room temperature for 30 min. The absorbance was recorded at 517 nm by using a spectrophotometer. The results were given as mmol trolox equivalent (TE)/kg.

2.2.9. Identification and Quantification of Phenolic Compounds

Analysis and chromatographic separation of phenolic compounds of the lupine seed samples was carried out with HPLC (Shimadzu, SCL-10A VP-Shimadzu, Kyoto, Japan) equipped with a PDA detector and an Inertsil ODS-3 (5 µm; 4.6 × 250 mm) column. The phenolic compounds used in this study were gallic acid, 3,4-dihydroxybenzoic acid, catechin, caffeic acid, syringic acid, rutin, p-coumaric acid, ferulic acid, resveratrol, quercetin, cinnamic acid, and kaempferol. The mobile phase was a mixture of 0.05% acetic acid in water (A) and acetonitrile (B) with a flow rate of 1 mL/min at 30 °C. The injection volume was 20 µL. The peaks were taken at 280 using a PDA detector. The elution program was employed: 0–0.10 min 8% B; 0.10–2 min 10% B; 2–27 min 30% B; 27–37 min 56% B; 37–37.10 min 8% B; 37.10–45 min 8% B. The total running time per sample was 60 min.

2.3. Statistical Analysis

JMP version 9.0 was used for analysis of variance (ANOVA). The results are mean ± standard deviation (MSTAT C) of two independent lupine types and sonication times.

3. Results and Discussion

3.1. The Oil Contents, Total Phenol and Flavonoid Contents and Antioxidant Activity Values of Lupine Seeds and Oils

3.1.1. Oil Content of Bitter and Sweet Lupine Seeds

Table 1 presents the oil content of lupine seeds and oils subjected to sonication for varying durations. The findings varied depending on the type of lupine and the sonication time. The oil contents of bitter and sweet lupine seeds ranged from 8.60% (control) to 9.90% (after 5 and 10 min) and up to 11.0% (after 25 min) and 13.00% (after 5 min), respectively. Overall, the highest oil extraction from lupine seeds occurred at 5 min of sonication, likely due to the weakening of the cell walls surrounding the oil droplets under ultrasonic wavelengths.

3.1.2. Total Phenol and Flavonoid Contents of Lupine Seeds and Oils

The total phenolic contents in bitter and sweet lupine seeds treated with different sonication times were characterized to be between 124.32 mg GAE/100 g (at 15 min) and 142.0 mg GAE/100 g (control) to 4.21 mg GAE/100 g (at 15 min) and 6.61 mg GAE/100 g (control), respectively. Additionally, the total flavonoid contents of bitter lupine seeds varied between 35.00 mg/100 g (control) and 52.30 mg/100 g (15 min), whereas sweet lupine seeds exhibited values between 5.00 mg/100 g (15 min) and 9.92 mg/100 g (control). The influence of sonication time on total oil content, total phenolic content, total flavonoid content, and antioxidant activity in bitter and sweet lupine seeds demonstrated significant differences between the two lupine varieties, suggesting a pronounced interaction between seed composition and the duration of ultrasonic treatment. Sonication significantly influenced the total oil content of both lupine types, although the response patterns differed. In bitter lupine seeds, sonication increased oil content from 8.60% in the control to 9.90% after 5 and 15 min of treatment, suggesting that ultrasonic cavitation effectively disrupted cellular structures and enhanced oil release. However, extending the sonication time to 25 min resulted in a slight decrease in oil content, indicating that prolonged ultrasonication did not further improve extraction efficiency. This may be attributed to oil re-emulsification or structural collapse of the seed matrix at longer exposure times. In sweet lupine seeds, a similar initial increase in oil content was observed, with the highest value recorded at 5 min (13.00%). Nevertheless, longer sonication times led to a progressive reduction in oil content, highlighting that excessive ultrasonication negatively affected oil recovery in sweet lupine. These results suggest that short sonication times are sufficient to maximize oil extraction, particularly in sweet lupine seeds. Total phenolic content (TPC) exhibited a contrasting trend compared to oil yield, especially in bitter lupine seeds. The highest TPC was observed in the control sample (142.00 mg/100 g), while sonication resulted in a significant reduction in phenolic content with increasing treatment time. The decline in TPC may be associated with the susceptibility of phenolic compounds to ultrasonic-induced degradation, possibly due to localized heating, free radical formation, or structural breakdown of phenolic molecules during prolonged sonication. In sweet lupine seeds, TPC values were considerably lower than those of bitter lupine across all treatments. Although minor fluctuations were observed, sonication generally caused a decrease in phenolic content, indicating limited extractability and low phenolic stability in sweet lupine seeds. In contrast to phenolic compounds, total flavonoid content (TFC) in bitter lupine seeds showed a clear enhancement at moderate sonication durations. The highest flavonoid concentration was recorded after 15 min of sonication, representing a substantial increase compared to the control. This suggests that moderate ultrasonication was effective in releasing flavonoids from the seed matrix without causing extensive degradation. However, the decrease in TFC observed at 25 min indicates that excessive sonication may lead to flavonoid breakdown. In sweet lupine seeds, TFC values were markedly lower and generally decreased following sonication, further emphasizing the limited potential of ultrasonic treatment to enhance flavonoid extraction in this lupine type.

3.1.3. Antioxidant Activity of Lupine Seeds and Oils

Antioxidant activity closely reflected changes in bioactive compound content. Bitter lupine seeds exhibited the highest antioxidant activity after 5 min of sonication, which coincided with increased oil yield and enhanced flavonoid content. However, prolonged sonication, particularly at 15 min, resulted in a significant reduction in antioxidant activity, despite elevated flavonoid levels at this duration. This discrepancy suggests that antioxidant capacity is not solely dependent on flavonoid concentration but also on the integrity and synergistic interactions of various antioxidant compounds. In sweet lupine seeds, antioxidant activity was not detected in any treatment, consistent with their extremely low phenolic and flavonoid contents. Overall, these findings demonstrate that bitter lupine seeds respond more favorably to ultrasonic treatment than sweet lupine seeds in terms of enhancing oil extraction and certain bioactive components. Nevertheless, sonication time plays a critical role, as excessive exposure may lead to degradation of phenolic compounds and reduced antioxidant activity. Therefore, short to moderate sonication durations (5–15 min) appear optimal for bitter lupine seeds, while sweet lupine seeds benefit minimally from sonication and are more susceptible to quality deterioration at longer treatment times. Interestingly, sweet lupine seeds and oils did not show antioxidant activity. In addition, antioxidant activities of the bitter lupine seeds and oils were characterized to be between 0.86 (15 min) and 1.11 mmol/kg (5 min) to 0.91 (control) and 0.92 mmol/kg (5, 15 and 25 min), respectively. Sonication time significantly influenced the total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity of bitter and sweet lupine seed oils, with marked differences between the two varieties. Bitter lupine oil exhibited substantially higher TPC and TFC values than sweet lupine oil under all conditions. Ultrasound treatment enhanced phenolic extraction in bitter lupine oil, with TPC increasing from 12.87 mg/100 g in the control to a maximum of 45.25 mg/100 g at 15 min, whereas prolonged sonication (25 min) led to a decline, likely due to phenolic degradation. In contrast, sweet lupine oil showed very low TPC values (≤1.27 mg/100 g) and no consistent improvement with increasing sonication time, indicating limited phenolic availability and stability. Total flavonoid content decreased with sonication in both oils, particularly in bitter lupine oil, where TFC dropped sharply from 24.29 mg/100 g in the control to 2.38 mg/100 g at 25 min, suggesting high flavonoid sensitivity to ultrasonic conditions. Antioxidant activity was detected only in bitter lupine oil and remained relatively stable (0.91–0.92 mmol/kg) across sonication treatments, despite reductions in TFC, implying that non-flavonoid phenolic compounds contributed predominantly to antioxidant capacity. Overall, moderate sonication effectively enhanced phenolic recovery in bitter lupine oil, whereas extended sonication negatively affected flavonoid stability, and sweet lupine oil showed limited responsiveness to ultrasound-assisted extraction. While the total oil content of bitter lupine was found to be low compared to sweet lupine, the total phenol, total flavonoid contents and antioxidant activity values of bitter lupine were found to be high. In contrast, bioactive properties of bitter and sweet lupine were found to be low. In addition, the total phenol, flavonoid contents and antioxidant activity values of sweet lupine oils were found to be low compared to bitter lupine oil. The low bioactive compound and antioxidant activity values of sweet lupine seeds and oils may probably be due to the removal of a significant portion of the phenolic components with water during debittering. While an increase in the total phenol and flavonoid contents of bitter lupine was observed with the increase in sonication time, a decrease was observed in sweet lupine. No antioxidant activity value was detected in sweet lupine berries and oils. This may be due to the removal of phenolic components showing antioxidant activity with water during debittering. Antioxidant activity values of bitter lupine oils subjected to different sonication times were found to be similar and statistically insignificant. According to previous studies, the oil contents of different types of lupine seeds were determined as 15–20.1% [47], 5–11% [48], 11.34 and 14.64 g/100 g [49], 10.5 g/100 g [50], 15.42 and 18.9 g/100 g, respectively [51,52]. Lupinus albus seeds contained 212.12 and 271.25 mg GAE/100 g total phenol and 1100 mg Catechin/g (dw) total flavonoid [53,54]. Oil contents, total phenol and flavonoid contents and antioxidant activity values of bitter and sweet lupine grains were observed to differ from the results of Erbaş et al. [47], Kahajdova et al. [49], Martinez-Villaluenga et al. [53] and Barba et al. [54]. These differences may have resulted from lupine varieties, whether they are bitter or sweet, sonication, drying, harvest time, cultivation and climatic factors, extraction methods and analytical processes.

3.2. The Phenolic Compounds and Their Amounts of Bitter and Sweet Lupine Seeds and Oils

Table 2 and Table 3 present the phenolic components of bitter and sweet lupine seeds and their oils after varying sonication times. The phenolic profiles of bitter and sweet lupine seeds differed, with bitter lupine exhibiting slightly higher levels than sweet lupine. In bitter lupine seeds, the main phenolic compounds identified were gallic acid, 3,4-dihydroxybenzoic acid, catechin, caffeic acid, p-coumaric acid, and quercetin. In contrast, sweet lupine seeds primarily contained gallic acid, 3,4-dihydroxybenzoic acid, caffeic acid, and rutin. Among all detected phenolics, catechin and rutin were the most abundant in both lupine types. In bitter lupine seeds, catechin and rutin ranged from 1.50 mg/100 g (25 min) to 1.79 mg/100 g (5 min), and from 0.54 mg/100 g (15 min) to 1.98 mg/100 g (5 min), respectively. For sweet lupine seeds, catechin levels varied between 0.13 mg/100 g (25 min) and 0.47 mg/100 g (control), while rutin ranged from 0.12 mg/100 g (control) to 0.40 mg/100 g (5 min). Additionally, 3,4-dihydroxybenzoic acid amounts are measured to be between 0.58 mg/100 g (25 min) to 0.67 mg/100 g (control and 5 min) in bitter lupine, 3,4-dihydroxybenzoic acid amounts of sweet lupine seeds were assessed to be between 0.06 mg/100 g (25 min) to 0.11 mg/100 g (15 min). Overall, the levels of phenolic compounds in both bitter and sweet lupine seeds decreased progressively with sonication compared to the control. Nevertheless, bitter lupine showed a slight rise in catechin, rutin, p-coumaric acid, quercetin, and kaempferol after 5 min of sonication, followed by a decline at longer sonication times. Similarly, sweet lupine exhibited a modest increase in rutin, ferulic acid, and kaempferol at 5 min, which then decreased with continued sonication.

The main phenolic constituents detected in bitter and sweet lupine seed oils treated for different sonication durations were gallic acid, catechin, rutin, and quercetin (Table 3). Gallic acid levels in bitter and sweet lupine oils ranged from 0.93 mg/100 g (5 min) to 1.98 mg/100 g (15 min) and from 0.32 mg/100 g (5 min) to 0.75 mg/100 g (25 min), respectively. Catechin in bitter lupine oils varied between 1.31 mg/100 g (control) and 2.97 mg/100 g (15 min), while sweet lupine oils contained catechin from 0.36 mg/100 g (25 min) to 1.29 mg/100 g (control). During sonication, catechin, rutin, and quercetin increased in bitter lupine oil, whereas these compounds decreased in sweet lupine oil (Table 3). In general, catechin content was lower in sweet lupine seed oil than in bitter lupine seed oil. Rutin levels ranged from 0.78 mg/100 g (25 min) to 0.91 mg/100 g (15 min) in bitter lupine oil and from 0.28 mg/100 g (15 min) to 0.78 mg/100 g (control) in sweet lupine oil. Quercetin levels were between 0.42 mg/100 g (control) and 0.93 mg/100 g (5 min) in bitter lupine oil, and between 0.21 mg/100 g (15 min) and 0.39 mg/100 g (5 min) in sweet lupine oil. Overall, the phenolic content of sweet lupine seed oils was slightly lower than that of bitter lupine seed oils. Similar to the phenolic compounds found in lupine seeds, both bitter and sweet lupine seeds generally exhibited a gradual reduction in phenolic compound levels during the sonication process. This decline is believed to result from the substantial release of phenolic compounds within the first 5 min of sonication, followed by the increased resistance of phenolic-compound-containing cell walls to the sonication frequency at later stages. No statistically significant differences were detected in the p-coumaric acid and ferulic acid levels of sweet lupine oil sonicated for 5 and 15 min compared to the control. Bitter lupine seeds contained 0.64–1.44 mg/kg quercetin, 443–766 mg/kg caffeic acid, 2.47–6.55 mg/kg p-coumaric acid, 5.39–17.8 mg/kg p-hydroxybenzoic acid, and 5.18–11.8 mg/kg ferulic acid [55]. Tirdilová et al. [56] reported that bitter lupine (L. luteus) seeds contained 0.13–0.53 mg/kg hesperidin, 0.17 kaempferol, 0.40 rutin, 0.10 caffeic, 0.56–4.47 p-coumaric acid, 0.54–4.55 p-hydroxybenzoic acid, 0.18–0.33 mg/kg gallic acid. In other studies, protocatechuic, vanillic, p-coumaric, and ferulic acids have been identified in lupine seed flour [57]. Brandolini et al. [58] stated that debittered lupine seeds (L. mutabilis) contained 99–125 mg/kg genistein, 5.94–7.42 naringenin, 5.75–14.36 cinnamic acid, 0.87–8.02 p-hydroxybenzoic acid, 0.54–9.11 vanillic acid. The phenolic compound contents of bitter and sweet lupine grains were lower than those determined by Vollmannova et al. [55], Tirdilová et al. [56] and Brandolini et al. [58]. When the phenolic compound amounts of lupine seeds and oils were compared with the literature data [55,56,57,58], the monitored changes may be due to the applied process and the lupine variety, climatic factors, harvest time, storage conditions, and analytical conditions.

3.3. Fatty Acid Compositions of Bitter and Sweet Lupine Oils Sonicated for Different Times

Table 4 presents the fatty acid profiles of bitter and sweet lupine oils subjected to sonication for various durations. Linoleic acid contents of the bitter and sweet oils were assessed to be between 21.52% (control) and 49.08% (25 min) to 18.88% (15 min) and 19.46% (control), respectively. Notably, the levels of linolenic and behenic acids in lupine oils were higher than those typically found in most commercially available edible seed oils. Oleic acid was the predominant fatty acid in both types of lupine oil, with concentrations varying from 36.78% (25 min) to 52.47% (15 min) in bitter lupine oil, and from 55.89% (15 min) to 56.69% (control) in sweet lupine oil. Linolenic acid levels in bitter lupine oil were observed between 9.87% (5 min) and 10.26% (control), whereas in sweet lupine oil they ranged from 9.23% (control) to 9.61% (15 min). Behenic acid content was measured between 3.20% (5 min) and 3.35% (control) in bitter lupine oil, and between 2.17% (control) and 2.91% (15 min) in sweet lupine oil. Additionally, palmitic acid amounts were recorded between 7.47% (15 min) and 9.52% (25 min) for bitter lupine oil, and between 7.80% (15 min) and 8.66% (control) for sweet lupine oil. No statistically significant difference was observed between the stearic acid contents of sweet lupine oil sonicated for 15 and 25 min, and the control. Myristic, arachidic, linolenic, behenic and erucic acids were not found in bitter lupine oil sonicated for 25 min. With the sonication time, the contents of stearic and oleic fatty acids in bitter and sweet lupine oils increased compared to the control (except oleic for 25 min in bitter lupine oil), while a decrease in palmitic acid content was observed (except palmitic for 25 min in bitter oil). The linoleic acid quantity of bitter lupine oil increased with sonication times compared to the control, while the linoleic acid amount of sweet lupine oil partially decreased. The amounts of some fatty acids of lupine oils showed fluctuations depending on sonication times. These changes may be due to oxidation and structural deterioration of fatty acids. White lupine seed oils contained 0.09–0.10% myristic, 6.54–7.31% palmitic, 1.59–1.85% stearic, 57.46–58.11% oleic, 14.23–16.18% linoleic, 7.22–7.58% linolenic, and 5.46–6.02% eikosenoic acids [59]. Lupin (L. albus) seed oil contained 11.6% palmitic, 1.9 stearic, 55.4 oleic, 22.4 linoleic, 8.7 linolenic acid [60]. Czubinski et al. [61] reported that lupine (L. mutabilis) seed oil contained 39.9% oleic acid, 36.7% linoleic acid, 12.6% palmitic acid, and 4.9% stearic acid. Interestingly, linolenic acid, which belongs to the group of n-3 fatty acids, had a share of 2.7%, while the n-6/n-3 acid ratio is estimated to be 13.6 [61]. Çoban et al. [4] stated that lupine seed oil contained 45.39% linoleic acid, 45.39% oleic acid, 8.27% palmitic acid and 5.61% stearic acids. Although the fatty acid profiles of sweet and bitter lupine oils in the present study were predominantly similar, significant changes were monitored in their amounts when compared with the literature. These changes may be due to sonication, lupine variety and cultivation conditions. Based on the data presented in Table 3, the effects of sonication time on the fatty acid composition of bitter and sweet lupine seed oils were evaluated in an integrated manner. In both lupine types, sonication time significantly influenced the relative distribution of fatty acids (p < 0.05); however, the extent and direction of these changes differed between varieties. In bitter lupine oil, short to moderate sonication (5–15 min) resulted in an overall reduction in saturated fatty acids and a gradual increase in oleic acid, whereas prolonged sonication (25 min) led to noticeable increases in stearic and arachidic acids. This suggests that extended ultrasonic treatment may promote the relative enrichment of long-chain saturated fatty acids. In contrast, sweet lupine oil exhibited a more stable saturated fatty acid profile, with palmitic acid decreasing with sonication time and only minor increases observed in stearic and arachidic acids. Oleic acid, the predominant monounsaturated fatty acid in both oils, responded differently to sonication in the two lupine types. In bitter lupine oil, oleic acid content increased significantly with increasing sonication time, reaching its maximum at 25 min. Conversely, sweet lupine oil showed only slight fluctuations in oleic acid levels, indicating greater resistance of its monounsaturated fatty acid fraction to ultrasonic treatment. Regarding polyunsaturated fatty acids, bitter lupine oil exhibited a marked decrease in linoleic and especially linolenic acids at prolonged sonication, highlighting the susceptibility of highly unsaturated fatty acids to ultrasonic cavitation and possible oxidative effects. Sweet lupine oil, however, maintained comparatively stable linoleic and linolenic acid contents across sonication times, with only minor variations. Overall, the results demonstrate that bitter lupine oil is more sensitive to changes in sonication duration than sweet lupine oil. Moderate sonication times (5–15 min) appear beneficial for both oils by enhancing oleic acid proportions while preserving polyunsaturated fatty acids, whereas extended sonication may compromise the nutritional quality of bitter lupine oil due to polyunsaturated fatty acid losses. Therefore, careful optimization of sonication time is essential in the ultrasonic extraction of lupine seed oils to maintain fatty acid quality. Although the fatty acid compositions of bitter and sweet lupine varieties show minor differences in general, the fatty acid results are partially similar to the results of Czubinski et al. [61], Çoban et al. [4], Grela et al. [59] and Erbaş et al. [47]. The main reason for the minor differences is probably due to the variety, climatic conditions and the oxidation of unsaturated fatty acids during extraction.

3.4. Lipid Index Values of the Oils Extracted from Bitter and Sweet Lupine Seeds

Table 5 shows some lipid index values of the oils obtained from bitter and sweet lupine seeds sonicated at different times. Differences in the index values of the oils were observed depending on the sonication times. Nutritive value index (NVI) results of bitter and sweet lupine seeds sonicated at different times were characterized to be between 6.41 (control) and 7.40 (5 min) to between 6.80 (control) and 7.54 (15 min), respectively. While calculated oxidizability values (Cox) of bitter lupine seeds change between 4.40 (25 min) and 4.96 (15 min), Cox values of sweet seeds were described to be between 4.54 (5 min) and 4.58 (15 min). Oleic desaturation ratio values of bitter and sweet seeds were calculated to be between 0.332 (25 min) and 0.381 (control) to between 0.334 (5 min) and 0.338 (control and 15 min), respectively. In addition, linoleic desaturation ratios of bitter and sweet seeds were calculated to be between 0.311 (5 min) and 0.326 (25 min) to between 0.290 (control) and 0.337 (15 min), respectively. In both lupine species, sonication generally increased NVI values. In bitter lupine seeds, NVI increased at 5 and 15 min of sonication compared to the control group, with the highest value obtained at 15 min (7.40); however, a slight decrease was observed at 25 min of sonication. Similarly, in sweet lupine seeds, NVI values increased with sonication duration, with the highest NVI recorded at 15 min (7.54) and largely maintained at 25 min. Overall, sweet lupine showed higher NVI values than bitter lupine at all durations. In bitter lupine seeds, Cox values fluctuated depending on the sonication duration, with a significant decrease (4.40) particularly at 25 min of sonication. This suggests that prolonged sonication may improve oxidative stability. In sweet lupine seeds, Cox values were not significantly affected by sonication time and remained at similar levels in all groups. This indicates that the oxidative stability of sweet lupine oils is more resistant to sonication. In bitter lupine seeds, ODR values generally tended to decrease as sonication time increased, with the lowest value obtained at 25 min (0.332). In contrast, in sweet lupine seeds, ODR values were not significantly affected by sonication time and remained quite stable in all groups. This suggests that the fatty acid composition of bitter lupine is more sensitive to sonication compared to sweet lupine. In bitter lupine seeds, LDR values did not show a significant change with sonication time, remaining at similar levels with small fluctuations. In sweet lupine seeds, sonication increased LDR values, especially in 5 and 15 min applications, and this increase showed a slight decrease at 25 min. These results indicate that sonication may promote linoleic acid formation in sweet lupine oils. Sonication positively affected the nutritional value index (NVI) in both lupine species; however, the effects on fatty acid desaturation rates and oxidative stability differed depending on the species and duration. Bitter lupine showed more pronounced changes with prolonged sonication, while sweet lupine seeds exhibited a more stable fatty acid profile. These findings suggest that the sonication duration should be optimized according to the lupine species. Since fat oxidation and the degree of saturation are evaluated using the calculated oxidizability (COX) or the saturated-to-unsaturated fat (S/P) ratio, oils with a higher S/P ratio demonstrate greater fatty acid stability compared to those with a lower ratio [62]. It has been reported that the average ODR value of sesame oil is 0.5 and the average LDR value is 0.01 [63]. The oxidizability value of sesame oil extracted from sesame seeds roasted in different environments has been reported to be between 4.97 and 5.06, and the ODR and LDR values changed between 0.5143–0.5172 and 0.0115–0.0136, respectively [64]. The Cox score of oils exhibits only minor variations, indicating that vegetable oils can effectively protect against oxidative degradation [65]. The differences in lipidomic values of lupine types may be due to differences in materials and extraction conditions, as these results differ from the literature [62,63,64,65].

4. Conclusions

In conclusion, the present study demonstrates that sonication time is a critical operational parameter in the ultrasound-assisted extraction of lupine seed oil. Among the conditions evaluated, a sonication duration of 5 min provided the most advantageous outcome, ensuring maximal oil recovery while maintaining the integrity of bioactive constituents. The absence of significant differences in antioxidant activity among bitter lupine oils subjected to varying sonication times indicates that, within the investigated range, ultrasonication does not exert a detrimental effect on overall antioxidant capacity.

With respect to phenolic composition, bitter lupine seeds exhibited slightly higher total phenolic contents than sweet lupine seeds. Catechin, kaempferol, and rutin were identified as the predominant phenolic compounds in both varieties. The data suggest that short-term ultrasonication facilitates the release of phenolic compounds, presumably through the disruption of cellular structures, whereas prolonged exposure may induce the partial degradation or structural modification of these thermolabile and oxidation-sensitive molecules. Accordingly, the highest phenolic content in bitter lupine seeds was observed at 5 min of sonication, while in sweet lupine seeds the optimal range was determined to be between 15 and 20 min. In the corresponding oils, maximum phenolic levels were detected at 15 min for bitter lupine oil and at 5 min for sweet lupine oil.

In terms of fatty acid composition, lupine oils were characterized by relatively elevated levels of linolenic and behenic acids compared with many conventional industrial edible oilseeds. Increasing sonication time resulted in elevated stearic and oleic acid contents in both bitter and sweet lupine oils, with the exception of oleic acid at 25 min in bitter lupine oil. Linoleic acid content increased progressively in bitter lupine oil, whereas a partial reduction was observed in sweet lupine oil under extended sonication. These findings indicate that ultrasound-assisted extraction not only enhances oil yield but also influences the distribution of individual fatty acids.

Collectively, the results confirm that a 5 min sonication period represents the most appropriate condition when both the extraction efficiency and preservation of bioactive compounds are taken into consideration. Extending the sonication time beyond this threshold yields limited additional oil recovery and may compromise phenolic stability. Future investigations should focus on alternative debittering strategies for bitter lupine, such as boiling or sonication in ash water, and comprehensively assess their effects on phytochemical composition, antioxidant activity, lipidomic indices, and overall nutritional quality.