1. Introduction
Grape processing generates large quantities of important agricultural and industrial wastes/by-products with potential to be reused for various purposes. It has been estimated that more than 0.3 kg of solid by-products is allocated per kg of mashed grape fruit during the processing [
1]. The waste streams of wine production contain organic waste, greenhouse gases (CO
2, vaporous compounds etc.) and non-organic waste (diatomaceous earth, bentonite clay, perlite). In particular, organic waste, as grape pomace with seeds, pulp and skins, grape stems and leaves, represents about two thirds of entire solid waste [
2,
3]. The handling and disposal of this great amount of waste/by-products is a large environmental problem [
3,
4]. Currently, new processes for the controlled waste removal are being searched, targeting the conversion of the waste material and its incorporation into new bio-products with added value.
Seeds are the major material of the industrial processing of grape berry (e.g., found in pomace) and constitute about 7–20% of the weight of grapes processed [
5]. Although grape seeds are mutually removed with the skins and vascular fruit tissues from the pomace, they can be easily separated through technological separation and sieving [
6]. Although grape peels and stems do not have an economic background for industrial utilization, seeds are rich in bioactive antioxidants, and can be raw materials for the development of new foods, as natural extracts, pharmaceutical products [
7,
8] and cosmetics. Therefore, the production of grape seed oil contributes to the advance of waste management that could increase the financial income of the primary industrial process and sustainability [
4].
The oil content of grape seeds from the literature was reported in the range of 13–15% depending on the variety and maturity of grapes [
9]. The interest in grape seed oil as a functional food product has increased, particularly because of its high levels of lipophilic ingredients, such as vitamin E, unsaturated fatty acids (UFAs), and phytosterols [
10] that possess greater antioxidant activity than hydrophilic ingredients [
9]. Namely, grape seed oil was identified as a rich source of tocopherols, tocotrienols and unsaturated fatty acids, especially in polyunsaturated fatty acids (PUFAs), whereas linoleic acid (C18:2) was found as predominant (49.0–78.2% of total PUFAs) [
4]. γ-tocotrienol was evidenced as the most abundant tocotrienol, followed by α-tocotrienol, while δ-tocotrienol was found in lower amounts [
11]. Tocopherols from seed oils are α-, β-, γ-, and δ-tocopherol, with α-tocopherol as one of the most potent intracellular fat-soluble antioxidants due to its activity in inhibiting the peroxidation of polyunsaturated fatty acids in biological membranes [
4]. A mixture of α-tocopherol and α- and γ-tocotrienol purified from grape seeds was more effective than other lipophilic grape seed fractions in neutralizing free and lipid peroxy radicals and in chelating prooxidant metals [
12]. Therefore, grape seed oil extract can be considered a valuable source of natural liposoluble antioxidants, having potential health benefits [
13,
14].
On the industrial level, grape seed oils are mainly produced by traditional oil extraction methods, such as cold-press and solvent extraction [
15,
16]. Traditional processing often leads to a higher solvent consumption, longer extraction times, lower yields and poorer extraction quality [
17]. As compared to Soxhlet extraction, cold pressing has potential for higher yields of fatty acids and tocopherols, without the assistance of heat and chemical treatment. Thus, cold-pressed oils are interesting raw materials for natural and safe food products favored by manufacturers and consumers [
18].
Commonly, Soxhlet extraction employs
n-hexane as solvent for grape oils, which is not selective and simultaneously removes non-volatile pigments and waxes. Consequently, the obtained extracts are dark, viscous and contaminated with the traces of toxic solvent [
19]. Many contemporary extraction technologies avoid the negative impacts of thermal degradation and meet the criteria for the “green“ extraction processes [
20]. Aside from that, they offer energy savings, either minimize or avoid the use of organic solvents, shorten the processing time, reduce the temperature, enhance the mass transfer process, and increase the extraction yield with high quality extracts [
21]. Extractions aided by ultrasound, microwave or high-pressure processing are being explored as alternative technologies for intensification of the extracted antioxidants from grape seeds.
Ultrasound-assisted extraction (UAE) is based on the acoustic cavitation phenomenon. The period of negative pressure during the ultrasound treatment causes bubbles, hence the origins of increased pressure and temperature with their subsequent collapse. When this happens, the resulting ”shock waves“ break the cellular walls and facilitate solvent penetration into plant materials which enhances extraction yields [
22]. Microwave-assisted extraction (MAE) uses microwaves that are nonionizing, electromagnetic waves with the frequency between 300 MHz and 300 GHz. Here, electromagnetic waves are transformed to thermal energy, which induces heating of the matrix on inside and outside without thermal gradient. If a sufficient amount of thermal energy is generated, this local heating damages cell wall of plant matrix and causes leakage of target compounds into extraction medium. In the literature, MAE was useful for extraction of biologically active substances with antioxidant properties from grape seeds [
23,
24]. Supercritical fluid extraction (SFE) represents another excellent alternative to conventional extraction with the potential to achieve comparable yields. Additionally, grape seed oils recovered by SFE are characterized by higher product quality that is similar to mechanical pressing [
25]. Supercritical fluids, especially CO
2, have the gas-like properties, e.g., viscosity and diffusivity, and liquid-like properties, e.g., density and solvation power [
26]. CO
2 is a green, low-cost, non-toxic and non-flammable solvent with critical pressure of 73 bar and temperature of 31 °C. It can be re-used in processing, hence its ability to reduce total energy costs in industry [
27]. In addition, the residues of the solvent do not remain in the final product, because supercritical CO
2 can be completely eliminated by pressure reduction [
9]. Co-solvents and modifiers (e.g., ethanol, methanol, acetone) may be added to improve the solubility of polar phytochemicals embedded in the cell wall [
27]. Moreover, supercritical CO
2 ensures selectivity in the extraction of certain target compounds by varying operating conditions (e.g., temperature and pressure) [
27], while an approximate economic estimate of industrial SFE scale-up from the laboratory is already available from the literature [
28].
Since there are no abundant data in the literature for comparison of alternative vs. conventional extractions of grape seed oils with potential for industrial applications, the aim of this study was to compare Soxhlet against UAE, MAE and SFE concerning efficiency for obtaining high-quality extracts. In this work, we postulated that SFE is an important green alternative to organic solvent extraction for recovery of lipophilic antioxidants from winery waste streams by considering extraction parameters, in-depth chemical profiling, functional qualities and bioactivity of samples. Samples were compared in terms of total extraction yields, fatty acid profiling, tocopherol content, and antioxidant properties.
4. Discussion
SFE is frequently used as modern technique for the isolation of grape seed oil, therefore it was chosen for optimization and further comparison with Soxhlet extraction and advanced extractions (e.g., UAE and MAE) accounting for yields and lipophilic antioxidant potential. The results of SFE at different pressures were well within the references in the literature, as similar findings were observed by Jokić et al. [
39] who performed SFE under experimental conditions of similar pressure (158.58–441.42 bar) and temperature (35.86–64.14 °C) and reached yields from 2.56 to 14.87% for the Cabernet Franc grape variety. In another study, Rombaut et al. [
16] investigated the influence of pressures (230–538 bar), temperatures (75–120 °C) and flow rates (5–17 kg h
−1) and observed total extraction yields from 5.7 to 17.2%. Moreover, Prado et al. [
40] observed yield of 13.42% at 350 bar, 40 °C and 0.46 kg CO
2 h
−1. The one-factor-at-a-time approach was applied in this work; therefore, after concluding that the highest yield was achieved at 350 bar, that parameter was kept constant for further experimentations. The temperature increase expedited the extraction kinetics by causing a crossover phenomenon. The increased temperature caused a decrease in CO
2 density resulting in reduced solubility which negatively affected extraction rates [
45]. Simultaneously, vapor pressure of the solute increased solubility and positively affected extraction yields. With isobaric increase in temperature, plots of solubility intersected, and these junctures were labeled as “lower and upper crossover points.” At pressures between these two points, solubility decreased with temperature increase, since solvent density overcome the vapor pressure effect. With vapor pressure outside the upper or lower crossover points, its effect become stronger than the density effect, thus the solubility increased with higher temperatures [
1].
Passos et al. [
19] performed supercritical extraction of Touriga Nacional grape seed oil samples at different pressures and temperatures and concluded that an enhanced extraction rate was achieved with increased pressure and decreased temperature. That was associated with their effects on oil’s solubility and mass transfer coefficients. Coelho et al. [
26] observed that higher yields may be achieved at lower pressures and temperatures, as with increased pressure, temperature influence becomes insignificant. Therefore, after conducted experiments, it was concluded that the optimal temperature for SFE was 60 °C, and this temperature was used for further SFE experiments.
A similar influence of solvent flow rate was observed by Duba and Fiori [
1], who concluded that increased supercritical CO
2 flow rates positively affected extraction rates due to external and internal mass transfer. For commercial usages, it was concluded that the solvent flow rates must be optimized in terms of the extraction time and solvent volume, since the increase of CO
2 flow rates increases specific solvent consumption. Molero Gómez et al. [
46] raised flow rates from 0.5 to 2.0 L min
−1 at the constant conditions (40 °C and 350 bar), and found no significant differences for the yields. The investigated flow rate reached maximum yield of 96% after 3 h of extraction, while lower flow rates took longer time to achieve maximum yields. Finally, the optimal CO
2 flow rate was defined at constant pressure and temperature at 0.4 kg h
−1.
Milling the samples facilitated a higher release of oil from the seed cells and shorter diffusion paths in a solid matrix [
45]. Molero Gómez et al. [
46] have shown that higher extraction yield was obtained with reduced particle size of the samples. As it can be seen from obtained results, the size of the milled grape seeds should be ≥ 350 µm to achieve better efficiency. Total extraction yields may be increased by reducing particle size, which allows higher release of oils from milled particles, due to the widening of the surface area [
47].
Coelho et al. [
26] compared yields for Soxhlet extraction with that of
n-hexane and supercritical CO
2 extractions. SFE was found to produce the higher yields (12.0–12.7%) in comparison with Soxhlet extraction (12.28%). Jokić et al. [
47] concluded that grape oil can be completely extracted by SFE at optimal operating conditions (
P = 400 bar and
T = 41 °C), resulting with the oil yield of 14.87%. However, here the yield of Soxhlet extraction with
n-hexane was 14.96%. Bravi et al. [
44] used a seed mixture of different red (Merlot, Cabernet Sauvignon, Cabernet Franc and Raboso) and white (Prosecco, Verduzzo, Pinot Grigio, Chardonnay, Pinot Bianco, Bianco) grape varieties in Soxhlet and SC-CO
2 extraction. The content of the oil that can be extracted with SC-CO
2 was 14.4% and was slightly lower than with hexane (15.4%). Prado et al. [
28] achieved yield of 13.42% using SFE technique at 350 bar, 40 °C and 0.46 kg CO
2 h
−1. Da Porto et al. [
48] reported Soxhlet extraction using
n-hexane in a 1:12 ratio for 6 h and UAE in 1:8 ratio at 20 kHz and 50, 100 and 150 W for 30 min against the Soxhlet extraction of grape seeds of Raboso Piave variety. Here, an increase of the ultrasound power from 50 to 150 W caused the yield to jump from 11.42 to 14.08%. Anyhow, Soxhlet extraction had a higher yield of 14.64%, which can be explained by providing freshly condensed solvent for 6 h, while UAE was a batch system that lasted for 30 min. UAE at 150 W for 30 min increased the yield to approximately 14%, which is comparable to the yield of Soxhlet extraction at 70 °C for 6h [
48]. In the study conducted by de Menezes et al. [
17], oil from Burgundy variety grape seeds was obtained by the Soxhlet technique and UAE technique with hexane. The oil content was 16.28% for the Soxhlet technique and 11.60% for the UAE technique. Böger et al. [
22] showed that UAE is useful technique for increasing of the oil yields while reducing the durations of the extractions, while using less solvent and obtaining the high quality oils.
It is important to note that fatty acid composition in grape seed oils may be highly influenced by the grape variety and growing conditions [
49]. Since the SFE was selected as the technique with the highest potential for the oil recovery, it was further examined how its operating parameters affect the fatty acid profiling (
Table S3). To that end, Prado et al. [
28] investigated both lab- and pilot-scale SFE grape samples for Malbec and Cabernet Franc varieties. Although SFE parameters differed among pilot and lab experiments, it was still found that all extracted oils contained the linoleic (71.20%) and oleic acids (15.10%) as the main components. When considering saturated fatty acids, palmitic (8.13%) and stearic (4.05%) acids were determined as the most abundant. Coelho et al. [
26] analyzed the fatty acid profiles of the SE/hexane samples and those extracted by SFE by varying different operating conditions. Highest percentages in samples accounted for linoleic (64.5–67.37%), oleic (19.18–20.64%), palmitic (7.38–8.22%) and stearic acids (4.33–5.61%). Judging by our data and the literature, it can be concluded that the fatty acid profile from all samples followed the expected content. However, notable differences in fatty acid profiling were found for seed samples recovered by different extraction techniques.
The obtained results of functional quality indices highlighted that grape seed oils recovered by SE and UAE expressed the best functional quality. On the other hand, it is important to mention that the aforementioned extracts were obtained using n-hexane, which can be evaporated from the oils, but they can still be contaminated by the traces of this organic solvent. In conclusion, SFE stands out as a successful technique for isolation of solvent-free grape seed oil with proper functional quality and without any traces of extraction solvents.
When observing the impact of pressure, it can be concluded that the higher pressure reduced tocopherol content, although higher pressures gave higher extraction yields. The increase in temperature resulted in elevated tocopherol content. While higher temperatures promote higher solubility of the solute and enhance mass transfer of solute from matrix to the SFE solvent, the lowest tocopherol content was found at 50 °C. This can be related to the crossover phenomenon at aforementioned temperature. Bravi et al. [
44] have been observed for SFE extracts that an elevated temperature (40 vs. 80 °C) influenced increased α-tocopherol content, due to the higher solubility of α-tocopherol at 80 than at 40 °C. Samples with a reduced particle size (RGS315-SFE) exhibited higher tocopherol content than samples with larger particle size (7.57 vs. 4.60 mg 100 g
−1). This was expected, as particle size reduction leads to increased extraction efficiency, since the free surface area for mass transfer is increased and diffusional resistance in solid phase is decreased [
44].
Since the optimal SFE parameters gave the highest overall yield, after adjusting for total tocopherols, results showed that SFE at given conditions was the most successful for exhaustion of grape seeds. In summary, red grape seed oils are richer in α- and γ-tocopherol and higher amount of total tocopherols can be recovered by SFE, but considerable attention must be paid to the proper adjustment of an extraction parameters.
Based on the data, it can be concluded that the antioxidant capacity is strongly dependent upon the applied extraction parameters. Moreover, oils obtained from red grape seeds had a higher antioxidant capacity as compared to those from white grape, probably because higher content of α-tocopherols was also in red grape samples. Tangolar et al. [
47] have previously reported higher concentrations of α- and γ-tocopherol in the Cabernet Sauvignon variety than Chardonnay variety. Similar results were noted by Ben Mohamed et al. [
9], who evaluated the bioactive compounds and antioxidant activities of six different grape seed oils.
Although several in vitro and in vivo studies have shown that tocotrienol-rich fractions from grape seeds are more potent antioxidants, another study documented that α-tocopherol had higher free radical scavenging activity than tocotrienol-rich fractions, and consisted of a mixture of γ-tocopherol and α- and γ-tocotrienol [
12]. This was explained by the purity of tocotrienol-rich fractions, which contains approximately 6% tocols.
Among SFE and SE extracts, the SFE exhibited higher antioxidant capacity for both DPPH and ABTS assays. The opposite findings were reported by Wang et al. [
50], who evaluated and compared in vitro antioxidant activities of unsaponifiable fractions of 11 kinds of edible vegetable oils (flaxseed, olive, grape seed, corn, soybean, sunflower seed, walnut, perilla, rapeseed, sesame, and camellia) by DPPH, ABTS and FRAP assays [
50]. The authors identified grape seed oils with the lowest total antioxidant capability that might be attributed to the different processing techniques and different amounts of hydrophilic and lipophilic antioxidant content in the samples. Hence, confirming that oil extraction procedure is the crucial element that affect antioxidant activity and overall quality of extracts. A different study by Ben Mohamed et al. [
9] found higher ABTS values than our study for red grape oils. Oils were recovered by both SFE and SE with 7.5–8.2 µM and 5.9–6.5 µM Trolox g
−1, respectively. White grape varieties recovered by SFE had the ABTS values of 4.9–6.0 µM Trolox g
−1, while SE samples had lower values for antioxidative activity of 4.4–4.9 µM Trolox g
−1 [
9]. In the work of Konuskan et al. [
15], the highest DPPH radical scavenging activity was noted for Cabernet Sauvignon variety, which also makes up the highest part in red grape seed mixture used in this work. The authors also found that hydrophilic antioxidant values were unaffected by the extraction method, while lipophilic values were higher for the super critical CO
2-extracted oils. This suggested that the type of extraction, as well as the corresponding parameters, should be thoroughly considered for the isolation of oil from grape seeds in order to obtain desired antioxidant potential.
5. Conclusions
Grape seeds, as a by-product of wine industry, can be successfully valorized as a raw material for recovering oils with high-quality bioactive antioxidants. Modern extraction technologies, such as UAE, MAE and SFE, were compared to Soxhlet extraction for obtaining red and white grape seed oils. The SFE was the best method with respect to extraction yield at optimum processing parameters (350 bar, 60 °C and 0.4 kg h−1), providing 12.23% and 11.86% yields for red and white grape seeds, respectively.
A fatty acid profiling of samples identified polyunsaturated fatty acids as dominating in this category of constituents (69.27–74.88%) with linoleic acid (68.61–74.15%) as major representative. Monounsaturated fatty acids were found in lower amounts (13.53–18.62%) where oleic acid was predominant compound (13.39–18.47%). Saturated fatty acids were detected in the lowest amounts ranging from 11.28–12.27%. Different extraction techniques did not alter fatty acid profiles in the samples; however, the application of SFE technology yielded appreciable quantities of tocopherols. The highest antioxidant potential (i.e., DPPH and ABTS) for red and white grape oils were observed for samples recovered by the UAE.
Based on the results, it can be concluded that the application of non-conventional extraction techniques was efficient for recovering of high-quality grape seed oils that were rich in lipid antioxidants. Thus, such extracts could be incorporated into different functional foods, pharmaceuticals or cosmetic products. Non-conventional techniques have environmental benefits, as they stand out as the “green” extractions, due to absence of organic solvents form the process. Hence, preventing numerous disadvantages of conventional alternatives, such as toxic residuals of organic solvents in the extracts, negative environmental impacts and flammability.