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Article

Valorization of Grape Seed Cake by Subcritical Water Extraction

by
Marko V. Malićanin
1,
Jaroslava Švarc-Gajić
2,
Steva M. Lević
3,
Vladislav A. Rac
3,*,
Ana S. Salević-Jelić
3,
Mirjana B. Pešić
3,
Danijel D. Milinčić
3,
Diana Pasarin
4 and
Vesna M. Rakić
3
1
Faculty of Agriculture Kruševac, University of Niš, 37000 Kruševac, Serbia
2
Faculty of Technology Novi Sad, University of Novi Sad, 21000 Novi Sad, Serbia
3
Faculty of Agriculture, University of Belgrade, 11080 Belgrade, Serbia
4
National Institute for Research & Development in Chemistry and Petrochemistry—ICECHIM, 060021 Bucharest, Romania
*
Author to whom correspondence should be addressed.
Processes 2025, 13(5), 1597; https://doi.org/10.3390/pr13051597
Submission received: 23 April 2025 / Revised: 14 May 2025 / Accepted: 19 May 2025 / Published: 20 May 2025
(This article belongs to the Special Issue New Advances in Green Extraction Technology for Natural Products)
Browse Figure
Versions Notes

Abstract

:
Agricultural waste originating from the wine industry presents an environmental and economic issue. Grape seeds, a major constituent of grape pomace, are only partially valorized through oil extraction. The cake remaining after oil production is rich in valuable bioactive compounds. In this study, an advanced extraction technique, which utilizes subcritical water, was employed for bioactive compound recovery from defatted grape seed cakes. Extraction was performed in a nitrogen atmosphere (10 bar) at 130 °C and 170 °C. The extracts were characterized in terms of the total polyphenols, flavonoids, proteins and antioxidant activity. Detailed polyphenol profiles were determined using UHPLC Q-ToF MS analysis. Quantification of the individual sugars was performed by HPLC. The amino acid profile was determined using ion chromatography. The yield of phenolic acids was found to be higher at 170 °C (883 vs. 557 mg/100 g at 130 °C), while the flavonoid content was favored at 130 °C (596 vs. 185 mg/100 g at 170 °C). The total protein, essential amino acid and xylo-oligosaccharide content was higher at 170 °C. The obtained results show that the use of water as the extraction solvent in subcritical conditions is a promising technique for the environmentally friendly valorization of grape seed cakes and biowaste in general.

1. Introduction

Agriculture and food production and processing are generating high quantities of waste, which presents an environmental problem; however, the waste can be utilized in different ways, by converting it into useful by-products or using it as raw material for other industries [1], in order to gain economic benefits and reduce unwanted residue. The winemaking industry produces large amounts of waste grape pomace, which presents some 20–30% of the total grape mass [2] and mainly consists of the seeds and skins. Grape seeds are most often valorized through the extraction of oil, which is rich in unsaturated fatty acids, with linoleic and oleic fatty acids being the most abundant [3,4,5,6,7]. However, both the seeds and the pomace as a whole can be used in various ways, most importantly for the recovery of bioactive compounds and dietary fibers [8], which has been successfully studied for the use of food fortification [2], but also can be used as a bioenergy feedstock [9]. Grape seeds consist of approximately 10–20% lipids, 10% protein and 40% dietary fiber, as well as polyphenols, sugars and minerals [8].
In the ever-growing search to formulate new, green and efficient ways for extracting bioactive compounds from plant material, subcritical water extraction (SWE) stands out for its versatility, efficiency and use of pure water as the solvent [10,11]. Subcritical water extraction, among other names by which it is also known as including pressurized liquid extraction, pressurized hot water extraction or high pressure high temperature extraction, employs water at high temperatures and pressures. The technique makes use of enhanced mass transfer during solid–liquid extraction at an elevated pressure/temperature, keeping water below its critical point, in a liquid state. High efficiency and selectivity, the reduction in extraction time, minimal sample pre-treatment and the preservation of compounds sensitive to oxygen are reported, which account for the recent growing number of publications related to SWE [11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26]. At normal conditions, water is not considered the solvent of choice for compounds of low polarity due to its high dielectric constant and polarity. However, at high temperatures/pressures, these parameters change, allowing its use as an efficient extraction solvent for substances of medium and low polarity [27]. For example, at 250 °C and 50 bar, the dielectric constant of water falls to a value close to ethanol [28]. Furthermore, by varying the temperature and pressure, the polarity can be changed to accommodate the targeted compound solubility [29]. The range of possible temperatures and water partial pressures is quite broad, limited only by its critical point (374 °C, 22.1 MPa). The apparatus usually used for SWE consists of a stainless steel reactor/extractor, which is pressurized, heated and stirred. The reactor can be pressurized with different gases, nitrogen being the most common, although other gases have been used, for instance CO2, in order to acidify the solvent [30,31]. Subcritical water extraction has been successfully applied in extracting polyphenols, polysaccharides and proteins from various plant material [10,11,30,31,32,33].
This paper presents the results of subcritical water extraction applied to defatted grape seed cake, performed with the aim to demonstrate the effectiveness and versatility of SWE when applied to an agricultural waste material of real economic and environmental significance. The extraction was performed in a nitrogen atmosphere at two temperatures, 130 °C and 170 °C. The extracts were characterized in terms of the total polyphenol and flavonoid content, antioxidative activity, detailed polyphenol profile, protein content and amino acid and sugar profiles. The two studied temperatures were chosen based on previous experience in the application of this extraction technique. Namely, it is known that the reactivity of hot compressed water increases with temperature. Many bioactive compounds are thermally decomposed at higher temperatures. Thus, the selected temperatures aimed to demonstrate differences in the extraction efficiency and reactivity of water heated to 130 °C and 170 °C, and balancing efficiency vs. water reactivity. When the goal of the treatment is to efficiently extract bioactive compounds while preventing their degradation, milder temperatures are usually chosen [10,30]. If the partial hydrolysis of macromolecules from the sample matrix is the aim, usually higher temperatures provide more intense decomposition processes [34]. The experimental design, thus, was based on the hypothesis that at 130 °C, the sample matrix would not decompose remarkably, but bioactive compounds would be extracted efficiently. A temperature of 170 °C should provide more intense matrix decomposition with an increased risk of thermal degradation of the bioactive constituents.

2. Materials and Methods

2.1. Grape Seed Cake

Grape seeds (Pinot Noir variety) were provided by the Zmajevac winery (Serbia), harvested in the 2022 season. The seeds were washed, dried to ˂9% moisture content and kept refrigerated in sealed bags. Prior to extraction, the seeds were crushed and subjected to cold pressing using a UM200 cold-press oil machine (Ulimac Machine, Ankara, Türkiye, ≈45 °C) in order to remove the majority of the lipids. The yield of the obtained seed oil was 10.6% w/w. The remaining grape seed cake was used for characterization and extraction in a powdered form.

2.2. Methods

2.2.1. Analytical Methods

Proximate Composition of the Grape Seed Cake

The proximate composition of the grape seed cake was determined using standard methods: moisture (NMKL 169:2002) [35], ash (NMKL 173, 2nd Ed.:2005) [36], proteins (ISO 16634-1:2008) [37], lipids (NMKL 160:1998) [38], carbohydrates (by difference, FAO) and insoluble and soluble fiber (AOAC 991.43:2000) [39]. The results are expressed as g/100 g of the grape seed cake ± SD.

Total Phenolics Content (TPC)

The Folin–Ciocalteu method [40] was used to estimate the total content of the polyphenols in the extracts. The procedure consisted of adding 2 mL of Folin–Ciocalteu (FC) reagent (diluted 1:10, v/v) to an aliquot of the extract (or a standard solution of gallic acid); after 4 min, Na2CO3 (1.6 mL, 7.5%, w/w) was added. After 90 min at room temperature, measurements were performed using a spectrophotometer at 765 nm. The procedure was performed in triplicate, and the results were presented as mg gallic acid equivalent per g of grape seed cake (mean ± SD mg GAE/g).

Total Flavonoids Content (TFC)

Determination of the flavonoid content (TFC) was measured using the standard spectrophotometric AlCl3 assay [41], with rutin trihydrate as the standard. Aliquots of the extracts (2 mL) were mixed with the same volume of AlCl3 solution (2%), and measurements were performed at 423 nm after a ten-minute period. The procedure was performed in triplicate for each sample, and the results were presented as mg rutin equivalent per g of grape seed cake (mean ± SD mg RE/g).

Total Antioxidant Capacity

The total antioxidant capacity of the extracts was determined by the phosphomolybdenum method [42]. An aliquot of 0.3 mL of the aqueous extract or standard solution was mixed with 3 mL of the reagent solution consisting of 0.6 mol/L sulfuric acid, 28 mmol/L sodium phosphate solution and 4 mmol/L ammonium molybdate. The mixtures were incubated at 95 °C for 90 min. After cooling to room temperature, the absorbance was measured at 695 nm. All measurements were performed in triplicate. Ascorbic acid (10–100 mg/L) was used as a standard. Results were presented as mg ascorbic acid equivalent per g of grape seed cake (mean ± SD mg AA/g).

DPPH Radical Scavenging Activity

Antioxidant activity was also estimated using the DPPH assay [34] with ascorbic acid as the standard. Aliquots of the extracts (0.25 mL) were added to the DPPH solution (2 mL, 0.04 mg/mL diluted in ethanol), and the spectrophotometric measurements were performed at 517 nm after the reaction solution was kept in the dark for 0.5 h. Results were presented as mg ascorbic acid equivalent per g of grape seed cake (mean ± SD mg AA/g).

Polyphenol Profile

Polyphenol profiles of the extracts were analyzed using UHPLC Q-ToF MS, following a method previously described in detail [43]. Sample preparation involved solid-phase extraction (SPE) using C18 cartridges (500 mg/6 mL, Hillium Group), elution with 0.1% HCl in methanol and filtration through 0.45 µm syringe filters. Both extracts were prepared and analyzed in duplicate. Analyses were performed on an Agilent 1290 Infinity UHPLC system coupled with a 653 °C Q-ToF MS (Agilent Technologies, CA, USA). Chromatographic separation was carried out at 40 °C using a Zorbax C18 column (2.1 × 50 mm, 1.8 µm), with the mobile phases consisting of ultrapure water with 0.1% formic acid (A) and 98% acetonitrile with 0.1% formic acid (B). A gradient program was applied as follows: 0–2 min (2% B) and 2–17 min (2–98% B), maintained at 2% B until 22 min. The mass spectrometer, equipped with a Dual Agilent Jet Stream electrospray ionization (ESI) source, operated in both ESI+ and ESI− modes. Ionization settings included the following: 45 psi nebulizer pressure, drying gas at 225 °C (8 L/min), sheath gas at 300 °C (10 L/min), 2500 V capillary voltage, 175 V fragmentor, 65 V skimmer and 750 V octopole RF Peak. Spectra were acquired in the m/z range of 100–1700 at a 2 Hz scan rate. Suspect screening was conducted via data-dependent acquisition (DDA) in Auto MS/MS mode with 30 eV collision energy. The data acquisition parameters were as follows: m/z = 100–1700, scan rate = 1 spectrum/s. Agilent MassHunter software was used for data processing (https://www.agilent.com/en/product/software-informatics/mass-spectrometry-software, accessed on 20 April 2025). Phenolic compounds were identified based on monoisotopic mass, MS/MS fragmentation patterns and comparison with the literature data [44], while quantification was performed using standards and expressed as g equivalents per 100 g of the grape seed cake powder. Accurate masses were calculated using ChemDraw software (version 12.0, CambridgeSoft, Cambridge, MA, USA). Results are presented as g/100 g of grape seed cake ± SD.

Amino Acid Profile

The amino acid composition of the extracts was determined using ion chromatography. Prior to the analysis, the samples were converted to a powder form using freeze-drying, and the dry extracts were hydrolyzed (6 M HCl + 0.1% phenol, 24 h at 110 °C). A Thermo Scientific ICS 5000 (Thermo Fisher, Watham, MA, USA) instrument was used, equipped with an AminoPac PA10 (2 × 250 mm) column and a gold electrode as the working electrode in the detector. The results were presented as g/100 g of dry extract ± expanded measurement uncertainty (p = 95%, k = 2).

Sugars

The Dionex Ultimate 3000 (Thermo Scientific, Waltham, MA, USA) HPLC system was used for the quantitative analysis of carbohydrates within the previously prepared samples (diluted and filtered). The system was equipped with an Agilent Hi-Plex Ca column (300 mm × 7.7 mm, 8 µm), operating at 80 °C. During the analysis, deionized water was used as the mobile phase, with an elution rate of 0.6 mL/min. Detection was performed using a RI detector (RefractoMax 520, ERC, Riemerling, Germany). All data acquisition and processing were performed using the Chromeleon™ 7.2 Chromatography Data System (Thermo Scientific). The results are expressed as g/100 g of the grape seed cake ± SD.

2.2.2. Subcritical Water Extraction

The defatted oil cake was subjected to subcritical water extraction using a home-built high temperature/high pressure reactor, described in [45]. The ratio of the grape seed cake to water was 1:20 w/v. Two different extraction temperatures were used, 130 °C and 170 °C, with a heating rate of approximately 10 °C/min. Prior to heating, the reactor was pressurized with 99.999% nitrogen (Messer, Belgrade, Serbia) to 10 bars. The extraction time was 40 min in both cases. A vibrational platform was used for mixing (3 Hz). After extraction, the temperature of the reactor was lowered to 20 °C using a flow-through water bath. The extracts were filtered and kept refrigerated (4 °C).

2.3. Statistical Analysis

Results, expressed as the mean ± standard deviation, were statistically analyzed by performing a t-test to determine significant differences at a 95% confidence level (p < 0.05) using Microsoft Excel 16 (Microsoft, Redmond, WA, USA).

3. Results and Discussion

3.1. Proximate Composition of the Grape Seed Cake

The powdered grape seed cake, obtained after cold pressing, was characterized in terms of its moisture, ash, total protein, total lipid, total carbohydrate and insoluble and soluble fiber content. The results, presented in Table 1, were in good agreement with the literature data. Grape seeds (prior to oil extraction) have been reported to approximately consist of fiber (40%), lipids (10–20%) and proteins (10%) [2,8,46,47]. Defatted grape seeds (obtained after hexane oil extraction) [48] were shown to contain approximately 11% protein, 2% lipids, 77% insoluble fiber, 4% soluble fiber, 11% moisture and 3% ash. In a different study [49], the proximate composition of the defatted grape seeds, obtained by hot pressing, was found to be approximately 9% protein, 1% lipids, 80% indigestible total fiber, 7% moisture and 2% ash. The differences between the data are due to the oil content (before and after an oil extraction), moisture content, the oil extraction method (hot pressing, cold pressing or hexane extraction) and, of course, grape variety, which all influence the exact percentages of the main constituents, but overall, the same trends were observed. Also, it is worth noting that, in this work, the composition of the grape seed cake from the specific vinery had been monitored for three consecutive years/harvests and that only small deviations from the presented values had been detected.

3.2. Characterization of the Subcritical Water Extracts

3.2.1. Total Polyphenols, Total Flavonoids and Antioxidative Activity

Although the more reliable quantification of the polyphenolic compounds in the extracts was performed by UHPLC Q-ToF MS (see Section 3.2.2), the more commonly used spectrophotometric methods for the assessment of total polyphenol and flavonoid content were applied as well, in order to compare the results and discuss the validity of the TPC and TFC assays.
Judging by the results of TPC and TFC (Table 2), the influence of the extraction temperature was present but not very pronounced; the total polyphenol content decreased with the increasing temperature, while, on the contrary, the flavonoid content increased. The slight decrease in TPC could be, at first glance, attributed to possible polyphenol degradation at a higher temperature, but this explanation is certainly not applicable to the TFC increase at 170 °C, especially because flavonoids are considered to be sensitive to temperature [50,51]. However, from the data in Table 2, no definite conclusions could be drawn and these comments will be discussed further in Section 3.2.2. For the evaluation of antioxidative activity of the extracts, two methods were used, the phosphomolybdenum method, often referred to as the total antioxidant capacity (TAA), and the DPPH assay. It is always recommended to assess antioxidative activity using more than one method, since the results are influenced by the different reaction mechanisms involved and, for that reason, they do not always correlate [52]. Indeed, in this work, the two applied methods indicated opposite influences of the temperature on antioxidative activity. In the case of TAA, significantly lower activity was measured for the extract obtained at 170 °C, while an increase in activity was observed using the DPPH method. It is reasonable to assume that the methods were sensitive to the different compounds and the specific types of polyphenols and their concentrations. Also, it is worth noting that the lower activity at the higher temperature of extraction, detected by the TAA method, corresponded to a lower TPC, while the DPPH results showed an opposite trend.

3.2.2. Polyphenol Profiles

Detailed polyphenolic profiles of the extracts were obtained using UHPLC Q-ToF MS analysis (Table 3 and Table S1). The MS base peak chromatograms of the extracts are depicted in Figure S1. Fragmentation patterns (MS/MS spectra) of all identified phenolic acid and derivatives (Figure S2a) and flavonoids and derivatives (Figure S2b) are given in the Supplementary Materials. The results (Table 3) showed that, in both extracts, the phenolic acids and their derivatives constituted the majority of the polyphenols. Lower amounts of the flavonoid derivatives (compared to phenolic acids) were observed, predominantly flavan-3-ols and pro(antho)cyanidins (and their derivatives). Anthocyanin and stilbenoid derivatives were also monitored, but were either not detected or were detected in very small concentrations (or below LOQ). Among the phenolic acids and their derivatives, gallic, dihydroxybenzoic and p-hydroxybenzoic acid were found in highest concentrations, followed by ellagic, caffeic, ferulic and p-coumaric acids and methoxycinnamate. Within the flavonoids and derivatives group, dominant species were catechin, gallocatechin, epigallocatechin and epicatechin 3-O-gallate. All these findings were in agreement with the commonly observed grape seed phenolic composition. According to [53], the major polyphenols in grape seed are gallic acid, catechin, epicatechin, gallocatechin, epigallocatechin and epicatechin 3-O-gallate. In a study using acetone as the extraction solvent [54], high amounts of p-hydroxybenzoic, gallic, syringic and vanillic acids were detected, together with caffeic, ferulic acids and p-coumaric acids; among the flavonoids, epicatechin and quercetin were dominant.
The influence of temperature on the polyphenol composition of the extracts was found to be very pronounced. Also, increasing the temperature evidently had a completely different effect on the yield of phenolic acids and flavonoids. The total yield of the phenolic acids increased significantly at a higher extraction temperature, from 557 mg/100 g to 883 mg/100 g. It was evident that raising the extraction temperature facilitated the extraction of phenolic acids, which can most probably be explained by the changes in polarity/the dielectric constant of water under high temperature/high pressure conditions [3], which leads to changes in solubility, as well as with an improved mass transfer. On the contrary, in the case of flavonoids and their derivatives, a large, more than threefold decrease in their total concentration was measured (596 vs. 185 mg/100 g) when the extraction temperature was changed from 130 °C to 170 °C. The reason for this can probably be found in the known sensitivity of flavonoids to high temperatures. It is also interesting to point out that epigallocatechin was an exception: it was detected at a high concentration (163 mg/100 g) at 170 °C, while it was not present at all at 130 °C. Considering the lowering of concentration or the disappearance of related structures (like gallocatechin, epicatechin 3-O-gallate) at 170 °C, it is possible that at a higher temperature, molecular transformations occurred yielding epigallocatechin. In addition, the solubility of epigallocatechin is better in organic solvents, like methanol and ethanol, in comparison to ambient water. Assuming that the solubility in water was modified by the reduced polarity, this could have also contributed to the noticeable increase in the compound content in the extract obtained at 170 °C. The fact that the two families of polyphenols require different extraction temperatures should not be regarded as a grave downside. In fact, this is where the versatility and selectivity of the high pressure/high temperature extraction using subcritical water comes so useful: the same plant material can be fully valorized in terms of specific polyphenol extraction by simply applying an optimal temperature.
Finally, the TPC and TFC results will be discussed, in relation to the presented polyphenol profiles. When the TPC values of the two extracts were compared to the total polyphenol content obtained using UHPLC Q-ToF MS (LC-MS for short), an interesting conclusion can be drawn. Firstly, the calculated contents by the different methods were, of course, different: 3550 mg/100 g (TPC—spectrophotometry) vs. 1154 mg/100 g (LC-MS) and 3300 mg/100 g (TPC—spectrophotometry) vs. 1068 mg/100 g (LC-MS) for 130 °C vs. 170 °C, respectively. These differences may come from the fact that the TPC values determined spectrophotometrically is often considered to overestimate the polyphenol content, because the FC reagent can also be reduced by other nonphenolic substances [55]. However, we must never disregard the fact that TPC is designed to give a relative value, i.e., the result is expressed as an equivalent (usually of gallic acid), not as the true polyphenol content. If we use the obtained TPC values as a relative value and compare the two extracts, we obtain a ratio of 3550/3300 ≈ 1.1 for TPC. Calculating the same ratio for LC-MS results, we obtain 1154/1068 ≈ 1.1 again, which is a remarkable agreement. So, it can be stated that TPC produced a good relative estimation. In the case of flavonoid content, TFC did not accurately depict the extracts; although the TFC value (156 mg/100 g) was not too far from the LC-MS value (185 mg/100 g) for 170 °C, the TFC method completely failed to detect the difference between the flavonoid concentrations of the two extracts.

3.2.3. Total Protein and Amino Acid Profile

The subcritical water extracts were also investigated in terms of their protein content and amino acid profile. The total protein in the extracts was found to be 3.6 ± 0.2 and 5.0 ± 0.1 g per 100 g of the defatted grape seed cake for extractions at 130 °C and 170 °C, respectively. Taking into account the protein content of the defatted cake before the extractions, 13.9 ± 0.2 g/100 g (Table 1), we obtained the percentage of the proteins that were extracted from the starting material, ≈26% and 36% for the extractions at 130 °C and 170 °C, respectively. These protein extraction yields were certainly very encouraging results, since they emphasized the potential use of SWE for the production of protein isolates and hydrolysates. In an investigation focused on defatted grape seed flour as a potential source of protein, using alkaline extraction followed by acid precipitation, the authors found the protein extraction yield to be between 12% and 53%, depending on the extraction conditions [48].
The content of individual amino acids in the two extracts is presented in Figure 1. Seventeen amino acids were detected, with the total amounts being 9.25 and 20.32 g/100 g of dry extract for 130 °C and 170 °C, respectively. Eight essential amino acids were found (Lys, Thr, Val, Ile, Leu, Met, His, Phe), with the total content being 1.77 and 4.86 g/100 g of dry extract for extractions at 130 °C and 170 °C, respectively. Among the non-essential amino acids, Gly, Glu and Arg were found in the largest concentrations in the extract obtained at 130 °C, while Gly, Ser, Pro, Glu and Arg were dominant in the extract obtained at 170 °C. At higher temperatures, higher concentrations were measured for the majority of the amino acids. The highest increase in concentration was detected for Hys (20-fold) and Cys (0.46 vs. ˂0.01 g/100 g), followed by Thr, Gly and Ser (approx. 5-fold). The change in the extraction temperature slightly influenced the percentage of essential amino acids vs. total amino acids, 19% (130 °C) and 24% (170 °C), which was mostly caused by the large concentrations of Gly, Ser and Pro in the 170 °C extract.

3.2.4. Sugars

The HPLC analysis of the sugars in the subcritical extracts was limited to molecules with up to six glycoside units. Among the detected molecules, the most important were those belonging to the group of xylo-oligosaccharides (XOS), xylose, xylobiose and xylotriose. XOS are considered to exhibit beneficial properties, anti-inflammatory, antioxidative, antitumor and antimicrobial, be able to reduce human health-related risks and positively impact growth and resistance to diseases in animals [56]. XOS are mostly prepared using chemical, physical or enzymatic degradation methods. Although this was not specifically the aim of this investigation, the results in Table 4 implied that subcritical water extraction should be further investigated as a possible method for XOS production, using agricultural waste as starting material. The amounts of XOS detected in the extracts clearly show that the higher temperature yielded more xylobiose and xylotriose, which is a good starting point for future work. Arabinose was also detected in both extracts, and it is, together with xylose, considered to have a positive effect on lowering the post-ingestive glycemic and insulinemic responses [57].

4. Conclusions

Subcritical water extraction was applied to defatted grape seed cake, at two temperatures, 130 °C and 170 °C, in a nitrogen atmosphere. The phenolic acids and their derivatives were found to be the majority of the polyphenols, while flavonoid derivatives were also detected, but in much lower concentrations, at both temperatures. The higher temperature favored phenolic acid extraction but resulted in the lowering of flavonoid content, probably due to thermal degradation. The TPC values determined spectrophotometrically correlated well with LC-MS results, while TFC data were not in agreement with the detailed flavonoid profile defined by LC-MS. Protein extraction yield was measured to be ≈26% and 36%, for extractions at 130 °C and 170 °C, respectively. Essential amino acids presented as 19% (130 °C) and 24% (170 °C) of the total amino acid amount in the dry extracts. Xylo-oligosaccharides (XOS), including xylose, xylobiose and xylotriose, were detected in the extracts, with higher yields at the higher extraction temperature. The results clearly support the potential of subcritical water extraction as an efficient extraction method. As assumed, applying a higher temperature led to higher yields of most of the targeted compounds, probably due to matrix decomposition and certainly to changes in water polarity and the subsequent solubility of the compounds. Our results also indicated the versatility of the method, seen here in the case of polyphenol extraction; simply by varying the temperature, the yields of different polyphenol groups were highly affected. Furthermore, it was shown that a significant percentage of protein was extracted from the grape seed cake, which strongly suggests the use of the method for the production of protein isolates/hydrolysates. With all this in mind, the subcritical water extraction method could play a significant role in the concept of circular economy, solving economic and environmental issues in agriculture and food production, waste management and the production of compounds for food fortification.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr13051597/s1, Figure S1: MS-Base peak chromatograms of phenolic compounds derived by subcritical water extraction of grape seed cake at: (a) 130 °C; (b) 170 °C; in negative ionization mode. The peaks of all identified compounds were extracted from these chromatographs based on monoisotopic masses of their precursor ions (see Table 3 (manuscript)); Figure S2. (a) Fragmentation patterns (MS/MS spectra) of all identified phenolic acids and derivatives, which are presented in Table 3. Compounds: (1) Gallic acid; (2) Gallic acid hexoside is. I; (3) Dihydroxybenzoic acid; (4) Gallic acid hexoside is. II; (5) Vanillic acid; (6) p-Hydroxyphenylacetic acid; (7) Homovanillic acid; (8) Ethyl gallate; (9) Ellagic acid; (10) Esculetin; (11) Caffeic acid; (12) Ferulic acid; (13) p-Coumaroyltartaric acid; (14) p-Coumaric acid; (15) Methoxycinnamate; (16) p-Coumaric acid methyl ester; (17) Caffeic acid ethyl ester. (b) Fragmentation patterns (MS/MS spectra) of all identified flavonoids and derivatives, which are presented in Table 3. Compounds: (18) Gallocatechin; (19) (Epi)catechin hexoside; (20) Catechin; (21) Epicatechin 3-O-gallate; (22) Epigallocatechin; (23) Amurensisin; (24) (Epi)catechin-(epi)afzelechin A-type; (25) (Epi)gallocatechin ferulate; (26) Ethyl (epi)catechin-(epi)catechin; (27) Afzelechin; (28) Procyanidin dimer B-type is. I; (29) Gambiriin A; (30) Procyanidin dimer B-type is. II; (31) Procyanidin dimer B type gallate is. I; (32) Procyanidin dimer B type gallate is. II; Table S1: UHPLC Q-ToF MS characterisation and identification of phenolic compounds derived from grape seed cake by subcritical water extraction. Identified compounds, expected retention time (RT), molecular formula, calculated mass, m/z exact mass and MS fragments.

Author Contributions

Conceptualization, M.V.M., J.Š.-G. and V.A.R.; investigation, M.V.M., J.Š.-G., V.A.R., S.M.L., M.B.P. and D.D.M.; resources, J.Š.-G., S.M.L., M.B.P. and V.M.R.; data curation, S.M.L., A.S.S.-J. and D.D.M.; writing—original draft preparation, M.V.M. and V.A.R.; writing—review and editing, J.Š.-G., A.S.S.-J. and D.P.; supervision, V.M.R.; project administration, V.M.R., S.M.L. and V.A.R.; funding acquisition, V.M.R. and S.M.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (project EUREKA E!13299 HEALTHYSEED and the contracts 451-03-137/2025-03/200116, 451-03-137/2025-03/200383, and 451-03-136/2025-03/200134) and by the Science Fund of the Republic of Serbia (#Grant No. 7744714, FUNPRO).

Data Availability Statement

The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Processes 13 01597 g001
Figure 1. The amino acid content of the subcritical water extracts, g/100 g of dry extract. Different letters indicate significant differences (p < 0.05) between the values for a specific compound.
Figure 1. The amino acid content of the subcritical water extracts, g/100 g of dry extract. Different letters indicate significant differences (p < 0.05) between the values for a specific compound.
Processes 13 01597 g001
Table 1. Proximate composition of the grape seed cake, g/100 g.
Table 1. Proximate composition of the grape seed cake, g/100 g.
Moisture8.8 ± 0.1
Ash3.8 ± 0.1
Lipids5.1 ± 0.1
Proteins13.9 ± 0.2
Carbohydrates68.2 ± 0.1
Insoluble fiber57.0 ± 2.0
Soluble fiber3.9 ± 0.4
Table 2. Total polyphenol and flavonoid content and antioxidative activity.
Table 2. Total polyphenol and flavonoid content and antioxidative activity.
SampleTPC
(mg GAE/g)		TFC
(mg RE/g)		TAA
(mg AAE/g)		DPPH
(mg AAE/g)
130 °C35.5 ± 0.3 a1.52 ± 0.01 a69.1 ± 0.4 a0.915 ± 0.004 a
170 °C33.0 ± 0.4 b1.56 ± 0.01 b55.4 ± 0.9 b0.979 ± 0.015 b
GAE—gallic acid equivalent; RE—rutin equivalent; AAE—ascorbic acid equivalent. Different letters indicate significant differences (p < 0.05) between the values within the same column.
Table 3. UHPLC Q-ToF MS identification and semi-quantification (mg/100 g of grape seed cake) of phenolic compounds derived by subcritical water extraction.
Table 3. UHPLC Q-ToF MS identification and semi-quantification (mg/100 g of grape seed cake) of phenolic compounds derived by subcritical water extraction.
Compound NameRTFormulam/z Exact MassMain MS FragmentSamples
130 °C170 °C
Phenolic acids and derivatives
Hydroxybenzoic acid and derivatives
Gallic acid b1.14C7H5O5−169.0162125.0256842 ± 2 A57 ± 4 B
Gallic acid hexoside is. I b1.75C13H15O10−331.0676169.016152.88 ± 0.02-
Dihydroxybenzoic acid b2.83C7H5O4−153.0215108.0227499 ± 5 A99 ± 12 A
Gallic acid hexoside is. II b3.13C13H15O10−331.0676125.0254914.6 ± 0.2<LOQ
p-Hydroxybenzoic acid b4.91C7H5O3−137.0248/318 ± 2 A464 ± 1 B
Vanillic acid b5.86C8H7O4−167.0372123.04583<LOQ4.9 ± 0.2
p-Hydroxyphenylacetic acid b6.33C8H7O3−151.0410108.02286<LOQ6.35 ± 0.05
Homovanillic acid b7.14C9H9O4−181.0513137.06204<LOQ7.12 ± 0.01
Ethyl gallate b7.48C9H9O5−197.0467124.018295.8 ± 0.2 A11.2 ± 0.5 B
Ellagic acid a7.88C14H5O8−301.0021301.0020617 ± 1-
∑499.28649.57
Hydroxycinnamic acid and derivatives
Esculetin c4.14C9H5O4−177.0216121.0309330 ± 13 A38 ± 5 A
Caffeic acid c4.44C9H7O4−179.0363179.0363211.0 ± 0.3 A44.5 ± 0.9 B
Ferulic acid c5.39C10H9O4−193.0523137.0622210.99 ± 0.01 A97.3 ± 0.8 B
p-Coumaroyl tartaric acid c6.12C13H11O8−295.0466119.05145<LOQ-
p-Coumaric acid a6.60C9H7O3−163.0417163.041691.76 ± 0.09 A11.4 ± 0.2 B
Methoxycinnamate c7.65C10H9O3−177.0576133.030961.80 ± 0.09 A38.8 ± 0.7 B
p-Coumaric acid methyl ester c8.42C10H9O3−177.0568162.029651.44 ± 0.02 A3.46 ± 0.04 B
Caffeic acid ethyl ester c9.74C11H11O4−207.0669133.030731.11 ± 0.02<LOQ
∑58.1233.46
Flavonoids and derivatives
Flavan-3-ols and derivatives
Gallocatechin d1.68C15H13O7−305.0683109.03057112 ± 3-
(Epi)catechin hexoside d5.18C21H23O11−451.1269289.0755324 ± 3-
Catechin a6.40C15H13O6−289.0739123.04661327 ± 5 A18.6 ± 0.9 B
Epicatechin 3-O-gallate d7.94C22H17O10−441.0843169.0161271.99 ± 0.09-
Epigallocatechin d8.49C15H13O7−305.0709137.02601-163 ± 2
Amurensisin d8.62C22H15O10−439.0721287.023182.2 ± 0.4-
(Epi)catechin-(epi)afzelechin A-type d8.69C30H23O11−559.1270269.049485.5 ± 0.2-
(Epi)gallocatechin ferulate is. I d8.73C25H21O10−481.1186289.075842.92 ± 0.03-
Ethyl (epi)catechin-(epi)catechin d 9.50C32H29O12−605.1703315.091112.38 ± 0.01-
Afzelechin d 10.51C15H13O5−273.0808124.017427.3 ± 1.8 A3 ± 1 B
∑555.29184.6
Pro(antho)cyanidins and derivatives
Procyanidin dimer B type is. I e6.07C30H25O12−577.1404289.0754412.9 ± 0.5-
Gambiriin A e6.34C30H27O12−579.1546289.0756818.73 ± 0.25-
Procyanidin dimer B type is. II e7.34C30H25O12−577.1403289.076106.0 ± 0.6-
Procyanidin dimer B type gallate is. I e7.35C37H29O16−729.1512407.083553.39 ± 0.06-
Procyanidin dimer B type gallate is. II e8.01C37H29O16−729.1521729.1692<LOQ-
∑41.02-
∑∑ total polyphenols1153.691067.63
Quantification results are shown as means ± standard error (n = 2). Abbreviations: “-“ nonidentified compounds. Compound quantities expressed using available standards a; Compounds expressed as a gentisic acid equivalent b; Compounds expressed as a coumaric acid equivalent c; Compounds expressed as a catechin equivalent d; Compounds expressed as a procyanidin B1 equivalent e; <LOQ—less than limit of quantification. Different letters (uppercase) indicate significant differences (p < 0.05) between the values within the same row.
Table 4. Content of detected sugars, g/100 g of defatted grape seed cake.
Table 4. Content of detected sugars, g/100 g of defatted grape seed cake.
130 °C 170 °C
glucose1.8 ± 0.1 a1.04 ± 0.04 b
galactose/xylose + mannose0.58 ± 0.03 a1.76 ± 0.04 b
arabinose2.1 ± 0.1 a1.62 ± 0.02 b
xylobiose0 a3.16 ± 0.04 b
xylotriose2.88 ± 0.06 a4.7 ± 0.1 b
sucrose2.7 ± 0.2 a1.72 ± 0.05 b
Different letters indicate significant differences (p < 0.05) between the values within the same row.
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Malićanin, M.V.; Švarc-Gajić, J.; Lević, S.M.; Rac, V.A.; Salević-Jelić, A.S.; Pešić, M.B.; Milinčić, D.D.; Pasarin, D.; Rakić, V.M. Valorization of Grape Seed Cake by Subcritical Water Extraction. Processes 2025, 13, 1597. https://doi.org/10.3390/pr13051597

AMA Style

Malićanin MV, Švarc-Gajić J, Lević SM, Rac VA, Salević-Jelić AS, Pešić MB, Milinčić DD, Pasarin D, Rakić VM. Valorization of Grape Seed Cake by Subcritical Water Extraction. Processes. 2025; 13(5):1597. https://doi.org/10.3390/pr13051597

Chicago/Turabian Style

Malićanin, Marko V., Jaroslava Švarc-Gajić, Steva M. Lević, Vladislav A. Rac, Ana S. Salević-Jelić, Mirjana B. Pešić, Danijel D. Milinčić, Diana Pasarin, and Vesna M. Rakić. 2025. "Valorization of Grape Seed Cake by Subcritical Water Extraction" Processes 13, no. 5: 1597. https://doi.org/10.3390/pr13051597

APA Style

Malićanin, M. V., Švarc-Gajić, J., Lević, S. M., Rac, V. A., Salević-Jelić, A. S., Pešić, M. B., Milinčić, D. D., Pasarin, D., & Rakić, V. M. (2025). Valorization of Grape Seed Cake by Subcritical Water Extraction. Processes, 13(5), 1597. https://doi.org/10.3390/pr13051597

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