Gayane
Hayrapetyan
ab,
Karen
Trchounian
b,
Laurine
Buon
c,
Laurence
Noret
a,
Benoît
Pinel
d,
Jeremy
Lagrue
d and
Ali
Assifaoui
*a
aUMR PAM, Equipe PCAV, Université de Bourgogne/Institut Agro, 1 Esp. Erasme, Agrosup, 21000 Dijon, France. E-mail: ali.assifaoui@u-bourgogne.fr
bDepartment of Biochemistry, Microbiology and Biotechnology, Yerevan State University, 1 Alex Manoogian St, Yerevan 0025, Armenia
cCERMAV (Centre de Recherches sur les Macromolécules Végétales), 38041 Grenoble, France
dSFE Process, 107 Bd Tolstoï, 54510 Tomblaine, France
First published on 4th September 2023
Inspired by the concept of organic waste valorisation and heading towards a sustainable economy, a green chemistry extraction technique involving supercritical carbon dioxide (SC-CO2) along with water as a co-solvent was employed for the main winery by-product (grape pomaces). The objective was to selectively extract high-value added molecules, in particular phenolic compounds and polysaccharides. The experimental design involved applying three distinct temperature conditions (40, 60, and 80 °C), and a constant pressure of 400 bar. Phenolic compounds and high-molecular weight isolates (449–478 kDa) containing low methoxyl (% DE = 23) pectic substances were detected in the SC-CO2 extracts accompanied by water as a co-solvent. The phenolic acids, namely, gallic (GA), protocatechuic (PCA), coumaric (CouA), and caftaric (CTA), and flavonoids, namely, procyanidin B1 (PRC B1), procyanidin B2 (+) (PRC B2), (+) catechin (CT), and (−) epicatechin (ECT) were found in all the extracts under the tested experimental conditions. The following study underscores the potential of the pressurized CO2/H2O medium as an effective solvent with minimal environmental impacts for the comprehensive valorisation of the main winery by-product, specifically targeting polysaccharides and phenolic compounds.
Sustainability spotlightThis study focuses on the extraction of valuable molecules such as polyphenols and fibers from grape pomace, a by-product of wine production, using a green chemistry technique called supercritical fluid extraction. These extracted molecules have potential applications as antioxidants in wine production. The research highlights the significance of specific United Nations sustainable development goals, including affordable and clean energy (SDG 7), industry, innovation, and infrastructure (SDG 9), and climate action (SDG 13). |
This work focuses on the SC-CO2 extraction, which is based on the application of carbon dioxide (CO2) as a solvent. The CO2 solvent under its supercritical condition (T = 31.2 °C, P = 74 bar) shows increased affinity towards non-polar substances. Thus, research on SC-CO2 recently reported in the literature primarily focuses on the extraction of oils.5–8 Moreover, the added values of the SC-CO2 process are its high recyclability rate compared to the conventional techniques and the overall reduced energy cost.9 However, CO2 alone is not a good solvent; to increase its solvating power and also attract hydrophilic molecules, the process is often associated with the implementation of additional co-solvents (ethanol/methanol).10,11
The valorization of by-products is often a challenge due to their complex nature. The grape pomace is the main winery by-product that makes up to 20–25% of total freshly pressed grape fruit.12 It is a potential source of high-value added products such as dietary fibres, oils, phenolic compounds and anthocyanins with various proven health benefits.13,14 It consists of skins (42.4%), seeds (22.5%), stalks (24.9%) and other minor components. The grape skin is rich in polysaccharides such as cellulose, hemicellulose, xyloglucan, arabinan, xylan, and mannan that constitute 30–35%, among which ∼20% are moderately methyl-esterified (62%) pectic substances, some structural proteins (<5%) and phenolic compounds.15
Despite its considerable socio-economic importance, contributing to a global wine production of approximately 260 million hectoliters in 2022,16 winemaking produces a substantial quantity of winery by-products. Although the pomace is a natural and completely biodegradable source, its huge amounts in vineyards generated within a short period of time are often associated with various environmental hazards such as soil (change in pH) and water pollution, unpleasant odors, insect attraction and spread of diseases.17 While in some countries the pomace is directly disposed in vineyards as a fertilizer, the European Union (EU) regulations request to send the winery by-products into the distillery sites for alcohol, spirits and piquette production only (Commission Delegated Regulation (Eu) 2018/273 of 11 December 2017, Article 14).
In the grape skin, phenolic compounds are cross-linked to pectic polysaccharides via hydrophobic interactions and hydrogen bonds and are later released upon fruit maturation.15,18 The phenolic compounds are secondary metabolites of plant-based organisms; these molecules are considered as natural alternatives to synthetic antioxidants and have been extensively studied using traditional and green chemistry extraction techniques over the years.10,18–20 Furthermore, water as a co-solvent in the SC-CO2 process is not applied commonly, and only some studies showed its potential application for the extraction of phenolic compounds. Da Porto et al.2 targeted mainly proanthocyanidins and phenols from the grape pomace by applying both water and/or ethanol 15% (w/w) as co-solvents in the SC-CO2 process under 40, 50, and 60 °C and 100 or 200 bar pressure experimental conditions.
Polysaccharides such as pectin in grapes are responsible for various physiological processes such as immune response to abiotic and biotic stresses.21 It has quite important economic value and is extensively used in food, cosmetic and pharmaceutical industries such as gelling, emulsifying and stabilizing agents. Some alternative extraction methods such as ultrasound22 and microwave-assisted (from citrus)23–25 were applied for pectin extraction in order to find better alternatives to the outdated conventional acid extraction. However, the alternative methods continue the use of harmful chemicals, and hence, they could not be considered “fully green”. In the context of finding sustainable solutions with the least environmental impact, the SC-CO2 together with water as a co-solvent is a promising medium and could potentially target polysaccharides and phenolic compounds. Furthermore, in contrast to SWE, which involves significantly higher temperatures (T = 100–374 °C), SC-CO2 operates at lower temperatures, thus safeguarding extracts from heat-induced degradation. There is indeed a huge gap in the research dedicated to the pectin extraction by fully green chemistry methods, especially by SC-CO2. To the best of our knowledge, only a study by the time of writing reported the extraction of bioactive compounds including pectin by SC-CO2 alone.26 The aim of this work is to valorise the main winery by-product (grape pomaces) by SC-CO2 along with water as a co-solvent. The extracted molecules were further characterized by various physical and biochemical methods.
The standards of white wine-related phenolic compounds such as gallic, protocatechuic, hydroxybenzoic, caftaric, gentisic, caffeic, coumaric, and chlorogenic acids and hydroxytyrosol, tyrosol, procyanidin B1, B2, (+) catechin and (−) epicatechin were purchased from Sigma-Aldrich (Steinheim, Germany). The monosaccharide standards (mannitol, L-fucose, L-rhamnose, L-arabinose, D-galactose, D-glucose, D-mannose, D-xylose, D-galacturonic and glucuronic acids) were purchased from Acros Organics and Sigma-Aldrich. Sodium hydroxide (NaOH) solution 46/48% (Fisher Bioblock), sodium acetate (NaOAc) (Sigma-Aldrish), trifluoroacetic acid (TFA) (Merck) and all the above-mentioned chemicals were of analytical grade.
The experimental plan was designed based on Moller's diagram and the experimental conditions were chosen to fall within the supercritical region of CO2, accompanied by the highest density values. An exact mass (26 g) of dry grape pomace powder was introduced into the extraction basket, the experiments were conducted twice for each condition and the extracts were collected in the corresponding containers. A sequential extraction process was applied to the matrix at a constant pressure of 400 bar, which was chosen to be efficient according to the preliminary experimental data. Moving forward the extracts will be labelled: SC-40, SC-60 and SC-80 which correspond to the applied temperatures 40, 60, and 80 °C and a constant pressure of 400 bar accordingly.
The supercritical condition of CO2 can be reached at different temperature and pressure values, where the minimal conditions are T = 31.2 °C and P = 74 bar. At the beginning of the process, CO2 exists in a mixed gas/liquid form, and then it undergoes decompression under high pressures. As it advances towards the pre-heating step and just before entering the extraction basket, it transitions into a liquid state. This marks the point of extraction. The process is further followed by the decompression step after the back pressure regulation. Eventually CO2 leaves the system by liberating into the atmosphere. It should be noted that Moller's diagram (enthalpy–entropy chart) is applicable when pure CO2 is applied and it changes depending on the co-solvents introduced.
The pomace was initially defatted using a continuous flow of CO2 (30 g min−1) for an hour under each tested experimental condition. Upon the termination of oil extraction, ethanol as a co-solvent was introduced into the system to collect the remaining oily substances. The characterization of the extracted oil will not be presented in this article.
Following the de-fatting process, water as a co-solvent (flow rate = 10 g min−1) was introduced into the system. The use of water as a co-solvent requires precise management of the temperature in the sapphire separator, which is a crucial factor for extract collection. For instance, higher temperatures (>30 °C) in the sapphire separator lead to the condensation of water and may initiate the formulation of CO2 ice blocks in the condenser, thus affecting the extract collection. Hence, it is important to maintain the separator temperature within the range of 20 to 30 °C, along with a pressure of 50 bar, to ensure that CO2 remains in liquid form for its uninterrupted circulation.
The extraction stopped manually following the color change in the transparent sapphire separator, when the extracts from deep burgundy tuned into pale pink. All the extracts were collected in the corresponding glass containers and stored at −4 °C until future characterizations. These liquid fractions were first characterized in terms of phenolic compounds by UHPLC and then alcohol insoluble fractions (AIF) were separated by alcoholic precipitation to target mainly pectic polysaccharides. The isolates were later characterized by FT-IR (Fourier-transform infrared) spectroscopy, HPAEC-PAD (high-performance anion-exchange chromatography/pulsed amperometric detection) and HPSEC (high-performance size-exclusion chromatography) for monosaccharide composition and molecular weight determination.
The SC-CO2 extracts prior injections were filtered using a PTFE filter (0.45 μm PTFE, Restek, Lisses, France) and 2 μL of each filtrate was loaded into a C18 acid-resistant column (Raptor ARS-18, 150 × 2.1 mm, 1.8 μm, 1000 bar, Restek, Lisses, France). Reverse-phase chromatography has been used. The stationary phase was composed of non-polar (hydrophobic) silica-grafted C18 alkyl chains. A binary solvent was run at a flow rate of 0.30 mL min−1 employing a mobile phase (A) H2O:methanol:trifluoroacetic acid (TFA) (95:5:0.28%) and (B) methanol (100%). The optimized elution system consisted of a stepwise gradient as follows: 3% of B for 5 min, 3–5% B in 3 min, 5% of B for 9 min, 5–9% B for 2 min, 9% B for 6 min, 9–14.3% for 10 min, 14.3–23% of B in 1 min and 23–100% B for 9 min. The column temperature was set at 35 ± 2 °C and the autosampler at 12 °C. Acquisitions, solvent delivery and detectors were controlled using the Empower 2 program.
The diode array detector was set at 320 nm for trans-caftaric, gentisic, trans-caffeic, chlorogenic, trans-coutaric, 2-S-glutathionylcaftaric (GRP), and trans-ferulic acids at 305 nm for salicylic, trans-coumaric, at 280 nm for gallic and at 260 nm for protocatechuic and hydroxybenzoic acids. The fluorescence detector was set at λex = 270 nm and λem = 322 nm for hydroxytyrosol, tyrosol, catechin, epicatechin, proanthocyanidin B1, and proanthocyanidin B2.
In this work, an HPSEC system (High Performance Size-Exclusion Chromatography – Shimadzu) coupled with a 3-angles light scattering detector (Mini Dawn Treos – WYATT) associated with a refractive index detector (Optilab – WYATT) and a UV detector (Shimadzu) was used.
The SEC columns used for the separation were two Shodex SB 806M HQ in series associated with a pre-column SB-G 6B thermostated at 30 °C. The solvent used was 0.1 M sodium nitrate (NaNO3) including 0.3 g L−1 sodium azide (NaN3) (pH: 6) and the flow rate was 0.5 mL min−1 with an elution time of 70 minutes.
The value of the refractive index increment dn/dc used for this analysis is 0.146 mL g−1, which is a value commonly used for polysaccharide detection (pectin). The lyophilized isolates were dissolved under stirring during 24 h at a concentration of 1 mg mL−1 in an eluent composed of 0.1 M sodium nitrate + 0.03% sodium azide (NaN3). Afterwards, they were filtered through a 0.2 μm filter and 100 μL of each was injected. Softer package Astra 6 (Wyatt Technology Co., USA) has been used for the data treatment.
The column was thermostated beforehand at 28 °C and the volume injected was 5 μL. Pulse potentials (E, volts) and durations (t, ms) were E1 = 0.1, t1 = 0, E2 = 0.1, t2 = 200, E3 = 0.1, t3 = 400, E4 = −2.0, t4 = 410, E5 = −2.0, t5 = 420, E5 = 0.6, t5 = 430, E6 = −0.1, t6 = 440, and E7 = −0.1, t7 = 500.
The elution was carried out at 0.18 mL min−1 under the following conditions: −t = 0 to t = 27 min: NaOH 0.016 M, t = 27 to t = 29.1 min: NaOH 0.016 M + linear gradient in NaOAc from 0 to 0.02 M, t = 29.1 to t = 37 min: linear gradient in NaOH from 0.016 M to 0.1 M and linear gradient in NaOAc from 0.02 M to 0.5 M, t = 37 to t = 43.1 min: a linear gradient in NaOH from 0.1 M to 0.2 M + NaOAc 0.5 M. A calibration curve of the following monosaccharides was carried out with respect to the following elution order: mannitol, fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, galacturonic acid and glucuronic acid.
High-molecular weight polyphenols such as procyanidines (B1 and B2) were also detected in the liquid extracts, which reached their highest value at 80 °C under 400 bar.
Solubility itself is a complex process, which depends on various factors such as the nature of molecules targeted and the extraction parameters. A number of studies in the related literature mainly focused on the experimental part of SC-CO2 with ethanol or methanol as a co-solvent.10,31,32 In an earlier work, (+) catechin and (−) epicatechin were extracted from grape skin by SC-CO2 with 20% ethanol as the co-solvent under the optimal extraction condition at 60 °C and 250 bar and the values ranged from 53.6 to 89.7 and 8.94 to 233.6 mg/100 g depending on the grape skin origin.10 Arce et al. reported even lower values of total polyphenol content from different grape pomaces measured by the Folin-Ciocalteu reagents, which varied between 13 and 30 mg/100 g under the conditions of 5% MeOH, 300 bar, and 50 °C (ref. 32) while much higher recovery was observed in this study using water as a co-solvent. In the present study, when water was used as a co-solvent, the affinity of carbon dioxide towards hydrophilic molecules was significantly enhanced. This enhancement facilitated the extraction of predominantly hydrophilic phenolic compounds. Similar patterns were also reported by other authors working on the SC-CO2 process.2,33
The interactions of carbon dioxide with water molecules under higher pressure conditions lack theoretical understanding. According to the model proposed by Galanakis et al.,34 phenols are generally better solubilized in polar protic media such as ethanol and methanol or hydroalcoholic mixtures; however, some of them such as gallic, cinnamic, and coumaric acids have preference over water instead.
Several thermodynamic properties such as tunable density, high diffusivity and low viscosity are typical to compressible solvents such as carbon dioxide and may have an impact on interfacial properties. Rocha et al.35 Investigated water and CO2 interactions at two pressure (200 and 280 bar) and two temperature values (45 and 65 °C) via molecular dynamics computer simulations. They have shown similarities between CO2 in water (C/W) interface and other conventional oil-in-water (O/W) interfaces, which can be adapted to extract amphiphilic molecules. It must be considered the non-negligible solubility of CO2 in water (24 molecules of CO2 in 1000 water molecules under 200 bar and at 45 °C). The increasing attraction of CO2 towards hydrophilic compounds (SC-80) at higher temperatures observed in this work is in alliance with the theoretical approach discussed.
The SC-40 + H2O liquid extract in this work after alcohol precipitation and separation steps, exhibited no isolates. This outcome is not surprising, considering that 40 °C is still relatively low for the extraction of targeted pectic polysaccharides. The isolates appeared at higher temperatures SC-60 and SC-80 + H2O; therefore, for polysaccharide extraction, the temperature here is a key factor. The extraction with SC-CO2 manually stopped when the burgundy extract became paler, indicating that CO2 under this condition reached its limits of extraction. The extraction yield exhibited an inverse correlation with the extraction time, displaying higher yields during the initial stages of extraction. Subsequently, the yield reached a plateau as the matrix became depleted. This observation aligns with findings from the existing literature.31
The yield of freeze-dried isolates after purification steps was estimated to be 1.4 g/100 g (on dry weight) and 3.2 g/100 g (on dry weight) for the samples SC-60 and SC-80, respectively. The CO2 amount used during the essays overall varied from 1870 g for SC-40, 2690 g for SC-60 and 3540 g for SC-80 respectively. This information could be important for scaling up the SC-CO2 process. The experimental design included three temperature conditions 40, 60, 80 °C at a constant pressure of 400 bar, which corresponds to the density of supercritical fluid of 1000, 900 and 800 kg m−3 respectively when only CO2 is applied. However, once modifiers such as ethanol or water are applied, the density of pure CO2 changes; therefore, for the reproducibility of the results, it is misleading to consider the density values.
It is well known that at higher temperatures, the density of CO2 is low, thus its solubility decreases, while the contrary effect was observed with water as the co-solvent. Furthermore, it should be noted that when CO2 is mixed with water, it is no longer under a supercritical condition. This change is not necessarily a disadvantage, especially when polysaccharides are the target. In addition, CO2 coupled with water as the co-solvent also changes the pH of the medium,36 consequently high pressure and lower pH conditions are naturally favorable for breaking down the cell-wall components and liberating polysaccharides and phenolic compounds.
Temp. [°C] | Rha | Ara | Gal | Glc | Man | Xyl | GalA | GlcA | % Total |
---|---|---|---|---|---|---|---|---|---|
a Fucose: not included-traces. | |||||||||
60 | 2.24 ± 0.13 | 3.26 ± 0.08 | 3.51 ± 0.09 | 1.55 ± 0.03 | 3.51 ± 0.14 | 1.00 ± 0.02 | 6.75 ± 0.13 | 2.02 ± 0.03 | 23.84 ± 0.65 |
80 | 1.18 ± 0.02 | 2.04 ± 0.02 | 2.08 ± 0.04 | 2.04 ± 0.02 | 2.35 ± 0.02 | 0.82 ± 0.02 | 5.31 ± 0.03 | 1.04 ± 0.02 | 16.86 ± 0.19 |
Fig. 3 HPAEC-PAD chromatogram obtained for the SC-CO2 + H2O isolate from the grape pomace. Waveform used: gold, carbo, quad = carbohydrates, gold on PTFE disposable working electrodes. |
The GalA content is highly dependent on various factors such as source quality, extraction and hydrolysis methods. The pectin backbone is composed of repeating sugar units, primarily D-galacturonic acid, linked together by α-1,4-glycosidic bonds. Therefore, the GalA content is an important indicator of pectin in the isolates. The data provided in the Table 1 show that the GalA content decreased by increasing the temperature from 6.75 ± 0.13 to 5.31 ± 0.03 for SC-60 and SC-80 + H2O respectively.
Low values of uronic acid from the grape pomace indicate the presence of small amounts of pectic substances in the samples.37 It is also possible that CO2 may have too much acidified the medium and had degraded pectin's backbone.
The existence of pectic substances in the SC-CO2 + H2O isolates could be explained by the dissolution of CO2 in water under high-pressure conditions. While conventional pectin extraction methods rely on hot acid treatment in the pH range of 1–3, SC-CO2 + H2O under high pressures is a naturally acidified medium, which effectively targets polysaccharides by breaking down the cell wall components. Moreover, the acidified medium does not pose an environmental concern, as liberated CO2 by the end of the process returns to the atmosphere, elevating the medium's pH.36
The pectic substances in the isolates were of lower degrees of methylation: 23.8 ± 0.5% for SC-60 and 22.8 ± 1.0% for SC-80 + H2O isolates accordingly. Similar results were found in the work of Colodel et al.,12 on acid-extracted low methoxyl pectin (DE = 18%, Mw 154 kDa) from white grape variety (Chardonnay) pomaces. We can suppose here that the SC-CO2 + H2O medium activated some natural enzymes present in the grape pomace.
Commercial pectin extraction often yields high-methoxyl pectin (HMP), and low-methoxyl pectin (LMP) needs to further undergo chemical transformations for the groups to be hydrolysed (de-esterification). This is usually achieved by acidic, alkaline or enzymatic treatments (methylesterase). The process augments the production cost and is harmful to the environment. Despite the low GalA content, pectic substances in the isolates obtained in this work are of lower degrees of methylation (LMP) without additional chemical modification.
Fig. 5 HPSEC-MALS-RI elution profile of isolates from the extracts SC-60 and SC-80 + H2O. Light scattering (LS-90° – dashed line) and refractive index (RI – solid line) detectors. |
Fig. 6 HPSEC-MALS-RI elution profile of isolates from the extracts SC-60 and SC-80 + H2O. Light scattering (LS-90° – dashed line) and refractive index (RI – solid line) detectors. |
The absence of a signal in light scattering after 40 minutes of elution time indicates the existence of very small molecules with the molecular weight out of the detection capacity.
The combined average molecular weights (Mw) of pectic substances were estimated to be 478 ± 6 kDa and 449 ± 3 kDa for the isolates from SC-60 and SC-80 + H2O, respectively. Moderately higher temperatures accelerated the thermal motion of molecules enabling better penetration of the solvent into the plant matrix and initiated the extraction of high-molecular weight components. A slightly lower molecular weight observed at 80 °C could indicate the beginning of an early degradation of the polymer chain. Pectin's molecular weight could range significantly based on the source material and extraction method applied (50 to 2000 kDa).38 Commonly used hot acid pectin extraction (HCl) results in lower to medium molecular weight of pectins. In the literature, several values have been reported for pectins isolated from sugar beet (70–355 kDa), banana (87 to 248 kDa) and orange, lemon and bigarade peels (88, 90 and 80 kDa) respectively.39–41
The average Mw of pectin extracted from the grape pomace by ultrasound-assisted methods ranged from 111 to 205 kDa (ref. 22) accordingly. Relatively low-molecular weight pectic substances were estimated by the subcritical water extraction (SWE) technique for citrus peel (CPP) to be 56 to 69 kDa and (APP) apple pomace pectin (APP) to be 28 to 65 kDa.42 The quality of the pectin is associated with high molecular weight. The lower values presented from the authors by SWE may be due to the hydrolysis of pectin's chain at high temperatures (120–130 °C). The results of this work highlight the potential application of the SC-CO2 process with water as the co-solvent not only for the successful extraction of various phenolic compounds but also high-molecular weight polysaccharides. Relatively low temperatures applied by this process put the extracts under lower risk to undergo heat-related degradations.
The UV absorption at 280 nm is characteristic of aromatic compounds such as proteins and polyphenol complexes in the isolates (Fig. 7). The supplementary DUMAS nitrogen analysis showed the nitrogen amount in the isolates to be ranging from 8.2 to 9.5% (on dry weight) while applying a pea protein factor of 5.4 to the SC-CO2 + H2O isolates. Fig. 7 illustrates the UV absorbance at 280, which confirms the existence of residual protein/polyphenol complexes in the isolates.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3su00183k |
This journal is © The Royal Society of Chemistry 2023 |