Pre-treatment of grape by-products for the enhancement of mass transfer phenomena: conventional and alternative techniques
Phenolic compounds exist in plants enclosed in particular structures such as the vacuoles of plant cells and lipoproteins bilayers.15 In intact cells, the membrane envelope restricts the exchange between the intracellular media and the surrounding solvent. Consequently, conventional solvent extraction techniques such as maceration or diffusion require long extraction time, due to the slow diffusion of solvent and solute through the solid.16 Thus, the degradation of cell-wall and of intracellular components is a fundamental step to improve the release of these compounds from the grape tissues. Extraction processes can be enhanced by several pre-treatments of the plant materials that are able to physically damage the cells, such as: grinding, pulsed electric field, high voltage electric discharges and ultrasound.
Pulsed electrical field (PEF) assisted extraction
Electroporation phenomena: When subjected to an external electric field, the charge accumulation on the membrane surfaces induces the increase of transmembrane potential of the cell membrane, initiating pore formation.26 Typically, electroporation phenomena requires some threshold value of transmembrane potential around 0.5 – 1.5 V.27 Above the critical value of transmembrane potential, the expansion of pores present in weak areas of the membrane will induce drastic increase of permeability 28,29 and will facilitate the leakage of intracellular compounds.30,31 Thus, Pulsed Electric Field (PEF) treatment increases transmembrane transport of molecules.31,32 For cellular tissues of 60-120 µm in diameter, initiation of pore formation can be achieved using electric field strengths of 0.1 – 0.5 kV/cm and treatment times of very short duration (within 10-4 – 10-2 s)33 without any significant temperature increase.34,35
Pulsed electric field pre-treatment of winery by-products: PEF treatment prior to conventional extraction allowed a better recovery of phenolic compounds from different winery by-products (Table 1). In most of these studies, the raw materials were submerged into water in order to improve electrical contacts between electrodes.
Treatment with liquid-to-solid ratio above 5 required high electric field strength (i.e. E > 13 kV/cm) to be effective for the enhancement of polyphenols extraction. As a consequence, specific energy consumptions were relatively high (i.e. 272 < W < 762 kJ/kg of treated raw material). On the contrary, pre-treatment by PEF combined with an accurate densification of wet pomace or wet skins can be achieved at lower electric field strengths (i.e. E ≈ 1.2 kV/cm) and lower energy requirements (i.e. 18 < W < 30 kJ/kg of treated raw material). The treatment of compacted wet winery by-products requires less output current, which can be advantageous for the industrial implementation of PEF.36 Pulse forms used were of different shape (monopolar, bipolar or exponential). However, no comparison of the effect of pulse shape on the extractability of phenolic bio-components is available in the existing literature.
Interestingly, a previous study showed that PEF treatment causes irreversible perforations in the cell wall of the outer hypodermis and distention of the fiber cell wall polysaccharides at the inner hypodermis.37 This electroporation phenomenon may allow the specific recovery of anthocyanins that are particularly located in the upper cell layers of the hypodermis. For instance, High Intensity Pulsed Electrical Field (Hi-PEF) treatment of fermented grape pomace (13.3 kV/cm, W = 272 kJ/kg) allowed the selective recovery of anthocyanins and the production of extracts with a high ratio anthocyanins/Total Phenolic Compounds (TPC). This reflects an increase of 40 % that cannot be achieved by conventional extraction procedure, such as grinding combined to diffusion (ratio anthocyanins/TPC < 5 %).38 Moderate PEF treatments (E < 3.0 kV/cm, W < 20 kJ/kg) were also effective in enhancing anthocyanins extraction from grape skins (+ 17 %)39 and grape pomace (+ 19 %)36. Consequently, PEF can replace conventional pre-treatments of grape by-products (e.g. dehydration and grinding), which have impacts on product quality and are more energy consuming, with the combined objectives of cost reduction and selectivity of extraction.
Conventional extraction experiments
Polyphenols extraction from grape pomace (100.0 ± 0.1 g) was carried out in a mixture of ethanol and water (50/50, v/v), maintained at the ambiant temperature in a cylindrical extraction cell. The liquid-to-solid ratio was maintained at the value of 5. A gentle agitation at 160 rpm (16.8 rad·s− 1) was provided using a round incubator of 12.5 mm shaking throw (Infors HT Aerotron, Bottmingen, Switzerland). For untreated samples, the same protocol of extraction was used. Regular sampling was carried out during 420 min of extraction. At the end of extraction, the juice was separated from grape pomace by centrifugation (Model 3-16P, Sigma Laborzentrifugen GmbH, Germany) at 3076 g during 10 min, and stored at − 18 °C for further analysis.
Total polyphenols content
The total phenolic content was spectrophotometrically measured according to a modified Folin Ciocalteu method to be applied in 96-well microplates. Stock solutions (10 mg/mL) of the grape pomace extracts were prepared in EtOH/H2O (25:75, v/v), and a microplate spectrophotometer (MultiSkan Spectrum, Thermo Scientific) was used for the incubation and measurement. Briefly, each well was filled with 184 μL of distilled water and 24 μL of the sample solution, followed by 12 μL of the Folin Ciocalteu reagent and 30 μL of 20% (w/v) Na2CO3 solution. Prior to the measurement of the absorbance at 765 nm, the mixture was incubated for 1h under dark conditions at 25°C. Gallic acid (0−24 mg/L) was used as a standard for calibration. Results, expressed as milligrams of gallic acid per 100 g of grape pomace sample (on a dry matter basis, DM), were a mean of six determinations.
Polyphenols extracted from grape are well-known for their antioxidant capacity. This antioxidant activity is not a single reaction but comprises a wide range and multiple mechanisms. It usually recommended using several techniques since no single technique is able to take into consideration all antioxidant mechanisms.
Therefore, four different antioxidant capacity assays were used: the fluorometric ORAC assay, which is based on hydrogen transfer and spectrophotometric ABTS, CUPRAC, and FRAP assays, which are based on electron transfer. FLUOstar Optima (BMG LabTech) was used for the first essay and an automated microplate reader (MultiSkan Spectrum (Thermo Scientific) for the other three analyses. As for the total phenolics assessment, for the antioxidant capacity spectrophotometric methods, solutions of the stem extracts (4 mg/10 mL) were prepared in EtOH/H2O (25:75, v/v). More diluted stock solutions of the sample extracts (20 mg/1L) were prepared in 75 mM phosphate buffer (pH 7.4) for the ORAC measurement. The difference in absorbance between a final reading and the reagent blank reading was correlated with Trolox standard curves in all assays. Because the moisture level of each pomace extract sample was quite different, antioxidant capacity was reported on a dry weight basis to enhance comparison with the literature. Thus, the results were expressed as milligrams of Trolox per gram of grape sample (DM). Each result value was a mean of six determinations
In 96-well microplates, the ABTS radical cation (ABTS•+) was prepared by the reaction of equivalent volumes (1:1) of both aqueous solutions of 7 mM ABTS and 2.45 mM potassium persulfate. This stock solution was allowed to react for 12–16 h at room temperature in the dark and used within the two following days stored in the same thermal and light conditions. At the moment of the analysis 8 mL of the ABTS solution was diluted with EtOH/H2O (25:75, v/v) in a 100 mL volumetric flask to obtain an absorbance of 1.00 ± 0.02 unit at 734 nm. In a 96-well microplate, extract solutions and ABTS reagent (190 μL in each well) were prewarmed at 25 °C for 20 min. Then, a reagent blank reading was taken at a wavelength of 734 nm. The reaction was carried out by adding 10 μL of the pomace extract solution to each well. After 3 min of shaking, the mixture was incubated at the same temperature for a 30 min period, and then the absorbance decrease was measured at the same wavelength. Trolox standard solutions were prepared at a concentration ranging from 0 to 0.8 mM (R2 = 0.995), by using EtOH/H2O (25:75, v/v) as a solvent.
Flavan-3-ols and gallic acid analyses
The HPLC system used for flavan-3-ols and gallic acid analysis was an Agilent 1200 HPLC Series (Agilent Technologies, Germany) equipped with a diode array detector. The samples were diluted (ratio 1/10) in water and then filtered through PTFE filters (Ø = 0.45 μm). A volume of 60 μL was injected in a Prontosil C18AQ column (4.6 × 250 mm, 5 μm, Bischoff Chromatography, Germany), operated at 25 °C in reverse phase. Solvent A, 0.1% trifluoroacetic acid (TFA) in water and solvent B, 0.1% TFA in acetonitrile, were used for elution at the flow rate of 1 mL·min− 1. The elution gradient had the following profile: t0 min B (7%), t2 min B (7%), t10 min B (16%), t40 min B (31%), t45 min B (50%), t48 min B (100%), t53 min B (100%), t54 min B (7%), and t59 min B (7%). The detection wavelength was 280 nm. Individual flavan-3-ols and gallic acid were identified using the corresponding standard compounds. Results were expressed as g of catechin equivalent/100 g RM for monomers and g of procyanidin B1 equivalent/100 g RM for oligomers.
Total Proanthocyanidins Content
The total proanthocyanidins contents of grape pomace by-products, obtained by Bate-Smith reaction, are shown in Table 2. Similar to phenolic content quantification by Folin Ciocalteu method, Dunkelfelder subcritical water extraction at 200°C showed the highest content of 72.52 ± 2.43 mg/g DM, whereas Cabernet Franc pomace presented the lowest value 11.67±1.67 mg/g DM. Significant differences (p < 0.05) were observed among the grape varieties investigated, the years of harvest and the temperature of extraction.
Temperature of extraction had a high influence on the extracted total proanthocyanidins. In our case for example polyphenols extracted from Chardonnay pomace showed an increase from 54.20±1.33 to 68.37±4.17 mg/g DM content of total proanthocyanidins by increasing the temperature from 100°C to 200°C and the polyphenol content above conventional solvent extraction at temperatures higher than 100°C.
Additionally the year of harvest had an important influence. Dunkelfleder variety with the three extraction temperatures significant differences were shown for the proanthocyanidin content, for example at 150°C extraction of 2012 harvest was 68.76 ± 2.55 and 2013 harvest 52.31 ± 0.59 g of tannins/100g DM.
Total proanthocyanidin Bate-Smith test is a coloration method to detect the presence of condensed tannins; which is important fraction in the extract that is usually overlooked for quantification subcritical water pomace extracts. For this reason it was difficult to compare results to other subcritical water extracts. Nonetheless results obtained in the present study were similar to values previously reported in the literature for pomace by-products from white and red grape varieties extracted using an organic solvent (Rockenbach et al. 2011; Mandic et al. 2008; Obreque-Slier et al. 2010; Travaglia et al.; González-centeno et al. 2012). Nonetheless, observed total tannins values 68 mg/g DM for Chardonnay pomace 2.2-fold higher than those obtained by González-centeno et al. 2012 using a solvent of MeOH/water (60:40, v/v) extraction. These differences could be attributed to the different vintage and viticulture conditions of the samples.
As previously observed in several studies (Mandic et al. 2008; Lorrain et al. 2013; Ky et al. 2014) a high significant correlation was found between the total phenolic and total proanthocyanidin contents of the grape pomace extracts (r = 0.94, p < 0.05).
HPLC Analysis of Monomeric and Oligomeric Flavan-3-ols
The monomeric and oligomeric flavan-3-ol composition of the grape pomace by-product from the four grape varieties extracted by subcritical water at different temperatures were investigated and described in Table 2. All the extracts were analyzed by HPLC to identify and quantify the flavan-3 ols procyanidin B1, (+)- catechin, (− )-epicatechin, and the trimer C1, in this order of elution.
The combined amount of the above flavan-3-ols in grape pomace by-products ranged from 27.90 to 198.86 mg/100 g DM, for Merlot (SWE 100°C) and Dunkelfelder (2012, SWE 200°C) varieties, respectively. These results are in accordance to results previously published with the total flavan-3-ol range (29−199 mg/100 g DM) proposed by Luque-Rodríguez et al. 2007 for red grape pomace by-product (8 MPa, 120 °C, 1:1 (v/v) ethanol, 0.8% (v/v) HCl). Significant differences were found among the four varieties considered, the year of harvest, and temperature of extraction (p < 0.05), both Dunkelfelder and Chardonnay exhibiting the highest total flavan-3-ol content of 198 mg/100 g DM and 97 mg/100 g DM respectively at 200°C. Temperature of subcritical water extraction was the most important factor yielding to a varied amount of Flavan-3-ols, for example increasing the temperature from 100°C to 200°C lead an increase of 1.37 to 1.91 folds of extracts Flavan-3-ols. Temperature had a differential influence on individual compounds, (+)-catechin and (- )-epicatechin were optimally extracted at 200°C for all grape pomaces. While Proanthocyanidins B1 and C1 were optimally extracted at 150°C for Cabernet Franc, Merlot and Chardonnay.
Table of contents :
1. CHAPTER 1: STATE OF THE ART ALTERNATIVE PROCESS OF EXTRACTION AND PURIFICATION OF HIGH ADDED VALUE COMPOUNDS FROM GRAPE BYPRODUCTS
1.2. EXTRACTION AND PURIFICATION OF HIGH ADDED VALUE COMPOUNDS FROM BYPRODUCTS OF THE WINEMAKING CHAIN USING ALTERNATIVE/NON-CONVENTIONAL PROCESSES/TECHNOLOGIES
1.2.2. Pre-treatment of grape by-products for the enhancement of mass transfer phenomena: conventional and alternative techniques
220.127.116.11. Pulsed electrical field (PEF) assisted extraction
18.104.22.168. High voltage electrical discharges (HVED) assisted extraction
22.214.171.124. Ultrasound (US) assisted extraction
126.96.36.199. Comparison of pre-treatment processes
1.2.3. Solid-to-Liquid extraction (SLE) of high added value compounds
188.8.131.52. Conventional extraction technique: Low pressure extraction using organic solvents
184.108.40.206. High-pressure extraction
220.127.116.11.1. High temperature and high-pressure extraction/ Subcritical water extraction (SWE)
18.104.22.168.2. Supercritical fluid extraction (SFE)
22.214.171.124. Comparison of extraction processes
1.2.4. Purification and fractionation of the extract
126.96.36.199. Solid phase extraction
188.8.131.52. Resin adsorption
184.108.40.206. Membrane processes
2. CHAPTER 2: SUBCRITICAL WATER EXTRACTION OF HIGH ADDED VALUE COMPOUNDS FROM FERMENTED GRAPE POMACE
2.2. CHARACTERIZATION OF POLYPHENOLS AND ANTIOXIDANT POTENTIAL OF RED AND WHITE POMACE BY-PRODUCT EXTRACTS USING SUBCRITICAL WATER EXTRACTION
2.2.2. Material and methods
220.127.116.11. Raw material
18.104.22.168. Process of extraction and parameters
22.214.171.124. Conventional extraction experiments
126.96.36.199.1. Total polyphenols content
188.8.131.52.2. Antioxidant activity
184.108.40.206.2.1. ABTS Assay
220.127.116.11.2.2. CUPRAC Assay
18.104.22.168.2.3. FRAP Assay
22.214.171.124.2.4. ORAC Assay
126.96.36.199.3. Anthocyanins analyses
188.8.131.52.4. Flavan-3-ols and gallic acid analyses
2.2.3. Results and discussion
184.108.40.206. Total Polyphenol Content.
220.127.116.11. Total Proanthocyanidins Content
18.104.22.168. HPLC Analysis of Monomeric and Oligomeric Flavan-3-ols
22.214.171.124. HPLC Analysis of Anthocyanins for red grape by-products
126.96.36.199. Antioxidant Capacity
2.3. SUBCRITICAL WATER EXTRACTION AND NEOFORMATION OF ANTIOXIDANT COMPOUNDS FROM DUNKELFELDER GRAPE POMACE
2.3.2. Material and methods
188.8.131.52. Raw material
184.108.40.206. Process of extraction and parameters
220.127.116.11. Conventional extraction experiments
18.104.22.168.1. Total polyphenols content
22.214.171.124.2. Antioxidant activity
126.96.36.199.3. Anthocyanins analyses
188.8.131.52.4. Flavan-3-ols and gallic acid analyses
184.108.40.206.5. Analysis of Hydroxymethylfurfural (HMF) and Furfural
2.3.3. Results and discussion
220.127.116.11. Influence of operating parameters
18.104.22.168. Flow rate/hydraulic retention time
22.214.171.124. Temperature influence on the extract composition
126.96.36.199. Antioxidant Capacity of the Extracts
188.8.131.52. Maillard and Caramelization Reactions
3. CHAPTER 3: FRACTIONATION OF DIFFERENT PHENOLIC CLASSES FROM GRAPE POMACE EXTRACTS BY MEMBRANE PROCESSES
3.2. SELECTING ULTRAFILTRATION MEMBRANES TO FRACTIONING HIGH ADDED VALUE COMPOUNDS FROM GRAPE POMACE EXTRACTS
3.2.2. Materials and methods
184.108.40.206. Subcritical water extraction
220.127.116.11. Experimental analysis and membranes
18.104.22.168. Membrane performance
22.214.171.124. Hydraulic resistance, using Darcy’s law
126.96.36.199. Contact angle
188.8.131.52. Chemical analysis
184.108.40.206.1. pH, Total sugars, Polysaccharrides
220.127.116.11.3. Total polyphenols content
18.104.22.168.4. Antioxidant activity – ORAC
22.214.171.124.5. Phenolic classes
3.2.3. Results and discussion
126.96.36.199. Grape subcritical extract composition
188.8.131.52. Membrane performance
184.108.40.206.1.1. Water permeability determination
220.127.116.11.1.2. Influence of operating conditions on the permeate flux
18.104.22.168.2. Retention of compounds
22.214.171.124. Retention of macromolecules
126.96.36.199.1. Retention of polysaccharides
188.8.131.52.2. Retention of proteins
184.108.40.206.3. Retention and fractioning of polyphenols
3.3. THE USE OF NANOFILTRATION MEMBRANES FOR THE FRACTIONATION OF POLYPHENOLS FROM GRAPE POMACE EXTRACT
3.3.2. Materials and methods
220.127.116.11. Experimental equipment and membranes
18.104.22.168. Subcritical water extraction
22.214.171.124. Filtration experiments
126.96.36.199. Analytical methods
188.8.131.52.1. Contact angle
184.108.40.206.2. pH and Total sugars
220.127.116.11.3. Total polyphenols content
18.104.22.168.4. Antioxidant activity – ORAC
22.214.171.124.5. Phenolic classes:
3.3.3. Results and discussion
126.96.36.199. Water permeability determination
188.8.131.52. Influence of operating conditions on the permeate flux
184.108.40.206. Fouling resistance
220.127.116.11. Phenolic compounds fractionation