Food Chemistry 133 (2012) 308–314

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Changes in polyphenol and polysaccharide content of grape seed extract and grape pomace after enzymatic treatment S. Chamorro a, A. Viveros b, I. Alvarez a, E. Vega a, A. Brenes a,⇑ a b

Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN-CSIC), José Antonio Novais, 10, Ciudad Universitaria, 28040 Madrid, Spain Departamento de Producción Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Ciudad Universitaria, 28040 Madrid, Spain

a r t i c l e

i n f o

Article history: Received 4 October 2011 Received in revised form 30 December 2011 Accepted 13 January 2012 Available online 28 January 2012 Keywords: Grape by-products Polyphenols Monosaccharides Cellulase Pectinase Tannase

a b s t r a c t Grape seed extract and grape pomace are rich sources of polyphenols. The aim of this study was to evaluate the release of polyphenols, the solubilisation of carbohydrate, and the antioxidant capacity of these grape by-products after enzymatic reaction with carbohydrases (cellulolytic and pectinolytic activities) and tannase for 24 h. The use of tannase in these by-products, and pectinase in grape pomace changed the galloylated form of catechin to its free form, releasing gallic acid and increasing the antioxidant activity. In grape pomace, cellulase treatment was not efficient for phenolic release and antioxidant activity improvement. The addition of carbohydrases to grape pomace, either alone or in combination, degraded the cell wall polysaccharides, increasing the content of monosaccharides. These results provide relevant data about the potential of pectinase, tannase and combinations of enzymes on the release of polyphenols and monosaccharides from grape by-products, improving the antioxidant capacity and the nutritional value. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Over the past few years, by-products of wine and grape juice processing have attracted considerable attention as a potential source of bioactive phenolic compounds, which have antioxidant properties and could be used in the pharmaceutical, cosmetic and food industries (Gonzalez-Paramás, Esteban-Ruano, Santos-Buelga, Pascual-Teresa, & Rivas-Gonzalo, 2004). After grapes are pressed and the juice is collected, the remaining material containing seeds, skins and stems is known as pomace. Grape seeds can be separated, extracted, and purified into grape seed extract (GSE). Catechins and their isomers and polymers are the main components in the seed. These phenolics have been reported to be linked to cell-wall polysaccharides. The grape pomace (GP) cell wall is a complex network composed of 30% of neutral polysaccharides (cellulose, xyloglucan, arabinan, galactan, xylan and mannan), 20% of acidic pectin substances, 15% of insoluble proanthocyanidins, lignin and structural proteins and phenols, these two latter crosslinked to the lignin–carbohydrate framework (Pinelo, Arnous, & Meyer, 2006). Cell wall polysaccharides contain hydrogen groups as well as aromatic and glycosidic oxygen atoms that have the ability to form hydrogen bonds and hydrophobic interactions with polyphenols (Le Bourvellec, Guyot, & Renard, 2004).

⇑ Corresponding author. Tel.: +34 91 5434545. E-mail address: [email protected] (A. Brenes). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.01.031

The growing interest in the exploitation of these bioactive compounds, especially flavonoids and phenolic acids from processed plant residues, has encouraged research on the application of cell-wall-hydrolysing enzymes to various wine by-products (Kammerer, Claus, Schieber, & Carle, 2005; Meyer, Jepsen, & Sorensen, 1998). The use of these enzymes, such as pectinase, cellulase, hemicellulase and glucanase, has been introduced to release cell wall complex polyphenols, facilitating the release of certain nutrients entrapped by the cell wall structure (Landbo & Meyer, 2004; Maier, Göppert, Kammerer, Schieber, & Carle, 2008). Enhancement of polyphenol bio-activities by enzyme reactions has also been reported by different authors (Kapasakalidis, Rastall, & Gordon, 2009; Zheng, Hwang, & Chung, 2009). Another enzyme implicated in the release of polyphenols is tannase. Tannase or tannin acyl hydrolase (EC, 3.1.1.20) catalyses the hydrolysis of the ester and depside bonds present in hydrolysable tannins or gallic esters, such as epigallocatechin O-gallate (EGCG) or epicatechin O-gallate (ECG), releasing gallic acid (GA) or glucose (Lekha & Lonsane, 1997). Tannase is an enzyme produced by various filamentous fungi, mainly Aspergillus and Penicillium in the presence of tannic acid. The major commercial applications of tannase are the elaboration of instant tea and the production of GA. Tannase application in food and beverages (beer and wines) might contribute to the removal of the undesirable effects (turbidity) of tannins (Belmares, Contreras-Esquivel, Rodriguez-Herrera, Ramirez, & Aguilar, 2004). The release of GA by the action of tannase on galloylated tannins would be beneficial, since this compound is supposed to have great antioxidant power (Netzel, Shahrzad, Winter, & Bitsch, 2000).

S. Chamorro et al. / Food Chemistry 133 (2012) 308–314

Studies on the effect of enzymatic treatment on grape polyphenols are of significant importance for the recovery of phenolics from grape by-products, in order to obtain an extract that may be used as a food supplement or as a novel functional ingredient. The hydrolysis of the complex polysaccharides and polyphenols into more simple sugars and phenols might increase the amount of bioactive substances available. Monomeric and some oligomeric polyphenols have been found to be absorbed (Shoji et al., 2006), while polymeric forms are poorly absorbed (Donovan et al., 2002; Gonthier et al., 2003). Thus, it is important to evaluate the effect of enzymatic treatment of grape by-products on the structure of phenolic compounds and on the antioxidant activity. To our knowledge there has been little or no study on the effect of carbohydrases or tannase addition on the polyphenolic components and on cell wall carbohydrate degradation of grape seed by-products. The purpose of this work was to examine the release of phenolic compounds and the cell wall carbohydrate degradation of GP after treatment with tannase, cellulase and pectinase (individually or in combination) and its relationship with the antioxidant capacity. The release of phenolic compounds after the treatment of GSE with tannase was also evaluated. 2. Materials and methods

309

in the work of Thomas and Murtagh (1985). Enzymatic hydrolysis of the samples was carried out in a thermostatically controlled shaking water bath with gentle agitation. To investigate the enzymatic release of polyphenols and monosaccharides of GP, one gram of sample was incubated with 6.75 and 13.5 U equivalent activity of PektozymeÒ (pectinase) and 157.5 and 315 U of equivalent activity of LaminexÒ in a final volume of 10 mL 0.1 M Na acetate buffer (pH 5.5) for 24 h under agitation (35 °C, 100 rpm). Grape seed extract (0.250 g) and GP (1 g) were incubated with 500 and 1000 U equivalent tannase in a final volume of 10 mL of 0.1 M Na acetate buffer (pH 5.5) for 24 h under agitation (35 °C, 100 rpm). Combinations of LaminexÒ, PektozymeÒ and tannase were also used in GP at similar concentrations. After 24 h of enzymatic hydrolysis, the samples were centrifuged (3500 rpm, 10 min), and the resulting supernatants were collected, filtered through a Technokroma filter (0.45 lm) and used to analyse the polyphenolic content and the monosaccharide contents. For the GP samples, the residue remaining after the enzymatic hydrolysis was collected and subjected to a subsequent extraction by adding ten millilitres of acetone/water (70:30 v/v), shaking and repeating the centrifugation. This supernatant was collected, filtered through a 0.45-lm filter and used to analyse the polyphenolic content and the antioxidant activity. All supernatants were stored at –18 °C until analysed.

2.1. Materials 2.5. Analytical methods Grape pomace was obtained from Alcoholeras Reunidas, SA (Argamasilla de Alba, Ciudad Real, Spain). Grape powdered seed extract (GSE) was purchased from NOR-FEED Sud (Angers, France) and it was obtained with water extraction and spray dried. The pomace, consisting of stems, skins and seeds from red grapes, was dried in a convection oven at 60 °C and semi-finely pulverised using a milling machine (passing through a 1-mm mesh sieve). 2.2. Solvents and reagents All solvents used for HPLC analysis were of liquid chromatography grade and were obtained from Sigma–Aldrich (St. Louis, MO), and all water was ultrapure. Catechin kit containing catechin (C), epicatechin (EC), epicatechin O-gallate (ECG), epigallocatechin (EGC) and epigallocatechin O-gallate (EGCG) was purchased from Extrasynthèse (Genay, France). Gallic acid (GA), gallocatechin (GC), gallocatechin O-gallate (GCG), procyanidin dimers B1 (PB1) and B2 (PB2), D-arabinose, and D-xylose were purchased from Sigma–Aldrich. D-Glucose, L-rhamnose and D-galactose were purchased from Merck, Steinheim, Germany. Acetone and methanol were obtained from Panreac (Castellar del Vallés, Barcelona, Spain).

2.5.1. Total polyphenols Total polyphenols were determined in supernatants obtained after enzymatic treatments by Folin–Ciocalteau procedure (Montreau, 1972), using gallic acid as standard. A mixture of 0.5 mL of extract, 0.5 mL of Folin–Ciocalteau reagent and 10 mL of Na2CO3 1 M were introduced in a 25 mL volumetric flask. After reacting for 1 h, absorbance was measured at 750 nm using an ultraviolet–visible spectrophotometer (Hitachi U-2000; Hitachi, Ltd., Tokyo, Japan). The results were expressed as g of gallic acid equivalents per 100 g of GP and GSE dry matter (DM).

2.4. Enzymatic treatment

2.5.2. Polyphenolic content analysis Analyses were performed using an Agilent 1100 series HPLC, comprising a quaternary pump with integrated degasser, autosampler, thermostated column compartment and diode array detector (DAD), coupled with an Agilent G1946D quadrupole mass spectrometer (Agilent Technologies, Waldbronn, Germany). Ten microlitres of filtered samples were separated on a Gemini C18 5 lm, 250 mm  4.6 mm i.d. column, (Phenomenex, Torrance, CA), eluted with a mobile phase made up of a mixture of deionised water (solvent A) and acetonitrile (solvent B), both containing 0.1% formic acid, at a flow rate of 1 mL/min. The solvent gradient changed according to the following conditions: from 90% A to 70% in 30 min, to 65% in 5 min, to 55% in 5 min, and then back to initial conditions in 10 min. Ionisation was achieved by atmospheric pressure electrospray ionisation (ESI) source, operated in negative ion mode, with the electrospray capillary voltage set to 3500 V, fragmentor 150 V, a nebulising gas flow rate of 12 L/min at 50 psi, and a drying temperature of 350 °C. Selected ion monitoring (SIM) was used for quantification with each standard: m/z 169 [M H] for GA, m/z 289 for C and EC, m/z 305 [M H] for GC and EGC, m/z 441 for ECG, m/z 457 for EGCG and GCG, and m/z 577 [M H] [M H] for PB1 and PB2. Data acquisition and analysis were carried out with Agilent ChemStation Software. Phenolics yields were expressed as mg per 100 g of GP and GSE dry matter.

The selected hydrolysis conditions (pH 5.5 and 35 °C) were based on evaluations of temperature and pH activity curves for the enzymes, as given on the enzyme suppliers’ data sheets, and

2.5.3. Antioxidant assay procedure The DPPH (2,2-diphenyl-1-picrylhydrazyl; Sigma–Aldrich) assay measuring the radical-scavenging activity of GSE and GP

2.3. Enzymes Three different types of enzymes were selected on the basis of the structural composition of grape seeds. PektozymeÒ (135 U/g; E.C. 4.2.2.10) is a pectolytic enzyme complex produced by fermentation with a selected strain of Aspergillus niger and LaminexÒ (3150 CMC-DNS U/g; E.C. 3.2.1.4) is a food enzyme complex hydrolysing b-glucans, pentosans and related carbohydrates produced by fermentation with a selected strain of Penicillium funiculosum. The enzymes were supplied by Danisco A/S (Denmark). Tannase (200 U/mg, E.C. 3.1.1.20.) from Aspergillus ficum was purchased from Sigma–Aldrich.

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extracts was done following the method proposed by Brand-Williams, Cuvelier, and Berset (1995), with modifications reported by Anastasiadi, Pratsinis, Kletsas, Skaltsounis, and Haroutounian (2010). More specifically, samples were diluted with ethanol to reach the linear range of a standard curve performed with known Trolox solutions. Then 1 mL of samples or Trolox standard were allowed to react with 1 mL DPPH solution (0.5 mM in absolute ethanol) at 30 °C for 30 min in the dark, and absorbance was measured at 510 nm using a Hitachi U-2000 ultraviolet–visible spectrophotometer (Hitachi, Ltd., Tokyo, Japan). Results were expressed as lmol of Trolox equivalents per g of dry matter.

2.5.4. Monosaccharides analysis Monosaccharides were analysed by ion chromatography using an 817 Bioscansystem (Metrohm, Herisau, Switzerland) equipped with a Hamilton RCX-30 column, and a pulsed amperometric detector (PAD) with a gold electrode. A three-step PAD protocol was used with the following time intervals (ms) and potentials (mV): t1, 400/E1 = +0.05 (detection); t2, 200/E2 = +0.75 (cleaning); t3, 400/E3 = 0.15 (regeneration). Samples (1.5 mL) were injected using an autosampler (Model 838 Advanced Sample Processor, Metrohm, Herisau, Switzerland). Elution was carried out with 150 mM NaOH for the analysis of rhamnose and arabinose, and with 3 mM Na acetate in 30 mM NaOH for galactose and xylose. The flow rate through the column was 1 mL/min. Appropriate dilutions of standard mixtures were used for calibration. Chromatographic peaks were identified by comparing sample retention times with those of known standard mixtures and the data was acquired using ICNET Version 2.3 software. Monosaccharide contents were expressed as lg/g dry matter.

2.6. Statistical analysis All analyses were performed in triplicate. Data were subjected to a one-way analysis of variance (ANOVA) by using the general linear model procedure (Version 9.2, SAS Institute Inc., Cary, NC). When the effect was declared significant (p < 0.05), means were compared using a Tukey’s Studentised range test.

3. Results and discussion 3.1. Phenolic composition of GSE after the action of tannase Changes in concentrations of GSE before and after enzymatic hydrolysis at two concentrations of tannase are presented in Table 1. The relative concentrations of phenolic compounds in GSE changed during enzymatic treatment. Tannase treatment on GSE increased the total polyphenolic content up to 41%. Simultaneously, an increase in GA (up to 6 times), EC (up to 22%) and procyanidin B2 (up to 42%) compared to the corresponding control level were also observed. This was accompanied with a decrease in the concentrations of EGCG, GCG and ECG until they were negligible in GSE treated with tannase. The results showed that tannase was able to hydrolyse the ester bonds from natural substrates and the release of GSE catechins was increased as enzyme concentration rose. Tannase treatment hydrolysed ECG into EC and GA. This is caused because the ester bonds between gallic acid, EGC/ EC in EGCG and ECG are cleaved by the action of tannase (Lekha & Lonsane, 1997; Thomas & Murtagh, 1985), producing GA and EC. Similar results have been published by Lu and Chen (2008) and Lu, Chu, Yan, and Chen (2009) using green tea or epicatechin gallate extract from green tea. To our knowledge, there are no reports on the effect of tannase on the polyphenolic content of GSE.

Table 1 Effect of tannase treatment on the release of total polyphenols (g gallic acid equivalent/100 g dry matter), individual phenolic compounds (mg/100 g dry matter), and on the antioxidant activity (lmol Trolox equivalent/g dry matter) of grape seed extract (GSE). Enzyme dosageA

Total polyphenols Gallic Acid Gallocatechin Epigallocatechin Catechin Epicatechin Procyanidin B1 Procyanidin B2 Epigallocatechin O-gallate Gallocatechin O-gallate Epicatechin O-gallate Antioxidant Activity

Tannase (T) 0

T1

T2

15.7c 336c 5.10 14.09 823 648b 903 530b 13.3 33.0a 157a 3299b

18.7b 1760b 5.51 15.12 888 788a 952 754a nd nd 3.02b 3411a

22.2a 1920a 5.25 13.80 846 762a 936 737a nd nd 2.31b 3347a,b

SEMB

p-ValueC

0.28 0.150 0.211 0.724 33.8 31.6 19.1 15.7 0.421 0.612 2.12 24.8

** ***

ns ns ns *

ns *** *** *** *** *

nd, Not detected. A T1 and T2, 2000 and 4000 U tannase/g GSE dry matter, respectively. B SEM, standard error of means; number of replicates = 3. C ns, No significant effect (p > 0.05). * p < 0.05. ** p < 0.01. *** p < 0.001. a,b,c Mean values within a row with different superscript letters are significantly different.

3.2. Phenolic composition of GP after the action of carbohydrases and tannase Polyphenols have both hydrophobic aromatic rings, and hydrophilic hydroxyl groups with the ability to bind to polysaccharides and proteins at several sites on the cell wall (Hanlin, Hrmova, Harbertson, & Downey, 2010; Pinelo et al., 2006). The release of phenolic compounds attached to the cell wall of GP after a treatment with degrading enzymes was determined. Commercial enzymes preparations of cellulolytic (LaminexÒ), pectinolytic (PektozymeÒ), and tannase activities, either alone or in mixture were suited for this purpose (Table 2). According to Table 2, LaminexÒ used alone did not result in an enhancement of phenol release except in the case of ECG, which was reduced (up to 71%). These results are similar to those reported by Meyer et al. (1998), and by Zheng et al. (2009), using grape pomace and apple. Different factors may explain the low activity of LaminexÒ on GP. The plant cell wall of GP is composed of cellulose, hemicellulose, pectin, and lignin arranged in a complex network (Valiente, Arrigoni, Esteban, & Amado, 1995). Lignin and cellulose combine to produce a material very resistant to chemical and biological degradation (Düsterhöft, Engels, & Voragen, 1993). Furthermore, the presence of lignin strengthens the hydrogen bonds in the cellulose and hemicellulose network (Brett & Waldron, 1996, chap. 2). The formation of covalent linkages by lignin and low molecular weight phenolic compounds with sugar residues may also affect the accessibility of hydrolytic enzymes (Düsterhöft et al., 1993). Likewise, enzymatic hydrolysis may be retarded by a non-productive adsorption of enzymes to lignin or polysaccharides in the wall matrix (Converse, Ooshima, & Burns, 1990). Thus, pre-treatments for removing lignin and hemicellulose are required, to make the cellulose accessible to enzymatic hydrolysis. In this sense, the use of technologies for pre-treatment of various lignocellulosic biomasses has been extensively reviewed by Kumar, Barrett, Delwiche, and Stroeve (2009). The course of the release of the phenol compounds was quite similar when pectinase and tannase were used separately (Table 2). PektozymeÒ and tannase used separately resulted in a substantial increase in GA content (up to 34% and 78%, respectively) and a

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Table 2 Effect of enzymatic treatment of grape pomace (GP) with LaminexÒ, PektozymeÒ, tannase and a mixture of these enzymes on the release of total polyphenols (g gallic acid equivalent/100 g dry matter), individual phenolic compounds (mg/100 g DM) and on the antioxidant activity (lmol Trolox equivalent/g dry matter). Enzyme treatment Enzyme dosage

A

Total polyphenols Gallic acid Gallocatechin Epigallocatechin Catechin Epicatechin Procyanidin B1 Procyanidin B2 Epicatechin O-gallate Antioxidant Activity

Treatments Control

Cellulase (C)

Pectinase (P)

Tannase (T)

Mixture (C + P + T)

0

C1

C2

P1

P2

T1

T2

C1 + P1 + T1

C2 + P2 + T2

0.11 b 5.74e 0.028 0.075c 2.96 2.03 4.63 2.62c 0.029a 16.5a

0.10 b 4. 04e 0.028 0.086b,c 2.77 2.08 4.56 2.63c 0.021b 17.1a,b

0.11 b 5.39e 0.028 0.087a,b,c 2.74 2.10 4.53 2.62c 0.008c 17.4a,b

0.11 b 6.90d 0.036 0.098a,b 2.73 2.14 4.93 2.87b,c 0.0214b 17.8b,c

0.12b 7.71d 0.022 0.099a 2.52 2.09 4.91 2.84b,c nd 18.5c

0.11 b 8.97c 0.026 0.076c 2.44 1.82 4.22 2.89b,c nd 18.2c

0.14 a 10.2b 0.023 0.077c 2.63 2.00 4.30 2.89b,c nd 19.8d

0.11 b 10.2b 0.031 0.098a,b 2.62 2.13 4.85 3.20b,c nd 20.9e

0.14 a 11.4a 0.022 0.097a,b 2.49 2.16 4.89 3.01a nd 21.7e

SEMB

p-ValueC

0.005 0.31 0.005 0.04 0.184 0.092 0.183 0.105 0.002 0.37

** ***

ns ***

ns ns ns *** *** ***

Nd, Not detected. A C1 and C2: 157.5 and 315 U LaminexÒ/g GP dry matter, respectively; P1 and P2 6.75 and 13.5 U PektozymeÒ/g GP dry matter, respectively; T1 and T2: 500 and 1000 U tannase/g GP dry matter. B SEM, standard error of means; number of replicates = 3. C ns, No significant effect (p > 0.05). ** p < 0.01. *** p < 0.001. a,b,c,d,e Mean values within a row with different superscript letters are significantly different.

reduction until a negligible amount of ECG. Pectinase also increased the release of EGC (up to 16%). These changes were only related with an increase in the total polyphenols content determined by Folin–Ciocalteau method in the case of tannase. Using the same methodology, Meyer et al. (1998) reported an increase in the recovery of phenols after treating GP with pectinase for a short time interval (less than 8 h), but in agreement with our results, did not detect any effect after 24 h of treatment. These authors reported that these effects were due to the degradation of phenolic compounds during long enzymatic hydrolysis. However, our results indicated that despite total phenol content being unaffected, individual phenolic compounds were modified by pectinase treatment. In a previous study, Kammerer et al. (2005) also reported an increase in the extraction yields of phenolic acids and flavan-3-ols in GP by the use of a selection of pectinolytic and cellulolytic enzymes. The release of individual phenolic compounds was only dosedependent in the case of tannase. Thus, when both enzymes were added separately, the maximum release of polyphenols was obtained with lower doses of PektozymeÒ (6.75 U/g of GP dry matter), and higher doses of tannase (1000 U tannase/g GP dry matter). When all three enzymes were used, a more complete breakdown was achieved. The combination of enzymes increased the release of GA (up to 99%), EGC (up to 29%), and procyanidin B2 (up to 22%) while ECG was fully hydrolysed. According to the manufacturer PektozymeÒ contains mainly pectinolytic activity. Pectinolytic preparations are widely used in food processing industry and in general are the most efficient at releasing phenols and degrading polysaccharides (Maier et al., 2008; Pardo, Salinas, Alonso, Navarro, & Huerta, 1999). Significant differences in the amount of total phenols have also been observed in GP (Costoya, Sineiro, Pinelo, Rubilar, & Nuñez, 2010; Meyer et al., 1998), wine samples (Bautista, Martínez, Ros, López, & Gómez, 2005), grape skins (Arnous & Meyer, 2010) and blackcurrant juice production (Landbo & Meyer, 2004), when commercial pectinolytic enzymes preparations were used. There is no available information concerning the release of polyphenolic compounds after tannase treatment of GP. The increase of GA and the reduction of ECG observed by the addition of tannase (Table 2) were similar to the results reported by Lu and Chen (2008) and Lu et al. (2009) using green tea. As previously mentioned, tannase cleaves the ester linkage between galloyl groups and various compounds. A possible application of

tannase in the wine industry has been proposed by Aguilar and Gutiérrez-Sánchez (2001). In this sense, tannase hydrolyses the ester bonds from polyphenols, preventing their polymerisation, giving a wine with a high content of aromatic compounds and appropriate colour and increased quality. The type of solvent is one of the most important factors that influence the efficiency of extraction. Because the enzymatic reaction was performed in aqueous solution appreciable amount of polyphenols may be retained in the residues. A previous work (Meyer et al., 1998) reported that extraction with 70% acetone after enzymatic hydrolysis of GP gave higher yields of phenols than extraction with pure water. Furthermore, as procyanidins tend to be more soluble in non-aqueous solvents, we used acetone to extract more efficiently the residue of GP. The polyphenol content of the residue remaining after the enzymatic hydrolysis of GP extracted with 70% acetone is reported in Table 3. In general, after the enzymatic hydrolysis a considerable amount of phenols were retained in the residue and the recovery was 7 times higher after its extraction with 70% acetone. This was more evident for ECG which was almost only recovered after the extraction with acetone. These findings agree with the results reported by Meyer et al. (1998) who obtained 4–5 times higher yield of phenols after the extraction with 70% of acetone. Despite these quantitative differences in the yield of polyphenols in both fractions, the efficacy of the enzyme treatments was similar, confirming that the use of the enzymes led to a higher release of GA, and a reduction of ECG. 3.3. Free radical scavenging Among the different methods used to characterise the total antioxidant capacity, the stable free radical DPPH has been extensively used to measure the free-radical-scavenging activity of pure antioxidant compounds extracted from fruits, plants, or food materials. The results in Table 1 indicate that radical-scavenging capacity was increased (4%) after the enzymatic hydrolysis of GSE with tannase. Also, the scavenging ability of GP treated with PektozymeÒ, tannase and combination of enzymes (including LaminexÒ) was increased by up to 12%, 20% and 32%, respectively (Table 2). We demonstrated that enzymatic treatments in both grape byproducts released more GA content than the untreated analogue. It has been proved that the scavenging capacity of GA is elevated. The antioxidant activity of phenolic acids increases with the

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Table 3 Total polyphenols (g gallic acid equivalent/100 g dry matter), individual phenolic compounds (mg/100 g DM), and antioxidant activity (lmol Trolox equivalent/g DM) of the enzymatic hydrolysis residues from grape pomace extracted with acetone:water (70:30). Enzyme treatment Enzyme dosage

SEM2

Treatments

1

Total polyphenols Gallic acid Gallocatechin Epigallocatechin Catechin Epicatechin Procyanidin B1 Procyanidin B2 Epicatechin O-gallate Antioxidant activity

Control

Cellulase (C)

Tannase (T)

Mixture (C + P + T)

0

C1

C2

P1

P2

T1

T2

C1 + P1 + T1

C2 + P2 + T2

0.71 2.07c 0.0282 0.0607b,c,d 2.40 1.27 2.60 1.78c,d,e 0.368a 72.5

0.69 2.28c 0.0271 0.0595b,c,d 2.44 1.30 2.67 1.62e 0.326b 73.5

0.72 2.28c 0.0260 0.0583c,d 2.33 1.28 2.60 1.74d,e 0.299b 72.2

0.69 2.93b 0.0272 0.0703a,b,c 2.23 1.27 2.76 1.87b,c,d 0.103c 69.1

0.71 3.20b 0.0286 0.0758a 2.17 1.26 2.84 1.92a,b,c,d 0.0445d 72.3

0.73 2.93b 0.0272 0.0479e 2.23 1.27 2.76 1.87b,c,d 0.103c 69.1

0.72 4.03a 0.0244 0.0508e 2.35 1.32 2.66 2.13a 0.0396d 69.6

0.72 4.18a 0.0280 0.0721a,b 2.28 1.29 2.78 2.15a 0.0302d 71.5

0.71 4.49a 0.0270 0.0723a,b 2.16 1.26 2.77 2.04a,b 0.0153d 71.3

A

Pectinase (P)

Ò

0.02 0.196 0.0003 0.004 0.094 0.054 0.196 0.080 0.011 1.66

p-Value3

ns ***

ns ***

ns ns ns *** ***

ns

Ò

C1 and C2: 157.5 and 315 U Laminex /g GP dry matter, respectively; P1 and P2: 6.75 and 13.5 U Pektozyme /g GP dry matter, respectively; T1 and T2: 500 and 1000 U tannase/g GP dry matter. B SEM, standard error of means; number of replicates = 3. C ns, no significant effect (p > 0.05). *** p < 0.001. a,b,c,d,e Mean values within a row with different superscript letters are significantly different.

Cellulase (C)

150

0

C1

Pectinase (P) 600

C2

0

P1

P2

µg / g DM µ

µg / g DM

100

400

50

200

0

0

Rhamnose

Arabinose

Xylose

Rhamnose

Galactose

Tannase (T) 0

T1

0

100

Xylose

Galactose

Mixture (M)

600

T2

µg / g DM µ

µg / g DM

150

Arabinose

M1

M2

400

200

50

0

0

Rhamnose

Arabinose

Xylose

Galactose

Rhamnose

Arabinose

Xylose

Galactose

Fig. 1. Monosaccharides yield (lg/g dry matter) of grape pomace (GP) after enzymatic treatment with different enzymes. Doses C1 and C2: 157.5 and 315 U LaminexÒ/g GP dry matter, respectively; P1 and P2: 6.75 and 13.5 U PektozymeÒ/g GP dry matter, respectively; T1 and T2: 500 and 1000 U tannase/g GP dry matter; M1: Doses C1 + P1 + T1, and M2: Doses C2 + P2 + T2.

number of hydroxyl groups, as is the case of GA, which shows high antioxidant potency (Balasundram, Sundram, & Samman, 2006). The increase in the release of polyphenols by the enzyme preparations suggest that these enzymes may contain activities that directly promote selective release of antioxidant phenols or modify released phenols to more potent antioxidant compounds. The antioxidant activity of the residue extracted with 70% acetone (Table 3) was higher than those obtained with GP after enzymatic treatment (Table 2). These results may indicate that a proportion of high-molecular-weight procyanidin with high antioxidant activity retained in the residue was extracted with acetone.

3.4. Grape pomace cell wall degradation after the action of carbohydrases and tannase Chemical analysis of GP revealed the presence of significant amounts of fermentable sugars that are retained in the pomace after pressing of the grapes. The monosaccharides release of GP after the enzymatic treatment with LaminexÒ, PektozymeÒ and tannase, added individually or simultaneously is reported in Fig. 1. Glucose could not be correctly quantified with this methodology because some analytes present in the commercial enzymes (PektozymeÒ and LaminexÒ) were found to co-elute at

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the same retention time as the glucose standard Grape pomace treated separately with LaminexÒ and PektozymeÒ resulted in a substantial increase in the monosaccharides content, whereas tannase did not modify its concentration. Although tannase is not a cell-wall-degrading enzyme, Garcia-Conesa, Ostergaard, Kauppinen, and Williamson (2001) reported that tannase may contribute to plant cell degradation. The release of monosaccharides, and hence the extent of cell wall degradation, was lower with LaminexÒ than with PektozymeÒ. These finding are in accordance with data reported by Arnous and Meyer (2010) that showed a degradation of the cell wall polysaccharides in skins of different varieties of grapes. Similarly, Mandalari et al. (2006) showed that the majority of the solubilised carbohydrates after the treatment of bergamot peel with PektozymeÒ and LaminexÒ during 24 h, were present as monosaccharide with a smaller proportion of oligosaccharides. In our study, the effect of LaminexÒ was dose-dependent and, at higher doses, the release of monosaccharides was increased 31 times for xylose, 17 times for galactose, 6 times for arabinose and 4 times for rhamnose. However, the release after PektozymeÒ treatment was higher at lower doses, increasing the yield of xylose (up to 133 times), followed by rhamnose (up 41 times), galactose (up to 38 times) and arabinose (36 times). Pectic substances are the main polymer-type constituents of the cell walls present in fresh grapes and GP (Pinelo et al., 2006). Pectin is a polymer of galacturonic units attached to units of glucose, xylose, arabinose and galactose. However, when the three enzymes were mixed the release of monosaccharides was not higher than when PektozymeÒ was used individually. In fact, the enzymatic cocktail caused a lower monosaccharide concentration than the sum obtained by the three individual enzymes. The extent of cell wall degradation observed in our study might explain the release of phenolic compounds obtained when PektozymeÒ was added to GP. Enzymatic degradation of polysaccharides breaks crosslinking machinery within the cell wall and its threedimensional structure, decreasing the molecular mass and size of the polysaccharides, and reducing their ability to bind tannins strongly (Hanlin et al., 2010). The ability to release polyphenols and monosaccharides by the action of polysaccharide degradative enzymes (mainly pectinase) is important in the wine industry since they contribute to the sensory attributes of wine. The use of exogenous pectinolytic enzymes, which help to break down the structural polysaccharides of the cell wall, accelerated the extraction of phenolic compounds, reducing the maceration time needed for high quality winemaking (Romero-Cascales, Ros-García, López-Roca, & Gómez-Plaza, 2012). Our results might also have applicability in nutrition. Polyphenols are thought to play important positive roles in human nutrition and health, but their functional effects depend on their bioavailability, which is largely influenced by their structure. Monomeric and some oligomeric polyphenols have been found to be absorbed (Shoji et al., 2006), while polymeric forms are poorly absorbed (Donovan et al., 2002; Gonthier et al., 2003). Strategies to improve the bioavailability of polyphenols need to be developed. The hydrolysis of the complex polysaccharides and polyphenols into more simple sugars and phenols obtained in the current experiment might increase the absorption of active substances. The applicability of these findings in nutrition needs to be supported by in vivo experiments. Grape by-products have been used as functional ingredients in animal feed (Brenes et al., 2008; Viveros et al., 2011) but the polyphenols bioavailability of these by-products is rather low and could be enhanced by the addition of pectinase and tannase. The addition of pectinase and tannase could improve the nutritional characteristics of grape by-products and revaluate these raw materials with little economical value.

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4. Conclusions The results obtained in this study demonstrated that the use of pectinase and tannase in grape by-products assisted the liberation and extraction of phenolic compounds. The products of these enzyme reactions showed higher antioxidant capacity than the untreated by-products. The use of tannase in grape seed extract and grape pomace, and pectinase in grape pomace changed the galloylated form of catechin to its free form, releasing gallic acid. Pectinolytic enzymes disintegrate the plant cell wall matrix of GP, releasing monosaccharides and facilitating the polyphenol extraction. A higher effect was obtained when both enzymes were combined. Cellulolytic enzymes neither affect the release of polyphenols nor the antioxidant activity of these grape byproducts. Tannase did not contribute to GP plant cell wall degradation Due to the scarcity of studies involving tannase in the phenol release of grape by-products, further studies are needed to explore the possible application of this enzyme in the food industry. The enzyme polyphenol extraction process might be applied in the food industry for enhancing bioactive compound production. Acknowledgements The authors wish to thank the Spanish Ministry of Science and Innovation (MICINN, Spain) and Comunidad Autónoma de Madrid (CAM) for financial support of this investigation, Projects AGL2009-07417/GAN and S2009/AGR-1704 (NEWGAN), respectively. References Aguilar, C. N., & Gutiérrez-Sánchez, G. (2001). Review: Sources, properties, applications and potential uses of tannin acyl hydrolase. Food Science and Technology International, 7, 373–382. Anastasiadi, M., Pratsinis, H., Kletsas, D., Skaltsounis, A. L., & Haroutounian, S. A. (2010). Bioactive non-coloured polyphenlos content of grapes, wines and vinification by-products: Evaluation of the antioxidant activities of their extracts. Food Research International, 48, 805–813. Arnous, A., & Meyer, A. S. (2010). Discriminated release of phenolic substances from red wine grape skins (Vitis vinifera L.) by multicomponent enzymes treatment. Biochemical Engineering Journal, 49, 68–77. Balasundram, N., Sundram, K., & Samman, S. (2006). Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chemistry, 99, 191–203. Bautista, A. B., Martínez, A., Ros, J. M., López, J. M., & Gómez, E. (2005). Improving colour extraction and stability in red wines: The use of maceration enzymes and enological tannins. International Journal of Food Science and Technology, 40, 867–878. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie, 28, 25–30. Belmares, R., Contreras-Esquivel, J. C., Rodriguez-Herrera, R., Ramirez, A., & Aguilar, C. N. (2004). Microbial production of tannase: an enzyme with potential use in food industry. LWT Food Science and Technology, 37, 857–864. Brenes, A., Viveros, A., Goñi, I., Centeno, C., Sáyago-Ayerdi, S. G., Arija, I., et al. (2008). Effect of grape pomace concentrate and vitamin E on digestibility of polyphenols and antioxidant activity in chickens. Poultry Science, 87, 307–316. Brett, C. T., & Waldron, K. W. (1996). Physiology and biochemistry of plant cell walls (2nd ed.). London: Chapman & Hall. Converse, A. O., Ooshima, H., & Burns, D. S. (1990). Kinetics of enzymatic hydrolysis of lignocellulosic materials based on surface area of cellulose accessible to enzyme and enzyme adsorption on lignin and cellulose. Applied Biochemistry and Biotechnology, 24–25, 67–73. Costoya, N., Sineiro, J., Pinelo, M., Rubilar, M., & Nuñez, M. J. (2010). Enzyme-aided extraction of polyphenols from grape pomace. Electronic Journal of Environmental, Agricultural and Food Chemistry, 9(4), 696–705. Donovan, J. L., Manach, C., Rios, L., Morand, C., Scalbert, A., & Remesy, C. (2002). Procyanidins are not available in rats fed a single meal containing grape seed extract or the procyanidin dimer B3. British Journal of Nutrition, 87, 299–306. Düsterhöft, E. M., Engels, F. M., & Voragen, A. G. J. (1993). Parameters affecting the enzymic hydrolysis of oil-seed meals, lignocellulosic by-products of the food industry. Bioresearch and Technology, 44, 39–46. Garcia-Conesa, M. T., Ostergaard, P., Kauppinen, S., & Williamson, G. (2001). Hydrolysis of diethyl diferulates by tannase from Aspergillus oryzae. Carbohydrate Polymers, 44, 319–324.

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