Recovery of added value compounds from cork industry by-products

Industrial Crops & Products 140 (2019) 111599

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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop

Recovery of added value compounds from cork industry by-products a,⁎

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Sofia F. Reis , Paulo Lopes , Isabel Roseira , Miguel Cabral , Nuno Mateus , Victor Freitas

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ICETA/REQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre, 687, 4169-007, Porto, Portugal Amorim & Irmãos S.A, Rua dos Corticeiros, 830, 4536-904, Santa Maria de Lamas, Portugal

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ARTICLE INFO

ABSTRACT

Keywords: Cork powder Ellagitannins Condensed tannins Response surface methodology HPLC-ESI/DAD-MS MALDI-TOF

The generated residues from cork industry are granulated and reused for the production of agglomerates, yet there is a percentage of those residues not valorised due to the small granulometry size denominated as cork powder. The extraction of high valuable compounds such as ellagitannins and condensed tannins from these cork residues were studied using different experimental approaches. For the extractions, a response surface methodology (RSM), using aqueous solutions of two green solvents such as acetone and ethanol, was used. Acetone was the best organic solvent due to the high yield of extraction achieved and less results variability. Using cork powder the extraction efficiency increased due to the increase on the yield of extraction (5.8 ± 1.0 mg ellagic acid equivalents/ g cork powder and 4.3 ± 0.8 mg proanthocyanidin fraction equivalents/ g cork powder) by using less solvent concentration (60% acetone) and extraction time (9h30). Twenty-three ellagitannins were tentatively identified by HPLC-DAD/ESI-MS in the tannin-rich extract of both corks granulate and powder, five of them being reported for the first time in cork extracts: vescalin, castalin, guajavin B/ eugenigrandinin A, vescavaloninic and castavaloninic acids. Other complex ellagitannins such as glycosylated structures up to DP3 of acutissimin A/B and guajavin B/ eugenigrandinin A, other oligomeric ellagitannins and a glycosylated dimer of gallocatechin linked to vescalagin/castalagin were tentatively identified by MALDI-TOF. These compounds recycled from undervalued cork industry residues may be further used in technological applications in different industrial fields, hence contributing for a circular economy. The results indicate that the recovery of these high valuable compounds using this methodology can add value and sustainability to this cork transformation chain.

1. Introduction Portugal is the world leader in cork exportation with a share of 63.2%. According to the latest data 184.8 thousand tonnes were exported in 2016 representing 90% of the Portuguese cork production (APCOR, 2018). The main target sector of cork products is the wine industry (72% in 2016) followed by the construction sector (25% in 2016). Cork stoppers industry is therefore the core of cork business with 44% of cork being used to produce natural cork stoppers and 28% to produce other type of stoppers (APCOR, 2018). There are no published data regarding generated residues from cork industry, however even knowing that in cork stoppers industry the generated residues are granulated and used for the production of agglomerates, there is a percentage of those residues which is turned into a granulometry fraction not suitable for the production of agglomerates. It is estimated that this fraction represents 20–30% of the cork used (Carriço et al., 2018; Santos et al., 2013, 2010), which reflects in cork stoppers industry a production of around 3–4 thousand tonnes in 2016. This fraction is denominated as cork powder and used for energy

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production representing 60–65% of the cork industry energy needs (APCOR, 2016). Cork powder is also recognised as a causative agent of suberosis, an occupational disease of cork workers. Suberosis is related to the inhalation of mould/fungi spores through the continuous exposition to biological dust particles. The clinical evidences of this disease are related to severe and usually irreversible lung lesions such as asthma, bronchitis and hypersensitivity pneumonitis (Oliveira et al., 2003; Villar et al., 2009). Looking to this clinical and environmental scenario it is urgent to call for the attention of cork industry to this issue. Some studies have been reported on the air quality of some industrial cork stopper facilities and indications on how to improve environmental hygienic conditions have been established (Oliveira et al., 2003). However, some of these improvements such as installation of ventilation systems, aspiration and personal protection equipment imply additional costs to the industry which delays their implementation or even makes it impossible to implement. Therefore, it is necessary to find an added value to cork powder so that the cork industry shows itself interested in this by-product as a profitable raw material for other technological applications.

Corresponding authors. E-mail addresses: [email protected], [email protected] (S.F. Reis), [email protected] (V. Freitas).

https://doi.org/10.1016/j.indcrop.2019.111599 Received 18 January 2019; Received in revised form 26 May 2019; Accepted 22 July 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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Cork contains bioactive compounds such as polyphenols that should not be disregarded due to their potential use as functional products and materials owing to their antioxidant, antimicrobial, anti-inflammatory, anticancer associated properties (Carriço et al., 2018; Fernandes et al., 2009; Granica et al., 2015; Moilanen et al., 2016; Santos et al., 2013, 2010; Touati et al., 2015). Cork polyphenols have been widely studied, the mostly reported are low molecular weight polyphenols (LMWP) namely phenolic acids, some aldehydes, lactones and flavanones (Conde et al., 1998, 1997; Fernandes et al., 2011; Santos et al., 2013, 2010; Touati et al., 2015), but also high molecular weight polyphenols (HMWP) mainly ellagitannins, gallotannins and condensed tannins in less quantity (Cadahía et al., 1998; Fernandes et al., 2011). The first studies reported focused on the cork phenolic composition and variability according to different trees, provenances and different steps of the industrial cork stoppers processing. These studies used ground cork (0.5–1 mm particle size) and the phenolic extract was obtained by a methanol/water extraction, which was used for the total quantification of polyphenols and each group of tannins. However, quantification by HPLC-DAD of individual compounds was only performed in the organic extracts obtained from an ethyl ether fractionation of the phenolic extract (Cadahía et al., 1998; Conde et al., 1998, 1997). Thus, the quantification was limited to the compounds present in the organic extract (mostly LMWP) and to the standards commercially available. Later studies, using slightly modifications of the extraction methods achieved a high number of phenolic compounds identified by the use of mass spectrometry techniques (Fernandes et al., 2011; Santos et al., 2010), however quantification was still limited as before (Santos et al., 2010). More recent studies focused the valorisation of cork by-products, detailing the characterisation of cork extractives such as phenolics as well as lipophilic compounds, and respective antioxidant and anti-microbial properties (Santos et al., 2013; Touati et al., 2015). These studies used a sequential extraction to obtain first the lipophilic fraction followed by the phenolic fraction, but no further improvements were achieved in the identification and quantification of the phenolic fraction. HMWP such as tannins, are still under evaluated. Indeed, according to the literature a wide distribution of tannins is expected on genus Quercus, however after two decades of the first study on cork polyphenol composition, there are few identified tannins most of them were never quantified and the ones quantified were underestimated. Castalagin was the major ellagitannin quantified in cork, followed by vescalagin, grandinin, roburin E and roburin A (Cadahía et al., 1998). Other ellagitannins were identified such as valoneic acid dilactone, HHDP-Glc, di-HHDP-Glc, mongolicain A/B and dehydrated tergallic-CGlc. Gallotannins were also identified such as trigalloyl-Glc, tetragalloyl-Glc, pentagalloyl-Glc, HHDP-galloyl-Glc, HHDP-digalloyl-Glc, di- HHDP-galloyl-Glc and trigalloyl-HHDP-Glc, however no quantification data was reported (Fernandes et al., 2011). Condensed tannins were reported as being present in 3-fold lower quantity than ellagitannins (Cadahía et al., 1998) but no structural characterisation has been reported regarding these compounds. These HMWP have a diversity use in cosmetic, pharmaceutic and food industries (Carriço et al., 2018), which may turn cork residues a potential raw material for the recovery of these compounds, being a possibility of profit to the cork industry. In order to encourage cork industry to adopt sustainable practices which reduces waste generation with no global greenhouse gas emissions simultaneously contributing to the prevention of their occupational disease, this work aims at the recovery of added value compounds such as tannins, particularly ellagitannins and condensed tannins, from cork granulate and cork powder.

had its origin in several cork plank residues from different geographical origins and distinct classifications and was treated with steam for the removal of volatile compounds. Cork powder was collected from different factory operations and was not submitted to any treatment. 2.2. Chemicals and compounds All reagents used in tannins extraction were of analytical grade: chloroform (Fisher Scientific, UK), acetone (LabChem, Portugal), ethanol (Aga, Portugal) and ethyl acetate (PanReac AppliChem, Germany). Sephadex LH-20 was purchased from Cambridge Chemicals LTD (Shangai, China). Solvents used on quantification of tannins were HPLC grade: methanol (ChemLab, Belgium), hydrochloric acid (Fluka, Austria) and phosphoric acid (Fisher Scientific, UK). Solvents used on mass spectrometry analysis were MS grade: formic acid (ChemLab, Belgium), acetonitrile (ChemLab, Belgium), acetone (Fisher Scientific, UK) and Trifluoro acetic acid (Merck, France). Castalagin, vescalagin, dehydrated tergallic-C-Glc (isomer 3) and grandinin were isolated by semi-preparative HPLC (Merck Hitachi Elite Lachrom) from tannins-rich fraction isolated from cork powder. The purity of the isolated compounds was confirmed by HPLC-DAD/ESI-MS in full scan and selected ion monitored (SIM) mode, where the respective ion and bordered ions were selected: castalagin (99.6%), vescalagin (95.9%), dehydrated tergallic-C-Glc (99.5%) and grandinin (76.2%). The structural characterisation of castalagin and vescalagin was confirmed by NMR, kindly provided by ICETA/REQUIMTE/LAQV – Faculty of Pharmacy, University of Porto (Porto, Portugal). Castalagin: δ 1H ppm/ δ 13C ppm [600 MHz, 100% D2O, 0.1% TSP, pH average = 7.4] 4.23 (d), 5.0 (br)/66.53, 5.08 (d)/69.02, 5.15 (t)/71.64, 5.19 (br)/76.35, 5.58 (d)/73.75, 5.76 (d)/68.37, 6.79 (s)/109.99, 6.85 (s)/111.24, 6.99 (s)/112.02. Vescalagin: δ 1H ppm/ δ 13C ppm [600 MHz, 100% D2O, 0.1% TSP, pH average = 7.4] 4.25 (d), 4.92 (br), 4.95 (br), 4.97 (br), 5.20 (t)/95.05, 5.39 (s)/80.02, 5.60 (d)/73.75, 6.77 (s)/109.76, 6.89 (s)/111.53, 6.98 (s)/112.02. 2.3. Tannins extraction from cork material Cork granulate and cork powder were defatted with chloroform during 8 h using Soxhlet system. After oven dried (37 °C, 48 h), cork material was storage in polyethylene bags at room temperature until further use. Maceration of cork defatted material was performed using 4 g of cork material and 100 ml of aqueous solution of organic solvents (ethanol or acetone). Cork granulate was macerated with ethanol and acetone in different concentrations (8, 25, 50, 75 and 92%) combined with different times (2, 32, 76, 120 and 150 h) and temperatures (23, 30, 40, 50 and 57 °C). Cork powder was macerated at room temperature (22–24 °C) only with acetone in different concentrations (15, 25, 50, 75 and 85%) over different times (9.5, 12, 18, 24 and 26.5 h). After each maceration, the supernatant was precipitated with 4 volumes of ethanol (96%) for removal of polymers such as pectic polysaccharides and polymerised tannins. After centrifugation at 10,000 rpm for 10 min at 4 °C, the precipitate was discarded and the supernatant, after removal of ethanol, submitted to a liquid-liquid extraction (LLE) with ethyl acetate. Aqueous phase was collected and purified by solid phase extraction (SPE) using Sephadex LH-20 and tannins fraction was eluted with a mixture of methanol/acetone/H2O (3:1:1, v/v/v). The tanninrich fraction was concentrated by evaporation at 40 °C, frozen at −70 °C and freeze-dried. 2.4. Quantification of total ellagitannins and condensed tannins

2. Material and methods

Ellagitannins and condensed tannins were determined after methanolysis of freeze-dried tannin-rich fraction (n = 3) using MeOH/HCl (84:16, v/v) over 2 h at 100 °C. Ellagitannins were estimated by HPLC quantification of ellagic acid before and after acid decomposition, considering one ellagic acid molecule liberated from one ellagitannin

2.1. Cork material Cork granulate (1–2 mm particle size) and powder (< 0.2 mm particle size) were gently supplied by Amorim & Irmãos. Cork granulate 2

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R2 = 0.9913; LQ = 1.5 μg/mL; LD = 0.1 μg/mL) were quantified with the respective calibration curve and the remaining compounds were quantified in equivalents of the compound of similar structure.

molecule (Cadahía et al., 1998). Ellagic acid (≥ 96%, HPCE, Fluka, Steinheim, Germany) quantification was done in a HPLC-DAD (Dionex Ultimate 3000, Thermo Scientific, Germering, Germany) using a reversed phase silica column RP-18 5 μm, LiChrospher® 100 (LiChroCART® 250-4). Column temperature was maintained at 25 °C and the solvents used were H2O/H3PO4 (99:0.1, v/v) (solvent A) and CH3OH/ H3PO4 (99:0.1, v/v) (solvent B). The flow rate was set at 1.0 ml/ min and the solvent gradient started at 100% solvent A to 0% in 20 min, kept for 5 min, reached 100% solvent A in 1 min and kept for additional 9 min. Condensed tannins were determined colorimetrically, measuring absorbance at 520 nm (Yanagida et al., 2003). Results were expressed as proanthocyanidin fraction equivalents of an extract rich in dimer, trimer and tetramer proanthocyanidins (Soares et al., 2015) obtained from Vitisol (VITISOL®, Berkem, Gardonne, France).

2.6.2. MALDI-TOF Tentative identification of compounds was performed by using an UltrafleXtreme mass spectrometer (Bruker, Bremen, Germany) operating in reflector positive ion detection mode with laser smartbeam2 and under Compass flex Series 1.4 software control (Bruker, Bremen, Germany). Mass spectra ranged from 200 to 4000 Da applying 3000 laser shots accumulated with 1000 Hz of frequency. Each freeze-dried fraction was dissolved with 30% acetone, 0.1% TFA to a 0.5 mg/ml sample solution. A mix of 5 μL of sample solution and 15 μL of matrix (10 mg/ml of Super-2,5-Dihydroxybenzoic acid-SDHB (Bruker)) was prepared and 1 μL mix was applied on the MALDI target.

2.5. Response surface methodology (RSM)

3. Results and discussion

A central composite rotatable design was used to investigate the effects of three independent variables: concentration (X1), extraction time (X2) and temperature (X3) on the ellagitannins and condensed tannins content of tannin-rich fraction obtained from cork granulate. The same design was used to investigate the effects of two independent variables: concentration (X1) and extraction time (X2) on the ellagitannins and condensed tannins content of tannin-rich fraction obtained from cork powder. Results from preliminary trials were used to select suitable values for the independent variables. A second order polynomial Eq. (1) for the dependent variables was established to fit the experimental data. An analysis of variance (ANOVA) was carried out using STATISTICA software (free trial) to determine the significance of the variables:

Cork powder is the cork by-product this study intends to valorise by showing that it is as much valuable as cork granulate, deserving to be reused into an added value product and not as biomass for energy production. This work studies the recovery of tannins from cork granulate and powder. Although cork granulate is already a by-product reused by cork industry the recovery of tannins from the granulate is here performed for comparison with other reported studies (with similar cork particle size) and also for comparison with the results obtained for cork powder. The expected results intend to encourage cork industry to adopt sustainable practices by collecting efficiently cork powder in the different factory sections (contributing to the prevention of their occupational disease) and reducing waste generation with less global greenhouse gas emissions.

Y = β0 + β1X1 + β2X2 + β3X3 … + β11X12 + β22X22 + β33X32 … + (1) β12X1X2 + β13X1X3

3.1. Optimum conditions for the recovery of tannins from cork granulate

where X1, X2, X3 …… X12, X22 …… X1X2 … are the independent variables with their linear, quadratic and interactive models, and β0, β1, β2 ……. β12 are the regression coefficients of responses.

The recovery of tannins from cork granulate was studied for two types of solvent, aqueous solutions of acetone and ethanol, and optimised concerning solvent concentration (25–75%), extraction time (32–120 h) and extraction temperature (30–50 °C), according to the experimental design presented in Table 1. A regression analysis was carried out to fit mathematical models to the experimental data (see Section 2.5) presented in Table 1. The regression coefficients for the uncoded variables are shown in Table 2. The models fitted to the acetone experimental design for each response explains 81% of the ellagitannins content variability (R2 = 0.8051) and 76% of the condensed tannins content variability (R2 = 0.7659). The p-values of regression and ANOVA analysis showed that only concentration was significantly (p < 0.05) affecting the ellagitannins content at 95% confidence level and none of the independent variables is significantly affecting condensed tannins content, in the range studied. The models fitted to the ethanol experimental design for each response explains 52% of the ellagitannins content variability (R2 = 0.5207) and 69% of the condensed tannins content (R2 = 0.6865). According to the regression analysis no independent variables were affecting significantly (p < 0.05) ellagitannins and condensed tannins content, in the range studied. The estimate response surfaces based on the experimental data show that by using acetone, ellagitannins content increases with increasing acetone percentage for all time range studied (Fig. 1a). However, along extraction time at high acetone percentage the ellagitannins content decreases and then increases, which means that the compounds extracted at the first hours of extraction end up decomposing being replaced a few hours later by new compounds. This fact suggests that a more efficient extraction of ellagitannins from cork granulate may be achieved by successive short periods of extraction time of the same cork granulate. Room temperature is required for the recovery of the highest content of ellagitannins combined with high acetone concentration and

2.6. Tannins characterisation 2.6.1. HPLC-DAD/ESI-MS Identification of hydrolysable tannins was performed on an LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fischer Scientific, Bremen, Germany), coupled to an Accela Thermo Fisher series HPLC system. The mass spectrometer was equipped with an atmospheric pressure ionization (API) source, using electrospray ionization (ESI) interface. Compounds were separated on a reversed phase silica column RP-18 5μm, Purospher®STAR (LiChroCART® 150-4.6) and the solvents used were H2O/HCOOH (99:1, v/v) (solvent A) and H2O/CH3CN/ HCOOH (79:20:1, v/v) (solvent B). Column temperature was maintained at 25 °C. A stepwise gradient from 0 to 100% solvent B was applied at a flow rate of 0.5 ml/min for 40 min, kept for 10 min and decrease to 20% B in 5 min and kept for 5 min. Electrospray mass spectra data were recorded on a negative ionisation mode for a mass range from m/z 150 to 2000. Capillary and vaporizer voltage were set at 27 V and 3 kV, respectively. Capillary temperature was set at 275 °C. Collision-induced dissociation (CID) of the analytes was achieved using helium as the collision gas with 45% of normalised collision energy. MS handling software (Xcalibur QualBrowser software, Thermo Fischer Scientific) was used to confirm the compounds structure by their m/z value and MS2 fragmentation pattern. Quantification of compounds was done using SIM mode, dehydrated tertgallic-C-Glc (0.2–61 μg/mL; R2 = 0.9908; LQ = 0.04 μg/mL; LD = 0.01 μg/mL), vescalagin (0.7–169 μg/mL; R2 = 0.9900; LQ = 0.6 μg/mL; LD = 0.2 μg/mL), castalagin (0.5–92 μg/mL; R2 = 0.9993; LQ = 0.3 μg/mL; LD = 0.1 μg/mL) and grandinin (1.8–91 μg/mL; 3

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Table 1 Experimental design and corresponding response values for cork granulate extractions with acetone and ethanol. Independent variables

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 a b

Ellagitannins

a

Condensed tannins

b

Concentration (%)

Extraction time (h)

Temperature (°C)

Acetone

Ethanol

Acetone

Ethanol

50 25 75 50 92 75 50 8 25 50 75 50 25 50 75 25

76 32 120 76 76 32 150 76 120 76 120 2 32 76 32 120

23 30 30 40 40 30 40 40 50 40 50 40 50 57 50 30

1.0 0.3 1.6 0.4 0.7 1.7 1.6 0.3 0.6 0.4 0.7 0.3 0.6 0.6 0.8 0.7

0.2 0.2 1.7 0.4 0.2 0.3 0.8 0.5 1.2 0.6 0.4 1.1 0.3 0.1 0.6 0.3

1.3 0.4 2.5 0.8 1.0 3.3 3.0 0.5 1.3 0.6 1.4 0.2 0.6 1.0 0.8 0.6

0.3 0.5 1.8 0.7 0.2 0.3 1.6 0.5 1.7 1.0 0.9 2.0 0.7 1.0 0.9 0.4

(0) (−1) (1) (0) (+2) (1) (0) (−2) (−1) (0) (1) (0) (−1) (0) (1) (−1)

(0) (−1) (1) (0) (0) (−1) (2) (0) (1) (0) (1) (−2) (−1) (0) (−1) (1)

(−2) (−1) (−1) (0) (0) (−1) (0) (0) (1) (0) (1) (0) (1) (2) (1) (1)

mg ellagic acid equivalents/ g cork. mg proanthocyanidin fraction equivalents/ g cork.

extraction time (Fig. 1b–c), which was expected when dealing with acetone, given its high volatility. Looking to the estimate response surfaces based on the experimental data using ethanol, the results are quite different. For short periods of extraction time, the increase in ethanol percentage does not affect ellagitannins content, while for long periods of extraction time the increase in ethanol percentage does increase ellagitannins content recovery (Fig. 1d). The recovery of ellagitannins is obtained by using room temperature and high ethanol percentage is the same as by using high temperatures and low ethanol percentage (Fig. 1e). Using high temperatures, low extraction time is required conversely to room temperature that required high extraction time (Fig. 1f). In the case of condensed tannins content, the estimate response surfaces based on the experimental data shows the same behaviour of ellagitannins when extracted with acetone (Fig. 2a). When using ethanol, the best values of condensed tannins are reached for 50% of ethanol and not for the highest percentages. However, high extraction time is also required (Fig. 2d) and the temperature positively affects their extraction in these conditions (Fig. 2e–f). In contrast, when using acetone, the highest values were obtained at room temperature (20–25 °C) and temperature higher than 35 °C affects negatively the extraction of condensed tannins (Fig. 2b). However, when using long periods of extraction, the content of condensed tannins increases with

increasing temperature (Fig. 2c). Combining this result with the previous (Fig. 2b–c), the change in the extracted compounds will be expected. Summing up, for the recovery of ellagitannins from cork granulate both acetone and ethanol required high extraction time and concentration. For the recovery of condensed tannins from cork granulate using acetone the same requirements were needed but when using ethanol, although low concentrations could be used, high extraction time and temperature were still required. High temperature was not efficient when using acetone while using ethanol the results suggested change in the compounds during long periods of extraction time. In this case, the same results could be achieved with low temperature by increasing extraction time and solvent concentration. In addition, using acetone the recovery of both tannin families was higher than using ethanol, this may be due to the lower relative polarity of acetone (0.355) when compared to ethanol (0.654). Also, the models obtained from the ethanol experimental data explain very little of the variability of the results, which suggest a great variability on the extraction when using this organic solvent, and consequently suggests an improved capacity of acetone to soak the cork matrix allowing for a greater reproducibility in the extraction. Thus, this work proceeds assuming aqueous solutions of acetone as the best solvent to extract tannins from cork.

Table 2 Regression coefficients and analysis of variance of uncoded units for ellagitannins and condensed tannins response after extraction of cork granulate with acetone and ethanol. Ellagitannins

Condensed tannins

Acetone

Ethanol

Acetone

Ethanol

estimate

p-value

estimate

p-value

estimate

p-value

estimate

p-value

β0 β1 β2 β3 β11 β12 β13 β23

1.2099 0.0452 0.0001 −0.0052 0.0001 −0.0905 0.0017 −0.0001

0.036 0.602 0.097 0.090 0.111 0.177 0.543

−3.3095 0.0429 0.0000 −0.0043 0.0001 0.1384 −0.0009 0.0000

0.791 0.905 0.379 0.271 0.928 0.589 0.888

1.1967 0.1078 0.0001 −0.0344 0.0002 −0.1105 0.0020 −0.0001

0.065 0.817 0.086 0.178 0.269 0.430 0.605

−3.0033 0.0608 −0.0003 −0.0217 0.0002 0.1348 −0.0008 0.0001

0.961 0.312 0.397 0.100 0.256 0.653 0.693

R2

0.8051

0.5207

0.7659

Bold value indicate p < 0.05. 4

0.6865

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Fig. 1. Estimated response surfaces for the effect of (a)(d) concentration and extraction time, (b)(e) concentration and temperature and (c)(f) extraction time and temperature, on ellagitannins content using acetone and ethanol.

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Fig. 2. Estimated response surfaces for the effect of (a)(d) concentration and extraction time, (b)(e) concentration and temperature and (c)(f) extraction time and temperature, on condensed tannins content using acetone and ethanol.

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The optimum conditions chosen, using the regression equation fitted to the data, to maximise both the ellagitannins and condensed tannins content, were: 90% acetone, 150 h of extraction time and 25 °C. The predicted ellagitannins and condensed tannins content by the regression Eq. (1) was 3.0 mg ellagic acid equivalents/ g cork granulate and 4.4 mg proanthocyanidin fraction equivalents/ g cork granulate. The experimental values observed (n = 4) were 2.9 ± 0.5 mg ellagic acid equivalents/ g cork granulate and 2.3 ± 0.5 mg proanthocyanidin fraction equivalents/ g cork granulate, which validate the model obtained for the ellagitannins content but not the model obtained for the condensed tannins. Other authors achieved similar quantities of ellagitannins in cork granulate of 0.5–1 mm particle size when using 80% methanol, during 24 h at room temperature, however the quantities obtained for condensed tannins were 3-fold less (Cadahía et al., 1998). The results achieved show that by changing conventional time and solvent of extraction high quantity of polyphenols is recovered, particularly affecting a family of phenols barely known in cork. However, the results indicate that the recovery of tannins from cork granulate is not a viable solution to cleaner production as too much energy will be expended to obtain an added value product. In addition, cork granulate is already reused by cork industry and hence the study follows with the recovery of tannins using cork powder as raw material, expecting improvements on the extraction conditions and towards a cleaner production.

Table 4 Regression coefficients and analysis of variance of uncoded units for ellagitannins and condensed tannins response after extraction of cork powder with acetone. Ellagitannins

a

Condensed tannins

b

estimate

p-value

estimate

p-value

β0 β1 β2 β11 β22 β12

5.2424 0.0758 −0.1013 −0.0008 −0.0017 0.0017

0.005 0.026 0.060 0.749 0.256

1.3975 0.1362 −0.1862 −0.0011 0.0019 0.0013

0.002 0.137 0.031 0.767 0.438

R2

0.9313

a b

0.9460

mg ellagic acid equivalents/ g cork. mg proanthocyanidin fraction equivalents/ g cork.

present a similar behaviour in the range studied. The recovery of tannins from cork powder increases with increasing acetone percentage. For ellagitannins the highest content was reached between 40–70% of acetone and for condensed tannins between 50–80% of acetone. Extraction time only influence tannins content when using the lowest acetone concentrations. The optimum conditions chosen, using the regression equation fitted to the data, to maximise both the ellagitannins and condensed tannins content, were 60% acetone and 9h30 of extraction time. The predicted ellagitannins and condensed tannins content by the regression Eq. (1) was 6.9 mg ellagic acid equivalents/ g cork powder and 4.7 mg proanthocyanidin fraction equivalents/ g cork powder. The experimental values observed (n = 3) were 5.8 ± 1.0 mg ellagic acid equivalents/ g cork powder and 4.3 ± 0.8 mg proanthocyanidin fraction equivalents/ g cork powder, which validate both models obtained for the ellagitannins and condensed tannins content. This result indicates that using cork powder the tannins extraction efficiency increases: the yield of extraction of both family of compounds doubles by using less solvent and extraction time. These results are promising to the use of cork powder as a raw material for the recovery of tannins. In 2016 the cork powder obtained from the cork stoppers production could provide a recovery of 180 tonnes of tannins-rich extract, despite being only 0.6% of the waste produced, the commercial value is very high. This alternative use of cork powder moves towards a cleaner production by increasing interest of cork industry to this waste leading to proper investments on the collection of cork powder through the different factory sections (decreasing occupational disease) and the reduction of cork powder combustion for the industry energy needs (reducing global greenhouse gas emissions). For better reduction of combustion other bioactive recoveries should be explored for the most use of cork powder.

3.2. Optimum conditions for the recovery of tannins from cork powder According to the results obtained previously with the acetone models, the recovery of tannins from cork powder was studied only using acetone and all the extractions were performed at room temperature (22–24 °C). Preliminary results demonstrated that by using 50% acetone during 16h30 the values obtained for ellagitannins and condensed tannins were 3-fold higher than the ones obtained using the optimum conditions used for cork granulate (data not shown). Consequently, the recovery of tannins from cork powder was optimised concerning solvent concentration (25–75%) and extraction time (12–24 h), according to the experimental design presented in Table 3. A regression analysis was carried out to fit mathematical models to the experimental data (see Section 2.5) presented in Table 3. The regression coefficients for the uncoded variables are shown in Table 4. The models fitted to the experimental design for each response explains 93% of the ellagitannins content variability (R2 = 0.9313) and 95% of the condensed tannins content variability (R2 = 0.9460). The p-values of regression and ANOVA analysis showed that concentration and extraction time were significantly (p < 0.05) affecting ellagitannins content and concentration and the quadratic factor of concentration were significantly affecting condensed tannins content, at 95% confidence level in the range studied. Estimate response surfaces based on the experimental data (Fig. 3) show that ellagitannins and condensed tannins

Table 3 Experimental design and corresponding response values for cork powder extractions with acetone. Concentration (%) 1 2 3 4 5 6 7 8 9 10 a b

25 50 75 50 15 50 50 85 25 75

Extraction time (h) (−1) (0) (1) (0) (−2) (0) (0) (2) (−1) (1)

24 18 12 9.5 18 18 26.5 18 12 24

Ellagitannins (1) (0) (−1) (−-2) (0) (0) (2) (0) (−1) (1)

mg ellagic acid equivalents/ g cork. mg proanthocyanidin fraction equivalents/ g cork. 7

3.9 6.1 5.9 6.8 4.5 6.5 5.9 6.7 5.7 6.0

a

Condensed tannins 1.3 3.9 3.7 4.9 1.2 3.8 3.6 4.2 2.0 4.3

b

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S.F. Reis, et al.

Fig. 3. Estimated response surfaces for the effect of concentration and extraction time on ellagitannins and condensed tannins content using acetone.

are easily explained by the lowest particle sizes consequently increasing the superficial area of contact between matrix and solvent when using cork powder which increases the extraction efficiency. The Bate-Smith reaction observed upon acid decomposition seem to indicate the presence of condensed tannins, however no condensed tannins were identified by HPLC-DAD/ESI-MS, which suggest that simple dimers and trimers are not present in this tannin-rich fraction. Thus, flow injection analysis (FIA) on LTQ Orbitrap XL mass spectrometer was performed to increase sensitivity, however the mass signals over 600 Da were too weak and fragmentation was not possible for most of them. Fragmentation was obtained in positive mode for the m/z ions 973-MS2 955 (-18 Da); 1105-MS2 1087 (-18 Da), 1015 (−90 Da) and 1141-MS2 1123 (-18 Da), 1105 (−36 Da), 528 (-613 Da). Their fragmentation pattern, loss of 18 Da is a characteristic fragmentation of a C-glycosidic ellagitannin molecule which turns these peaks as possible ellagitannins. MALDI-TOF analysis was the following approach in line with other authors which achieved a tentative identification of condensed tannins on softwood (Bianchi et al., 2015, 2014; Ucar et al., 2013) and hardwood bark extracts (Bikoro Bi Athomo et al., 2018) including Quercus species (Ricci et al., 2017). The MALDI spectrum obtained showed regular and continuous intervals between peaks of 16 Da, 22 Da, 38 Da, 132 Da and 168 Da which may be attributed to potassium salt adduct (15.97 Da; Na+↔ K+) or OH group (15.99 Da), sodium salt adduct (21.98 Da; Na+↔ H+), potassium salt adduct (37.96 Da; K+↔ H+), sugar pentose and gallic acid, respectively (Fig. 4). The peak observed at m/z 957 was assigned to the sodium adduct of vescalagin/castalagin (Table 6) which presents an interval of 132 Da to 1089 m/z peak assigned to a sodium adduct of grandinin/roburin E and an interval of 168 Da to 1125 m/z peak assigned to a sodium adduct of vescavaloninic/castavaloninic acid. It was also detected an interval of 272 Da to 1229 m/z peak and 288 Da to 1245 m/z peak, as 1229 m/z peak was attributed to the sodium adduct of acutissimin, which is vescalagin linked to catechin (Yoshida et al., 2010), then making 272 Da corresponding to a catechin linkage and 288 Da to a gallocatechin, and consequently 1245 m/z peak may be assigned to the sodium adduct of guajavin B/ eugenigrandinin A, being for the first time reported in cork extracts. From these two peaks, it can be observed increments of 132 Da and also 264 Da, which corresponds to two linked sugar pentoses and from these last m/z peaks other increments of 132 Da are observed. This result suggests that acutissimin A/B and guajavin B/ eugenigrandinin A may be linked to several sugar pentoses through the respective

3.3. Characterisation of tannin-rich fraction from cork granulate and powder The tannin-rich fraction obtained from cork granulate and powder using the optimum conditions chosen was characterised by HPLC-DAD/ ESI-MS. The same compounds were identified in both corks granulate and powder, although in different quantities (Table 5), which indicates that cork powder presents the same polyphenol composition as cork granulate. Twenty-three ellagitannins were tentatively identified based on exact mass, mass fragmentation and elution order of the literature data regarding characterization of Quercus species (Fernandes et al., 2011; García-Villalba et al., 2017; Moilanen et al., 2015; Muccilli et al., 2017; Navarro et al., 2017). Castalagin and vescalagin were identified on the same basis but also by NMR (results on Section 2.2). The three isomers of dehydrated tergallic-C-Glc, 2 isomers of HHDP-Glc and 4 isomers of Di-HHDP-Glc tentatively identified are included in the simple ellagitannins group. Vescalin, castalin, vescalagin, castalagin, ethylvescalagin, grandinin, roburin E, vescavaloninic and castavaloninic acids belong to the C-glycosidic ellagitannins group. Three flavano-ellagitannins such as mongolicain A/B, acutissimin A/B and guajavin B/ eugenigrandinin A and two oligomeric ellagitannins such as roburin A and D were also tentatively identified. The major compound present was castalagin, followed by isomer 1 and 3 of dehydrated tergallic-CGlc. Vescalagin, roburin A/D and vescavaloninic acid were the following compounds, between them the quantity is not significantly different due to their extraction variability. The remaining compounds were present in less than 100 μg/g in cork granulate and 200 μg/g in cork powder. Vescalin, castalin, guajavin B/ eugenigrandinin A, vescavaloninic and castavaloninic acids were tentatively identified for the first time in cork extracts. Cork powder presented 2 to 3-fold higher content of each compound than cork granulate, except for isomer 4 of Di-HHDP-Glc (1.3 fold). Cork powder also presented lower variation coefficients for each compound than cork granulate, which varied from 4% for isomer 1 of dehydrated tergallic-C-Glc to 37% for roburin A/D (first isomer) and 9% for castalin to 72% for acutissimin in the case of cork granulate. This fact is also reflected in the total quantity of ellagitannins calculated by the sum of all quantified compounds by HPLC-ESI/DAD-MS, 17% and 33% of variation coefficients in cork powder and granulate, respectively. These observations confirm the RSM results seen previously and 8

9 1101.077 1175.130 1205.141 1221.136 1850.145

C48H30O31

C55H36O30 C56H38O31 C56H38O32 C82H51O51

481.06241 481.06281 613.04852 613.04755 613.04797 631.05841 631.05939 783.07007 783.06866 783.07031 783.06946 933.06482 933.06512 961.06000 1065.10657 1065.10620 1101.07019 1101.07019 1175.12458 1205.12720 1222.12604 1850.12824 1850.12702

[M−H]− Obs. 301, 275, 421 523, 493, 465, 595, 301 493, 299 493, 523, 301 613, 569, 587, 441, 467, 479 569, 613, 587, 441, 425, 467 703, 721, 765, 659, 401, 845 481, 341, 299, 275, 419, 721, 765 301, 481, 275, 747 301, 481, 275, 249, 570, 651, 530 915, 871, 569, 613 631, 569, 897, 871, 915, 425 917, 873 975, 931, 987, 673, 945, 501, 1029 975, 1029, 931, 987, 843, 901 1039, 995, 1082, 915, 870, 461, 769 1057, 870, 932, 995, 631, 569, 1039 873, 495, 855 915, 871 915, 457, 551, 305, 589 915, 871, 569, 493, 613 631, 871, 569, 915, 897, 467, 507, 613

MS2 (m/z)

238 238 238 238 238

238

238 238

238

238

238, 373 238, 370 238, 373 238

238

λmáx (nm)

(c)

Total

(b)

(b)

(d)

(d)

(d)

(d)

0.9913

0.9900 0.9993

(a)

(b)

0.9908

(a)

(a)

(a)

R2

tr 15 ± 3.7 264 ± 133 51 ± 28 185 ± 75 tr 41 ± 3.5 16 ± 6 45 ± 14 14 ± 4 23 ± 31 130 ± 54 424 ± 105 16 ± 5 58 ± 26 77 ± 24 118 ± 26 67 ± 24 29 ± 14 62 ± 44 tr 103 ± 40 149 ± 74 1870 ± 625

μg/g cork granulate

tr 48 ± 13 440 ± 18 108 ± 6.0 380 ± 30 tr 118 ± 29 30 ± 7 60 ± 15 22 ± 6 75 ± 52 346 ± 123 842 ± 231 39 ± 6 193 ± 54 190 ± 38 257 ± 118 185 ± 55 44 ± 2.4 146 ± 13 tr 258 ± 96 324 ± 37 4056 ± 678

μg/g cork powder

NMR results described in material and methods; tr – traces; (a) quantified in equivalents of dehydrated tergallic−C−Glc isomer 3; (b) quantified in equivalents of castalagin; (c) quantified in equivalents of vescalagin; (d) quantified in equivalents of grandinin.

*

961.065 1065.114

C34H24O22

C42H26O27 C46H34O30

783.074

C27H20O18

Vescalin Castalin Di-HHDP-Glc

933.070

631.062

C27H18O17

Dehydrated tergallic-C-Glc

C41H26O26

613.051

C20H18O14

4,6-HHDP-Glc

Vescalagin* Castalagin* Ethylvescalagin Grandinin Roburin E Vescavaloninic acid Castavaloninic acid Mongolicain A/B Acutissimin A/B Guajavin B/Eugenigrandinin A Roburin A/D

481.066

Molecular formula

Compound name

[M−H]− Calc.

Table 5 Tentative identification and quantification of ellagitannins by HPLC-DAD/ESI-MS and corresponding MSn fragmentation profiles in cork granulate and powder.

S.F. Reis, et al.

Industrial Crops & Products 140 (2019) 111599

Industrial Crops & Products 140 (2019) 111599 x10 5

In ten s . [a .u .]

Inte ns . [a .u.]

S.F. Reis, et al.

973 .208

x1 0 4

1 8 8 9.4 3 5 411.14

291.05

1.2 5

1.25

168.03 132.06

18 7 4 .4 72 1.0 0

290.04

274.08

1.00

0.7 5

257.08

243.10

132.07

2 1 6 4.50 9 167.03

0.5 0

168.04

0.75

20 2 1 .50 2 20 4 1 .5 02

1 9 29 .3 9 1

272.11

2 1 48 .5 4 7

2 0 5 7.47 7

132.04

21 1 7 .5 75

2 1 80 .4 9 5 2 3 00 .5 7 8

2 00 6 .5 1 3

0.2 5

288.11 0.0 0

614.18

1 90 0

2 00 0

2 1 00

2 2 00

2300

2400 m /z

0.50 917.24 132.33

167.03

132.30

1141.249

168.04

264.40

0.25

264.38

1245 .335

132.07

132.06

1889.43 5

132.07

1377.6 33

1509.716

287.61

158 7.382

17 73.845

1929 .391

2057.477

2164.509

2300.5 78

0.00 1000

1200

140 0

1600

1 80 0

2000

2200

2400 m /z

Fig. 4. MALDI-TOF spectrum of tannin-rich fraction obtained from cork powder.

Moreover, following the increments of 1245 m/z peak attributed to guajavin B/ eugenigrandinin A, it is possible to observe after m/z peak 1641 (guajavin + 3 sugar pentoses) an increment of 288 Da, corresponding to a gallocatechin which indicates the possible presence of a glycosylated dimer of gallocatechin linked to the vescalagin/castalagin. From the 957 m/z peak, it was also observed a mass increment of 613 Da possibly of a vescalin molecule and a mass increment of 915 Da attributed to another vescalagin/castalagin molecule corresponding to the 1874 m/z peak attributed to the sodium adduct of roburin A/D. It’s also possible to observe mass increments of 168, 242, 257, 274, 290 and 411 Da from the roburin A/D sodium adduct. These increments may be attributed to gallic acid, trihydroxyflavan, tetrahydroxyflavan, catechin, gallocatechin and gallocatechin 3-p-hydroxybenzoate, justifying the differences of 2H to the dense areas observed which may be due to signal overlap. These types of flavonoid structures were also observed as building blocks of condensed tannins of softwood and hardwood bark tannin extracts (Bikoro Bi Athomo et al., 2018; Ucar et al., 2013), however as far as we know their linkage to ellagitannins has never been reported. MALDI-TOF analysis allowed for the first time the tentative identification of complex glycosylated ellagitannins and a glycosylated dimer of gallocatechin linked to vescalagin/castalagin. Further work needs to be done to improve the detailed structural characterisation of these condensed tannins, despite the results indicate that they are linked to ellagitannins, the presence as common polymerised proanthocyanidins and/or prodelphinidins cannot be discarded as evidences of signal overlap have been observed.

Table 6 Tentative identification of experimental MALDI-TOF peaks. MW exp.

MW calc.

Proposed composition

Attempt identification

957.230 973.208 1089.298 1105.273 1125.270 1141.249 1229.336 1245.335 1261.348 1361.665 1377.633

957.068 973.063 1089.112 1105.107 1125.075 1141.070 1229.139 1245.134 1261.129 1361.254 1377.249

[M+Na]+ [M+K]+ [M+Na]+ [M+K]+ [M+Na]+ [M+K]+ [M+Na]+ [M+Na]+ [M+K]+ [M+Na]+ [M+Na]+

Vescalagin/Castalagin

1493.735 1509.716

1493.369 1509.364

[M+Na]+ [M+Na]+

1571.407 1587.382 1625.791 1641.782

1571.127 1587.122 1625.484 1641,479

[M+Na]+ [M+K]+ [M+Na]+ [M+Na]+

1773.845

1773.594

[M+Na]+

1874.472 1889.435 1929.391

1874.143 1890.138 1929.735

[M+Na]+ [M+K]+ [M+Na]+

2021.502 2041.502 2057.477 2117.575 2131.548 2148.547 2164.509 2180.495 2300.578

2022.253 2042.247 2058.242 2114.405 2130.404 2146.392 2162.399 2178.138 2298.500

+

[M+K] [M+Na]+ [M+K]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+Na]+ [M+K]+ [M+Na]+

Grandinin/Roburin E Vescavaloninic/Castavaloninic acid Acutissimin A/B Guajavin B/ Eugenigrandinin A acutissimin A/B+sugar pentose guajavin B/eugenigrandinin A+sugar pentose acutissimin A/B+2 sugar pentoses guajavin B/ eugenigrandinin A+2 sugar pentoses vescalagin/castalagin+vescalin acutissimin A/B+3 sugar pentoses guajavin B/ eugenigrandinin A+3 sugar pentoses guajavin B/ eugenigrandinin A+4 sugar pentoses Roburin A/D guajavin B/ eugenigrandinin A+gallocatechin + 3 sugar pentoses roburin A/D+sugar pentose roburin A/D+gallic acid roburin roburin roburin roburin

A/D+trihydroxyflavan A/D+tetrahydroxyflavan A/D+catechin A/D+gallocatechin

4. Conclusion This study shows that cork powder is a potential raw material for the recovery of high valuable compounds with a potential application in different industries such as cosmetic, pharmaceutic and food industries. This alternative use of cork powder can raise high profits to cork industry which may increase the interest on this waste moving towards cleaner production by proper investments on collection of cork powder through the different factory sections (increasing occupational disease prevention) and reducing cork powder combustion for the industry energy needs (decreasing global greenhouse gas emissions). This study also improves the knowledge on cork phenolic fraction through the tentative identification of new tannins, the quantification of most of the ellagitannins tentatively identified and the evidences of complex ellagitannins which may be linked to condensed tannins.

roburin A/D+gallocatechin 3-phydroxybenzoate

catechin/gallocatechin group, observed here up to 3. In softwood bark extracts increments of 132 Da were also observed and the presence of condensed tannins attached to carbohydrate chains was not discarded (Bianchi et al., 2015). Also, long carbohydrate chains attached to flavonoids were reported in a tannin extract of the shells of the pods of African locust bean tree (Drovou et al., 2015). However, ellagitannins linked to this type of structures have not been reported so far. 10

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Declaration of Competing Interest

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None. Acknowledgements This work was developed under the project 3310-CorkPlus“Contribution of cork stoppers to the chemical and sensory properties of bottled wine” co-financed by the European Regional Development Found (FEDER) through the Operational Competitiveness and Internationalization Program (COMPETE 2020) and UID/ QUI/50006/ 2013 - POCI/01/0145/FEDER/007265. The authors acknowledge Dr Joana Pinto from ICETA/REQUIMTE/LAQV – Faculty of Pharmacy, University of Porto, for providing NMR characterisation of castalagin and vescalagin. References APCOR, 2018. CORK_17/18. Bol. Estatístico 55. APCOR, 2016. CORK_2016. Bol. Estatístico 59. Bianchi, S., Gloess, A.N., Kroslakova, I., Mayer, I., Pichelin, F., 2014. Analysis of the structure of condensed tannins in water extracts from bark tissues of Norway spruce (Picea abies [Karst.]) and Silver fir (Abies alba [Mill.]) using MALDI-TOF mass spectrometry. Ind. Crops Prod. 61, 430–437. https://doi.org/10.1016/j.indcrop. 2014.07.038. Bianchi, S., Kroslakova, I., Janzon, R., Mayer, I., Saake, B., Pichelin, F., 2015. Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry 120, 53–61. https://doi.org/10.1016/ j.phytochem.2015.10.006. Bikoro Bi Athomo, A., Engozogho Anris, S.P., Safou-Tchiama, R., Santiago-Medina, F.J., Cabaret, T., Pizzi, A., Charrier, B., 2018. Chemical composition of African mahogany (K. ivorensis A. Chev) extractive and tannin structures of the bark by MALDI-TOF. Ind. Crops Prod. 113, 167–178. https://doi.org/10.1016/j.indcrop.2018.01.013. Cadahía, E., Conde, E., de Simón, B., García-Vallejo, M.C., 1998. Changes in tannic composition of reproduction cork Quercus suber throughout industrial processing. J. Agric. Food Chem. 46, 2332–2336. https://doi.org/10.1021/jf9709360. Carriço, C., Ribeiro, H.M., Marto, J., 2018. Converting cork by-products to ecofriendly cork bioactive ingredients: novel pharmaceutical and cosmetics applications. Ind. Crops Prod. 125, 72–84. https://doi.org/10.1016/j.indcrop.2018.08.092. Conde, E., Cadahía, E., García-Vallejo, M.C., Fernández de Simón, B., 1998. Polyphenolic composition of Quercus suber cork from different Spanish provenances. J. Agric. Food Chem. 46, 3166–3171. https://doi.org/10.1021/jf970863k. Conde, E., Cadahía, E., García-Vallejo, M.C., Fernández de Simón, B., González Adrados, J.R., 1997. Low molecular weight polyphenols in cork of Quercus suber. J. Agric. Food Chem. 45, 2695–2700. https://doi.org/10.1021/jf960486w. Drovou, S., Pizzi, A., Lacoste, C., Zhang, J., Abdulla, S., El-Marzouki, F.M., 2015. Flavonoid tannins linked to long carbohydrate chains – MALDI-TOF analysis of the tannin extract of the African locust bean shells. Ind. Crops Prod. 67, 25–32. https:// doi.org/10.1016/j.indcrop.2015.01.004. Fernandes, A., Fernandes, I., Cruz, L., Mateus, N., Cabral, M., de Freitas, V., 2009. Antioxidant and biological properties of bioactive phenolic compounds from Quercus suber L. J. Agric. Food Chem. 57, 11154–11160. https://doi.org/10.1021/ jf902093m. Fernandes, A., Sousa, A., Mateus, N., Cabral, M., de Freitas, V., 2011. Analysis of phenolic

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