Accepted Manuscript Title: Microwave superheated water and dilute alkali extraction of brewersˇı spent grain arabinoxylans and arabinoxylo-oligosaccharides Author: Elisabete Coelho M. Ang´elica M. Rocha Jorge A. Saraiva Manuel A. CoimbraThese authors contributed equally to the work. PII: DOI: Reference:
S0144-8617(13)00893-X http://dx.doi.org/doi:10.1016/j.carbpol.2013.09.003 CARP 8092
To appear in: Received date: Revised date: Accepted date:
20-2-2013 30-8-2013 3-9-2013
Please cite this article as: Coelho, E., Rocha, M. A. M., Saraiva, J. A., & Coimbra, M. A., Microwave superheated water and dilute alkali extraction of brewersˇı spent grain arabinoxylans and arabinoxylo-oligosaccharides, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Microwave superheated water and dilute alkali extraction of brewers´
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spent grain arabinoxylans and arabinoxylo-oligosaccharides
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Elisabete Coelhoa*, M. Angélica M. Rochaa, Jorge A. Saraiva, and Manuel A. Coimbra
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QOPNA, Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro,
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Portugal
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*Corresponding author. Tel.:+351 234 370706; fax: +351 234 370084.
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E-mail address: [email protected] (E. Coelho).
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These authors contributed equally to the work.
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Abstract
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Microwave superheated water extractions (MWE) were performed to evaluate the
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feasibility of this technology for quantitative recovery of the arabinoxylans (AX) or
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arabinoxylo-oligosaccharides (AXOS) from brewers´ spent grain (BSG). The
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AX+AXOS yield increased with the increase of the temperature in the range from 140
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to 210 °C during 2 min. The higher temperatures promoted depolymerisation,
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debranching, and deesterification of the polysaccharides, with formation of brown
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products. The conditions that promote a compromise between the yield and the structure
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obtained, minimizing the thermal degradation of the fractions extracted by MWE are the
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following: 1) 140 ºC, to remove the residual starch mixed with β-glucans; 2) Suspension
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of the residue left in water and treated at 180 ºC; 3) suspension of the residue in 0.1 M
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KOH and treated at 180 ºC. Using this sequential procedure, it was possible to extract
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62 % of BSG AX+AXOS, presenting degrees of polymerization ranging between 7 and
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24 xylose residues, and a degree of phenolic acids esterification between 5 and 21 %.
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The structural variability obtained by MWE allows defining specific types of
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compounds for different applications and uses depending on the extraction conditions
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used.
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Keywords: Brewer’s spent grain; polysaccharides; arabinoxylans; feruloylated
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arabinoxylo-oligosaccharides; microwave-assisted extraction; prebiotics; food
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ingredients; barley.
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1. Introduction During germination of the barley grain, cell walls, mainly of the endosperm, are
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enzymatically degraded. After filtration of the liquid extracted from the mashing,
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remaining unhydrolysed cell walls, which are derived mainly from the aleurone and
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some insoluble material present in the endosperm, are disposed of as the brewer’ spent
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grain (BSG). The BSG is composed exclusively by the material from barley husks, with
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75 % moisture, having a high percentage of polysaccharides, namely, arabinoxylans
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(AX, 22-23 %), cellulose (12-25 %) and small amounts of starch (4 %) and β-glucans
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(Aliyu & Bala, 2011; Kabel et al., 2002a). BSG represents up to 85 % of the total
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residues from the brewing process and it amounts to about 20 kg/hL beer. It is estimated
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that Europe produces 3.5 million ton per year of BSG (Jay et al., 2008). This by-product
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has been used for animal feed as a way to reduce the residue produced by these
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industries. The use of BSG as a source of polysaccharides with fibre properties and
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other biological activities that have been associated with AX and β-glucans, namely
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prebiotic activity (Broekaert, Courtin, Verbeke, Van de Wiele, Verstraete & Delcour,
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2011; Crittenden et al., 2002), has a great potential. These polysaccharides are
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considered as dietary fibre due to their resistance to hydrolysis by the digestive tract
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enzymes. Additionally, they can present immunomodulatory (Van Craeyveld et al.,
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2008), antioxidant (Ohta, Semboku, Kuchii, Egashira & Sanada, 1997) and
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anticarcinogenic effect (Broekaert et al., 2011), and reduce blood serum triglycerides
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and cholesterol levels (Broekaert et al., 2011).
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BSG AX are composed by a backbone of β-(1→4)-linked xylose residues
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containing single units of arabinose as side chains, phenolic acids, such as ferulic and p-
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coumaric acids are esterified to arabinofuranosyl residues (Bartolomé, Santos, Jiménez,
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del Nozal & Gómez-Cordovés, 2002; Mandalari et al., 2005). Feruloylated arabinoxylo-
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oligosaccharides (AXOS) are more potent antioxidants and radical scavengers than
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ferulic acid itself (Broekaert et al., 2011; Katapodis, Vardakou, Kalogeris, Kekos,
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Macris & Christakopoulos, 2003; Ohta et al., 1997). In vitro, feruloylated AXOS
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strongly inhibit copper mediated oxidation of low density lipoprotein (Katapodis et al.,
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2003). The antioxidant properties of feruloylated AXOS derived from wheat bran have
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recently been confirmed in an in vivo model of diabetic rats (Ou, Jackson, Jiao, Chen,
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Wu & Huang, 2007). In diabetic rats, feruloylated AXOS significantly increased total
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antioxidant capacity level, glutathione peroxidase, and superoxide dismutase activities,
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decreased blood glucose and malondiadehyde levels, and decreased xanthine oxidase
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activity in serum and liver. Feruloylated AXOS were, overall, more efficient in
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mitigating oxidative damage in diabetic rats than sodium ferulate and ascorbic acid.
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Furthermore, feruloylated AXOS showed greater antioxidant capacity in vivo than in
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vitro when compared with ascorbic acid (Ou et al., 2007). The ester linkage between
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ferulic acid and AXOS prevents its uptake until reaching the colon, where AXOS-
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inducible bacterial feruloyl esterases are present (Vardakou, Nueno Palop, Gasson,
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Narbad & Christakopoulos, 2007). Feruloylated AXOS is therefore a special type of
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antioxidant whose active principle is released in the colon (Broekaert et al., 2011).
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Moreover, formulations combining AXOS with AX potentiate their prebiotic activity
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(Broekaert, Courtin, Damen & Delcour, 2010).
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Microwave-assisted extraction is a process that uses microwave energy to heat the
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solvent that is in contact with a sample in order to partition analytes from the sample
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matrix into the solvent. The main advantage of this technique is the ability to rapidly
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heat the sample solvent mixture. By using closed vessels the extraction can be
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performed at elevated temperatures accelerating the mass transfer of target compounds
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from the sample matrix. Typically, microwave-assisted extraction procedure takes 15–
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30 min and uses small solvent volumes, about 10 times smaller than volumes used by
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conventional extraction techniques (Eskilsson & Bjorklund, 2000). Recently,
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microwave
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galactomannans from spent coffee grounds was proposed (Passos & Coimbra, 2013).
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The use of microwaves for AX extraction has also been proposed for some industry
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residues, such as barley husks (Roos, Persson, Krawczyk, Zacchi & Stalbrand, 2009),
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corn and corn stover (Benkő, Andersson, Szengyel, Gáspár, Réczey & Stålbrand, 2007;
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Pang, Xue, Yu, Zhang, Li & Kang, 2012; Rose & Inglett, 2010; Yoshida, Tsubaki,
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Teramoto & Azuma, 2010), wheat straw (Janker-Obermeier, Sieber, Faulstich &
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Schieder, 2012), and flax shives (Buranov & Mazza, 2010). These polymers can be
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obtained directly in water (Roos et al., 2009; Yoshida et al., 2010), dilute alkali and/or
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acid (Benkő et al., 2007; Janker-Obermeier et al., 2012) or in a hydroalcoholic solution
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(Buranov & Mazza, 2010). The aim of this work was to determine the yield and changes
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in the structure of AX that can be recovered from BSG when microwave technology is
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used to carry out the extraction using water at temperatures reaching 140-210 ºC during
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2 minutes. In addition a sequential extraction using water and diluted alkali solution was
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assayed.
extraction
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superheated
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2. Experimental
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2.1. Samples
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The BSG used in this study was provided by Unicer-Bebidas S.A., Leça do Balio,
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Portugal, with 76 % of moisture, being the dry material composed by carbohydrates (50
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%), proteins (30 %), fats (8 %), and ashes (5 %).
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2.2 Effect of temperature on the extraction of AX by microwave superheated water BSG (10 g) was suspended in 60 mL distilled water in a 100 mL Teflon-coated
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vessel at a liquid/solid ratio of 6:1 and the pH was measured before the microwave
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irradiation. The slurry was then treated in a microwave oven (MicroSYNTH Labstation
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for Synthesis), using temperatures of 140, 170, 180, 190, 200, and 210 ºC, during 2
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minutes under continuous stirring. The temperature and pressure were controlled with a
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thermocouple immersed in the slurry.
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After microwave irradiation, the pH was measured. The reactor was cooled at room
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temperature and centrifuged at 24,652 g for 15 min at 1 ºC and filtered to separate the
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water-soluble fractions from the cellulosic residues (CRs). The CRs were suspended in
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water, frozen, and freeze-dried. The MWE was performed in duplicate for each
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temperature.
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Absolute ethanol was added to the water-soluble fractions to perform 70 % (v/v)
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ethanol solutions, assuming additive volumes. These solutions were stirred for 2 h at 4
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ºC until formation of a precipitate and then were centrifuged to obtain the precipitate
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(Et70) that was removed from the supernatant solution (EtSn). To remove the ethanol
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completely, each precipitate and supernatant solution was dissolved in water,
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concentrated by rotary evaporation at 35 ºC under reduced pressure, frozen, and then
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freeze-dried.
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2.3 Sequential extraction of AX by microwave superheated aqueous solutions
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Based on the temperature conditions studied, the BSG was submitted to a sequential
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extraction. BSG (10 g) was suspended in 60 mL distilled water using a liquid/solid ratio 6 Page 6 of 32
of 6:1. The microwave treatment was performed at 140 ºC during 2 min under
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continuous stirring, reaching a pressure of 4 bar. The reactor was cooled at room
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temperature and the slurry was filtered. Absolute ethanol was added to the filtrate
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solution to reach 70 % (v/v) ethanol and the material precipitated was recovered by
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centrifugation (140_Et70). The residue obtained was suspended in water using the same
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liquid/solid ratio used previously for the BSG extraction. The microwave treatment was
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performed at 180 ºC during 2 min, reaching a pressure of 8 bar. The reactor was cooled
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at room temperature and the slurry was filtered. Absolute ethanol was added to the
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filtrate solution to reach 70 % (v/v) ethanol and the material precipitated was separated
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from the supernatant by centrifugation, giving origin to fractions 180_Et70 and
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180_EtSn, respectively. The residue obtained was re-suspended in an aqueous solution
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of 0.1 M KOH, using the same liquid/solid ratio and temperature. Under these MWE
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conditions a pressure of 27 bar was reached. The slurry was filtered and the residue was
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washed with water, frozen and freeze-died. The filtrate solution was neutralized until
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pH 5 with glacial acetic acid, leading to the formation of a precipitate, separated from
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the supernatant by centrifugation (180_KOH_pp). The material present in the
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supernatant was precipitated with ethanol 70 % (v/v), allowing to obtain the
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180_KOH_Et70 fraction (Figure 1). Each precipitate and supernatant solution was
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dissolved in water, concentrated by rotary evaporation at 35 ºC under reduced pressure,
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frozen, and then freeze-dried.
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2.4. Neutral sugar analysis
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Monosaccharides were released from cell wall polysaccharides by a pre-
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hydrolysis in 0.2 mL of 72 % H2SO4 (w/w) for 3 h at room temperature followed by 2.5
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h hydrolysis in 1 M H2SO4 at 100 ºC (Selvendran, March & Ring, 1979). For the
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hydrolysis of the cellulosic residue of the sequential extraction, a 2 M H2SO4 at 120 ºC 7 Page 7 of 32
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during 1 h was also used. Neutral sugars were analysed as their alditol acetates by gas-
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chromatography-flame ionization detection (Blakeney, Harris, Henry & Stone, 1983;
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Harris, Blakeney, Henry & Stone, 1988). The hydrolysis of all fractions was performed
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in duplicate. Results used have less than 5% variability in the major component sugars.
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2.5. Glycosidic-linkage composition of polysaccharide fractions
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Glycosidic-linkage composition was determined by gas chromatography-
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quadrupole mass spectrometry (GC-qMS) of the partially methylated alditol acetates
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(Ciucanu & Kerek, 1984; Coelho, Rocha & Coimbra, 2011; Harris, Henry, Blakeney &
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Stone, 1984).
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2.6. Determination of total phenolic compounds and antioxidant activity
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Total phenolic composition was determined by the Folin-Ciocalteu colorimetric
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method (Coelho et al., 2011; Folin & Ciocalteu, 1927), and measured as gallic acid
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equivalents. The antioxidant activity was measured by scavenging of the ABTS radical
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cation (Re, Pellegrini, Proteggente, Pannala, Yang & Rice-Evans, 1999). The results
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were expressed as trolox equivalents.
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2.7. Determination of the presence of starch In order to evaluate the presence of starch in the fractions obtained by the
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sequential MWE, the samples were treated with α-amylase from Bacillus subtilis (62
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U/mg) at 37 ºC overnight. During the enzymatic assay were used as positive control
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amylase, and as negative control cellulose. The sugars released by enzymatic hydrolysis
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were analysed as described in section 2.4.
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3. Results and Discussion
189 3.1. Influence of temperature on yield and sugar composition of the microwave
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extracted material from the BSG
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The microwave superheated water extraction (MWE) was applied to BSG using the
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following temperatures: 140, 170, 180, 190, 200, and 210 ºC. After MWE, the water-
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soluble fractions were precipitated with 70 % (v/v) of ethanol, obtaining the Et70
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fractions that were separated from the supernatant solutions (EtSn). Table 1 shows the
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results of the yield of soluble solids extracted from BSG for the different temperatures
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applied, e.g. calculated as the percentage of the mass of the fraction obtained in relation
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to the mass applied in the reactor for extraction. The amount of material extracted with
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hot water, on BSG dry basis, increased with the increase of temperature. The yield in
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the aggregate of fractions Et70 and EtSn recovered at 140 ºC was 2 %, increasing to 8
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% and 10 % at 170 ºC and 180 ºC, respectively. Taking into consideration the mass not
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solubilised (residue, Table 1), it is possible to infer that the use of these temperatures
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allowed a recovery of the totality of the BSG compounds. However, although the yields
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of extraction increased to 17, 23, and 29 % when the treatments were performed at 190,
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200, and 210 ºC, respectively, recovery of the BSG compounds was only 90%, showing
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the occurrence mass loss of the BSG components at these higher temperatures. These
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results suggest that the components of BSG were more thermolabile than those
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extracted from spent coffee ground, as at the temperature of 200 ºC no mass loss was
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noticed (Passos & Coimbra, 2013).
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3.1.1. Arabinoxylan recovery The Et70 fractions recovered at all temperatures were richer in sugars, ranging
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between 64.6 and 84.2%, than the EtSn fractions, which were composed by 40.6 to 49.0
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% of sugars. Although for Et70 a tendency to obtain fractions richer in sugars with the
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increase of the temperature of extraction, no tendency was observed for EtSn. This
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higher extraction yields of sugars at higher temperatures, seems to result in residues
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with lower content of sugars. Concerning the residue left upon extraction, the relative
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amount of xylose increased with the increment of the temperature. Based on the amount
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of xylose in the fractions and their yield, the amount of xylose recovered in the Et70
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fractions was estimated to range from 0.34 % for 140 ºC to 21.0 % for 210 ºC, whereas
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for EtSn the amount of xylose ranged from 0.15 to 24.4 %. The relative amount of
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arabinose increased with the increase of temperature applied until 180ºC. The further
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increase of temperature did not cause increment of the relative recovery of arabinose
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and at 210 ºC a decrease of arabinose in both Et70 and EtSn fractions was observed
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(Table 1). The relative amount of arabinose was much higher in EtSn than in Et70
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fractions.
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The amount of xylose+arabinose extracted increased with the increase of
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temperature, however, the lower temperatures (140, 170, and 180 °C) extracted only
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small amounts of these sugars (0.69, 6.9, and 13.3 %, respectively). As observed for the
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amount of material extracted, the best extraction conditions of xylose+arabinose were
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also achieved using higher temperatures (190, 200, and 210 °C), yielding 22.6, 34.5,
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and 43.4 %, respectively. Nevertheless, the increase of the yield when higher
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temperatures are used caused a loss of xylose+arabinose content (Figure 2). The
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temperatures until 180 ºC showed a recovery of 92 %, considering all the three fractions
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(Et70+EtSn+residue), but from 190 ºC to 210 ºC the recovery decreased from 88 % to
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72 %, respectively. At 210 ºC, 28 % of the total xylose+arabinose content was lost,
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probably due to the thermal degradation. A compromise between temperature and
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xylose+arabinose yield should be achieved. The pH of the BSG suspensions was measurement before and after the MWE. The
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initial pH value, before the treatment, was 5.93. Values of the final pH of the aqueous
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extracts ranged from 5.64 to 4.81. It was observed that the pH values decreased
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gradually with the increase of the temperature of MWE, which may contribute also for
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the higher amount of material extracted at higher temperatures. This pH decrease and
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the release of insoluble material into solution may be due to the occurrence of
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autohydrolysis of the insoluble BSG material upon microwave treatment (Benkő et al.,
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2007). The internal superheating of the cell water content, by absorbing the microwave
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radiation, should enable the cell disruption, allowing the solvent to access the
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polysaccharides (Kratchanova, Pavlova & Panchev, 2004). The pH decrease can be due
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to the formation of acids “in situ” by the hydrolysis of esters, namely those of AX with
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ferulic and acetic acids or by the thermal degradation of arabinose residues that can be
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converted into formic and levulinic acids (Carvalheiro, Esteves, Parajó, Pereira & Gı́rio,
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2004).
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3.1.2. Structural analysis of AX
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In order to evaluate the structure of the arabinoxylans (AX) and derived
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oligosaccharides (AXOS) obtained for the different temperatures, a methylation analysis
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was performed to all Et70 and EtSn fractions (Table 2). The main linkage observed in
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Et70 fractions was (1→4)-Xyl, with exception of the sample treated with 140 ºC, where
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the main linkage found was (1→4)-Glc, confirming the extraction of glucans at this
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lower temperature. The relative amount of (1→4)-Xyl in relation to total xylose
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recovered in the fraction increased gradually with the temperature range applied, from
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51 % to 80 %. Arabinose occurred mainly as terminally-linked for all temperatures (78
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– 90 % in relation to total arabinose). The content of arabinose decreased in relation to
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the xylose content with the increase of the temperature. This decrease is also in
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accordance with the relative decrease of the xylose branched residues, allowing to infer
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the debranching effect of temperature. These observations are in accordance with the
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temperature effect reported to occur in arabinoxylans (Benkő et al., 2007; Carvalheiro et
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al., 2004), as well as in arabinogalactans and galactomannans (Passos & Coimbra,
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2013).
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Concerning the EtSn fractions, the relative amount of (1→4)-Xyl was almost
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constant for the different fractions, varying from 50 to 74 % in relation to the total
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xylose. However, the amount of branched xylose residues decreased in relation to the
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amount of total xylose. Also, the amount of arabinose tend to decrease in comparison to
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the total amount of xylose, allowing to infer that the compounds obtained in EtSn are
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less branched as the temperature increased, as observed for the Et70.
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The branching degree calculated as the ratio of (2,4-Xyl+3,4-Xyl+2*2,3,4-
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Xyl)/total xylose of the fractions Et70 and EtSn decrease with the increase of the
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temperature (Table 2 and Figure 3). Figure 3 shows a linear decreasing (y = -44.315x +
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30.747, r2 = 0.894) of the ratio with the amount of AX recovered. The debranching of
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the AX promotes the increasing of arabinose in solution, converting it into furfural and
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caramelisation products (Carvalheiro et al., 2004). The amount of arabinose by itself
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does not justify all branching points present in the xylan backbone. Concerning the Et70
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fractions, the ratio of Ara/(2,4-Xyl+3,4-Xyl+2*2,3,4-Xyl) ranges between 0.5 and 0.6.
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This range of ratios were also achieved for arabinoxylans present in barley residue (left
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after removal of water extractable polysaccharides) (Izydorczyk, Macri & MacGregor,
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1998). Wheat bran arabinoxylans also showed a similar ratio when in native form.
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However, when treated with TFA, they decreased to a ratio of 0.2 due to the lability of
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arabinose in acidic media. In these experiments, the presence of galactose and
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glucuronic acid attached to the heteroxylan backbone was suggested (Brillouet,
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Joseleau, Utille & Lelièvre, 1982). Taking into account the contribution of terminal
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galactose and glucuronic acid, the ratio of the terminal to the branched residues [(t-
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Ara+t-Gal+t-GlcA)/(2,4-Xyl+3,4-Xyl+2*2,3,4-Xyl)] is 1.0. Accordingly, the presence
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of uronic acids has been also reported to occur in BSG (5-6 mol%), mainly in the water
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soluble solids from the alcohol insoluble residue, which accounted for 8 mol% (Kabel et
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al., 2002a), as well as in fractions retained in anion exchange chromatography (Kabel,
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Schols & Voragen, 2002b). These authors reported that the acid fractions were more
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difficult to degrade by endoxylanase, presenting characteristics like those of highly
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branched glucuronoarabinoxylan-like material (Kabel et al., 2002b). As the BSG used
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in this work presented 7 mol% of uronic acids and terminally-linked galactose, it is
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possible that these structures contain similar structural features as those observed
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previously by these authors.
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The amount of (1→5)-linked arabinose, when compared with the amount of total
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arabinose, was higher in all EtSn fractions. These residues may result from the ester-
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linked ferulic and p-coumaric acids (Bartolomé et al., 2002; Chanliaud, Saulnier &
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Thibault, 1995; Mandalari et al., 2005). These residues decreased with the increase in
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temperature for all fractions, from 44 % at 140 ºC to 9 % at 210 ºC for the EtSn
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fractions, and from 9 % to 2 %, respectively, for the Et70 fractions. The temperature
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increment also promotes the desterification of phenolic acids from the arabinosyl
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residues. Figure 4 shows that EtSn fractions present a decrease in the ratio 5-
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Araf/Aratotal from 44.4 to 30.5 at temperatures between 140 ºC and 180 ºC, to 16.2 at
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190 ºC, and to 9.3 at 210 ºC. The same behaviour was observed for the Et70 but the
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ratio 5-Araf/Aratotal was smaller than those observed for the EtSn fractions, ranging
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between 9.2 and 0.8, for 140 ºC and 190 ºC, respectively. The presence of higher
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contents of esterified phenolic acids should justify their solubility in the ethanol
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solutions.
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The analysis of the relative amount of total xylose divided by the amount of
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terminally-linked xylose offers a way to determine the degree of polymerization (DP) of
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the AX (DP≥10) and AXOS (1
S0144-8617(13)00893-X http://dx.doi.org/doi:10.1016/j.carbpol.2013.09.003 CARP 8092
To appear in: Received date: Revised date: Accepted date:
20-2-2013 30-8-2013 3-9-2013
Please cite this article as: Coelho, E., Rocha, M. A. M., Saraiva, J. A., & Coimbra, M. A., Microwave superheated water and dilute alkali extraction of brewersˇı spent grain arabinoxylans and arabinoxylo-oligosaccharides, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Microwave superheated water and dilute alkali extraction of brewers´
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spent grain arabinoxylans and arabinoxylo-oligosaccharides
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Elisabete Coelhoa*, M. Angélica M. Rochaa, Jorge A. Saraiva, and Manuel A. Coimbra
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QOPNA, Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro,
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Portugal
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*Corresponding author. Tel.:+351 234 370706; fax: +351 234 370084.
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E-mail address: [email protected] (E. Coelho).
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These authors contributed equally to the work.
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Abstract
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Microwave superheated water extractions (MWE) were performed to evaluate the
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feasibility of this technology for quantitative recovery of the arabinoxylans (AX) or
19
arabinoxylo-oligosaccharides (AXOS) from brewers´ spent grain (BSG). The
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AX+AXOS yield increased with the increase of the temperature in the range from 140
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to 210 °C during 2 min. The higher temperatures promoted depolymerisation,
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debranching, and deesterification of the polysaccharides, with formation of brown
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products. The conditions that promote a compromise between the yield and the structure
24
obtained, minimizing the thermal degradation of the fractions extracted by MWE are the
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following: 1) 140 ºC, to remove the residual starch mixed with β-glucans; 2) Suspension
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of the residue left in water and treated at 180 ºC; 3) suspension of the residue in 0.1 M
27
KOH and treated at 180 ºC. Using this sequential procedure, it was possible to extract
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62 % of BSG AX+AXOS, presenting degrees of polymerization ranging between 7 and
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24 xylose residues, and a degree of phenolic acids esterification between 5 and 21 %.
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The structural variability obtained by MWE allows defining specific types of
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compounds for different applications and uses depending on the extraction conditions
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used.
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Keywords: Brewer’s spent grain; polysaccharides; arabinoxylans; feruloylated
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arabinoxylo-oligosaccharides; microwave-assisted extraction; prebiotics; food
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ingredients; barley.
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1. Introduction During germination of the barley grain, cell walls, mainly of the endosperm, are
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enzymatically degraded. After filtration of the liquid extracted from the mashing,
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remaining unhydrolysed cell walls, which are derived mainly from the aleurone and
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some insoluble material present in the endosperm, are disposed of as the brewer’ spent
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grain (BSG). The BSG is composed exclusively by the material from barley husks, with
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75 % moisture, having a high percentage of polysaccharides, namely, arabinoxylans
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(AX, 22-23 %), cellulose (12-25 %) and small amounts of starch (4 %) and β-glucans
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(Aliyu & Bala, 2011; Kabel et al., 2002a). BSG represents up to 85 % of the total
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residues from the brewing process and it amounts to about 20 kg/hL beer. It is estimated
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that Europe produces 3.5 million ton per year of BSG (Jay et al., 2008). This by-product
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has been used for animal feed as a way to reduce the residue produced by these
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industries. The use of BSG as a source of polysaccharides with fibre properties and
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other biological activities that have been associated with AX and β-glucans, namely
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prebiotic activity (Broekaert, Courtin, Verbeke, Van de Wiele, Verstraete & Delcour,
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2011; Crittenden et al., 2002), has a great potential. These polysaccharides are
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considered as dietary fibre due to their resistance to hydrolysis by the digestive tract
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enzymes. Additionally, they can present immunomodulatory (Van Craeyveld et al.,
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2008), antioxidant (Ohta, Semboku, Kuchii, Egashira & Sanada, 1997) and
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anticarcinogenic effect (Broekaert et al., 2011), and reduce blood serum triglycerides
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and cholesterol levels (Broekaert et al., 2011).
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BSG AX are composed by a backbone of β-(1→4)-linked xylose residues
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containing single units of arabinose as side chains, phenolic acids, such as ferulic and p-
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coumaric acids are esterified to arabinofuranosyl residues (Bartolomé, Santos, Jiménez,
3 Page 3 of 32
del Nozal & Gómez-Cordovés, 2002; Mandalari et al., 2005). Feruloylated arabinoxylo-
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oligosaccharides (AXOS) are more potent antioxidants and radical scavengers than
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ferulic acid itself (Broekaert et al., 2011; Katapodis, Vardakou, Kalogeris, Kekos,
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Macris & Christakopoulos, 2003; Ohta et al., 1997). In vitro, feruloylated AXOS
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strongly inhibit copper mediated oxidation of low density lipoprotein (Katapodis et al.,
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2003). The antioxidant properties of feruloylated AXOS derived from wheat bran have
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recently been confirmed in an in vivo model of diabetic rats (Ou, Jackson, Jiao, Chen,
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Wu & Huang, 2007). In diabetic rats, feruloylated AXOS significantly increased total
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antioxidant capacity level, glutathione peroxidase, and superoxide dismutase activities,
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decreased blood glucose and malondiadehyde levels, and decreased xanthine oxidase
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activity in serum and liver. Feruloylated AXOS were, overall, more efficient in
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mitigating oxidative damage in diabetic rats than sodium ferulate and ascorbic acid.
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Furthermore, feruloylated AXOS showed greater antioxidant capacity in vivo than in
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vitro when compared with ascorbic acid (Ou et al., 2007). The ester linkage between
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ferulic acid and AXOS prevents its uptake until reaching the colon, where AXOS-
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inducible bacterial feruloyl esterases are present (Vardakou, Nueno Palop, Gasson,
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Narbad & Christakopoulos, 2007). Feruloylated AXOS is therefore a special type of
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antioxidant whose active principle is released in the colon (Broekaert et al., 2011).
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Moreover, formulations combining AXOS with AX potentiate their prebiotic activity
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(Broekaert, Courtin, Damen & Delcour, 2010).
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Microwave-assisted extraction is a process that uses microwave energy to heat the
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solvent that is in contact with a sample in order to partition analytes from the sample
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matrix into the solvent. The main advantage of this technique is the ability to rapidly
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heat the sample solvent mixture. By using closed vessels the extraction can be
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performed at elevated temperatures accelerating the mass transfer of target compounds
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from the sample matrix. Typically, microwave-assisted extraction procedure takes 15–
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30 min and uses small solvent volumes, about 10 times smaller than volumes used by
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conventional extraction techniques (Eskilsson & Bjorklund, 2000). Recently,
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microwave
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galactomannans from spent coffee grounds was proposed (Passos & Coimbra, 2013).
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The use of microwaves for AX extraction has also been proposed for some industry
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residues, such as barley husks (Roos, Persson, Krawczyk, Zacchi & Stalbrand, 2009),
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corn and corn stover (Benkő, Andersson, Szengyel, Gáspár, Réczey & Stålbrand, 2007;
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Pang, Xue, Yu, Zhang, Li & Kang, 2012; Rose & Inglett, 2010; Yoshida, Tsubaki,
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Teramoto & Azuma, 2010), wheat straw (Janker-Obermeier, Sieber, Faulstich &
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Schieder, 2012), and flax shives (Buranov & Mazza, 2010). These polymers can be
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obtained directly in water (Roos et al., 2009; Yoshida et al., 2010), dilute alkali and/or
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acid (Benkő et al., 2007; Janker-Obermeier et al., 2012) or in a hydroalcoholic solution
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(Buranov & Mazza, 2010). The aim of this work was to determine the yield and changes
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in the structure of AX that can be recovered from BSG when microwave technology is
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used to carry out the extraction using water at temperatures reaching 140-210 ºC during
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2 minutes. In addition a sequential extraction using water and diluted alkali solution was
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assayed.
extraction
(MWE)
of
arabinogalactans
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superheated
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2. Experimental
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2.1. Samples
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The BSG used in this study was provided by Unicer-Bebidas S.A., Leça do Balio,
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Portugal, with 76 % of moisture, being the dry material composed by carbohydrates (50
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%), proteins (30 %), fats (8 %), and ashes (5 %).
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2.2 Effect of temperature on the extraction of AX by microwave superheated water BSG (10 g) was suspended in 60 mL distilled water in a 100 mL Teflon-coated
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vessel at a liquid/solid ratio of 6:1 and the pH was measured before the microwave
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irradiation. The slurry was then treated in a microwave oven (MicroSYNTH Labstation
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for Synthesis), using temperatures of 140, 170, 180, 190, 200, and 210 ºC, during 2
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minutes under continuous stirring. The temperature and pressure were controlled with a
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thermocouple immersed in the slurry.
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After microwave irradiation, the pH was measured. The reactor was cooled at room
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temperature and centrifuged at 24,652 g for 15 min at 1 ºC and filtered to separate the
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water-soluble fractions from the cellulosic residues (CRs). The CRs were suspended in
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water, frozen, and freeze-dried. The MWE was performed in duplicate for each
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temperature.
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Absolute ethanol was added to the water-soluble fractions to perform 70 % (v/v)
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ethanol solutions, assuming additive volumes. These solutions were stirred for 2 h at 4
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ºC until formation of a precipitate and then were centrifuged to obtain the precipitate
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(Et70) that was removed from the supernatant solution (EtSn). To remove the ethanol
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completely, each precipitate and supernatant solution was dissolved in water,
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concentrated by rotary evaporation at 35 ºC under reduced pressure, frozen, and then
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freeze-dried.
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2.3 Sequential extraction of AX by microwave superheated aqueous solutions
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Based on the temperature conditions studied, the BSG was submitted to a sequential
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extraction. BSG (10 g) was suspended in 60 mL distilled water using a liquid/solid ratio 6 Page 6 of 32
of 6:1. The microwave treatment was performed at 140 ºC during 2 min under
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continuous stirring, reaching a pressure of 4 bar. The reactor was cooled at room
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temperature and the slurry was filtered. Absolute ethanol was added to the filtrate
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solution to reach 70 % (v/v) ethanol and the material precipitated was recovered by
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centrifugation (140_Et70). The residue obtained was suspended in water using the same
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liquid/solid ratio used previously for the BSG extraction. The microwave treatment was
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performed at 180 ºC during 2 min, reaching a pressure of 8 bar. The reactor was cooled
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at room temperature and the slurry was filtered. Absolute ethanol was added to the
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filtrate solution to reach 70 % (v/v) ethanol and the material precipitated was separated
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from the supernatant by centrifugation, giving origin to fractions 180_Et70 and
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180_EtSn, respectively. The residue obtained was re-suspended in an aqueous solution
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of 0.1 M KOH, using the same liquid/solid ratio and temperature. Under these MWE
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conditions a pressure of 27 bar was reached. The slurry was filtered and the residue was
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washed with water, frozen and freeze-died. The filtrate solution was neutralized until
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pH 5 with glacial acetic acid, leading to the formation of a precipitate, separated from
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the supernatant by centrifugation (180_KOH_pp). The material present in the
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supernatant was precipitated with ethanol 70 % (v/v), allowing to obtain the
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180_KOH_Et70 fraction (Figure 1). Each precipitate and supernatant solution was
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dissolved in water, concentrated by rotary evaporation at 35 ºC under reduced pressure,
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frozen, and then freeze-dried.
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2.4. Neutral sugar analysis
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Monosaccharides were released from cell wall polysaccharides by a pre-
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hydrolysis in 0.2 mL of 72 % H2SO4 (w/w) for 3 h at room temperature followed by 2.5
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h hydrolysis in 1 M H2SO4 at 100 ºC (Selvendran, March & Ring, 1979). For the
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hydrolysis of the cellulosic residue of the sequential extraction, a 2 M H2SO4 at 120 ºC 7 Page 7 of 32
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during 1 h was also used. Neutral sugars were analysed as their alditol acetates by gas-
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chromatography-flame ionization detection (Blakeney, Harris, Henry & Stone, 1983;
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Harris, Blakeney, Henry & Stone, 1988). The hydrolysis of all fractions was performed
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in duplicate. Results used have less than 5% variability in the major component sugars.
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2.5. Glycosidic-linkage composition of polysaccharide fractions
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Glycosidic-linkage composition was determined by gas chromatography-
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quadrupole mass spectrometry (GC-qMS) of the partially methylated alditol acetates
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(Ciucanu & Kerek, 1984; Coelho, Rocha & Coimbra, 2011; Harris, Henry, Blakeney &
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Stone, 1984).
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2.6. Determination of total phenolic compounds and antioxidant activity
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Total phenolic composition was determined by the Folin-Ciocalteu colorimetric
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method (Coelho et al., 2011; Folin & Ciocalteu, 1927), and measured as gallic acid
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equivalents. The antioxidant activity was measured by scavenging of the ABTS radical
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cation (Re, Pellegrini, Proteggente, Pannala, Yang & Rice-Evans, 1999). The results
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were expressed as trolox equivalents.
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2.7. Determination of the presence of starch In order to evaluate the presence of starch in the fractions obtained by the
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sequential MWE, the samples were treated with α-amylase from Bacillus subtilis (62
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U/mg) at 37 ºC overnight. During the enzymatic assay were used as positive control
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amylase, and as negative control cellulose. The sugars released by enzymatic hydrolysis
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were analysed as described in section 2.4.
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186 187 188
3. Results and Discussion
189 3.1. Influence of temperature on yield and sugar composition of the microwave
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extracted material from the BSG
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The microwave superheated water extraction (MWE) was applied to BSG using the
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following temperatures: 140, 170, 180, 190, 200, and 210 ºC. After MWE, the water-
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soluble fractions were precipitated with 70 % (v/v) of ethanol, obtaining the Et70
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fractions that were separated from the supernatant solutions (EtSn). Table 1 shows the
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results of the yield of soluble solids extracted from BSG for the different temperatures
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applied, e.g. calculated as the percentage of the mass of the fraction obtained in relation
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to the mass applied in the reactor for extraction. The amount of material extracted with
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hot water, on BSG dry basis, increased with the increase of temperature. The yield in
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the aggregate of fractions Et70 and EtSn recovered at 140 ºC was 2 %, increasing to 8
202
% and 10 % at 170 ºC and 180 ºC, respectively. Taking into consideration the mass not
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solubilised (residue, Table 1), it is possible to infer that the use of these temperatures
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allowed a recovery of the totality of the BSG compounds. However, although the yields
205
of extraction increased to 17, 23, and 29 % when the treatments were performed at 190,
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200, and 210 ºC, respectively, recovery of the BSG compounds was only 90%, showing
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the occurrence mass loss of the BSG components at these higher temperatures. These
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results suggest that the components of BSG were more thermolabile than those
209
extracted from spent coffee ground, as at the temperature of 200 ºC no mass loss was
210
noticed (Passos & Coimbra, 2013).
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211 212
3.1.1. Arabinoxylan recovery The Et70 fractions recovered at all temperatures were richer in sugars, ranging
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between 64.6 and 84.2%, than the EtSn fractions, which were composed by 40.6 to 49.0
215
% of sugars. Although for Et70 a tendency to obtain fractions richer in sugars with the
216
increase of the temperature of extraction, no tendency was observed for EtSn. This
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higher extraction yields of sugars at higher temperatures, seems to result in residues
218
with lower content of sugars. Concerning the residue left upon extraction, the relative
219
amount of xylose increased with the increment of the temperature. Based on the amount
220
of xylose in the fractions and their yield, the amount of xylose recovered in the Et70
221
fractions was estimated to range from 0.34 % for 140 ºC to 21.0 % for 210 ºC, whereas
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for EtSn the amount of xylose ranged from 0.15 to 24.4 %. The relative amount of
223
arabinose increased with the increase of temperature applied until 180ºC. The further
224
increase of temperature did not cause increment of the relative recovery of arabinose
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and at 210 ºC a decrease of arabinose in both Et70 and EtSn fractions was observed
226
(Table 1). The relative amount of arabinose was much higher in EtSn than in Et70
227
fractions.
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The amount of xylose+arabinose extracted increased with the increase of
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temperature, however, the lower temperatures (140, 170, and 180 °C) extracted only
230
small amounts of these sugars (0.69, 6.9, and 13.3 %, respectively). As observed for the
231
amount of material extracted, the best extraction conditions of xylose+arabinose were
232
also achieved using higher temperatures (190, 200, and 210 °C), yielding 22.6, 34.5,
233
and 43.4 %, respectively. Nevertheless, the increase of the yield when higher
234
temperatures are used caused a loss of xylose+arabinose content (Figure 2). The
235
temperatures until 180 ºC showed a recovery of 92 %, considering all the three fractions
10 Page 10 of 32
236
(Et70+EtSn+residue), but from 190 ºC to 210 ºC the recovery decreased from 88 % to
237
72 %, respectively. At 210 ºC, 28 % of the total xylose+arabinose content was lost,
238
probably due to the thermal degradation. A compromise between temperature and
239
xylose+arabinose yield should be achieved. The pH of the BSG suspensions was measurement before and after the MWE. The
241
initial pH value, before the treatment, was 5.93. Values of the final pH of the aqueous
242
extracts ranged from 5.64 to 4.81. It was observed that the pH values decreased
243
gradually with the increase of the temperature of MWE, which may contribute also for
244
the higher amount of material extracted at higher temperatures. This pH decrease and
245
the release of insoluble material into solution may be due to the occurrence of
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autohydrolysis of the insoluble BSG material upon microwave treatment (Benkő et al.,
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2007). The internal superheating of the cell water content, by absorbing the microwave
248
radiation, should enable the cell disruption, allowing the solvent to access the
249
polysaccharides (Kratchanova, Pavlova & Panchev, 2004). The pH decrease can be due
250
to the formation of acids “in situ” by the hydrolysis of esters, namely those of AX with
251
ferulic and acetic acids or by the thermal degradation of arabinose residues that can be
252
converted into formic and levulinic acids (Carvalheiro, Esteves, Parajó, Pereira & Gı́rio,
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2004).
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3.1.2. Structural analysis of AX
256
In order to evaluate the structure of the arabinoxylans (AX) and derived
257
oligosaccharides (AXOS) obtained for the different temperatures, a methylation analysis
258
was performed to all Et70 and EtSn fractions (Table 2). The main linkage observed in
259
Et70 fractions was (1→4)-Xyl, with exception of the sample treated with 140 ºC, where
260
the main linkage found was (1→4)-Glc, confirming the extraction of glucans at this
11 Page 11 of 32
lower temperature. The relative amount of (1→4)-Xyl in relation to total xylose
262
recovered in the fraction increased gradually with the temperature range applied, from
263
51 % to 80 %. Arabinose occurred mainly as terminally-linked for all temperatures (78
264
– 90 % in relation to total arabinose). The content of arabinose decreased in relation to
265
the xylose content with the increase of the temperature. This decrease is also in
266
accordance with the relative decrease of the xylose branched residues, allowing to infer
267
the debranching effect of temperature. These observations are in accordance with the
268
temperature effect reported to occur in arabinoxylans (Benkő et al., 2007; Carvalheiro et
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al., 2004), as well as in arabinogalactans and galactomannans (Passos & Coimbra,
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2013).
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Concerning the EtSn fractions, the relative amount of (1→4)-Xyl was almost
272
constant for the different fractions, varying from 50 to 74 % in relation to the total
273
xylose. However, the amount of branched xylose residues decreased in relation to the
274
amount of total xylose. Also, the amount of arabinose tend to decrease in comparison to
275
the total amount of xylose, allowing to infer that the compounds obtained in EtSn are
276
less branched as the temperature increased, as observed for the Et70.
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The branching degree calculated as the ratio of (2,4-Xyl+3,4-Xyl+2*2,3,4-
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Xyl)/total xylose of the fractions Et70 and EtSn decrease with the increase of the
279
temperature (Table 2 and Figure 3). Figure 3 shows a linear decreasing (y = -44.315x +
280
30.747, r2 = 0.894) of the ratio with the amount of AX recovered. The debranching of
281
the AX promotes the increasing of arabinose in solution, converting it into furfural and
282
caramelisation products (Carvalheiro et al., 2004). The amount of arabinose by itself
283
does not justify all branching points present in the xylan backbone. Concerning the Et70
284
fractions, the ratio of Ara/(2,4-Xyl+3,4-Xyl+2*2,3,4-Xyl) ranges between 0.5 and 0.6.
285
This range of ratios were also achieved for arabinoxylans present in barley residue (left
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12 Page 12 of 32
after removal of water extractable polysaccharides) (Izydorczyk, Macri & MacGregor,
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1998). Wheat bran arabinoxylans also showed a similar ratio when in native form.
288
However, when treated with TFA, they decreased to a ratio of 0.2 due to the lability of
289
arabinose in acidic media. In these experiments, the presence of galactose and
290
glucuronic acid attached to the heteroxylan backbone was suggested (Brillouet,
291
Joseleau, Utille & Lelièvre, 1982). Taking into account the contribution of terminal
292
galactose and glucuronic acid, the ratio of the terminal to the branched residues [(t-
293
Ara+t-Gal+t-GlcA)/(2,4-Xyl+3,4-Xyl+2*2,3,4-Xyl)] is 1.0. Accordingly, the presence
294
of uronic acids has been also reported to occur in BSG (5-6 mol%), mainly in the water
295
soluble solids from the alcohol insoluble residue, which accounted for 8 mol% (Kabel et
296
al., 2002a), as well as in fractions retained in anion exchange chromatography (Kabel,
297
Schols & Voragen, 2002b). These authors reported that the acid fractions were more
298
difficult to degrade by endoxylanase, presenting characteristics like those of highly
299
branched glucuronoarabinoxylan-like material (Kabel et al., 2002b). As the BSG used
300
in this work presented 7 mol% of uronic acids and terminally-linked galactose, it is
301
possible that these structures contain similar structural features as those observed
302
previously by these authors.
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303
The amount of (1→5)-linked arabinose, when compared with the amount of total
304
arabinose, was higher in all EtSn fractions. These residues may result from the ester-
305
linked ferulic and p-coumaric acids (Bartolomé et al., 2002; Chanliaud, Saulnier &
306
Thibault, 1995; Mandalari et al., 2005). These residues decreased with the increase in
307
temperature for all fractions, from 44 % at 140 ºC to 9 % at 210 ºC for the EtSn
308
fractions, and from 9 % to 2 %, respectively, for the Et70 fractions. The temperature
309
increment also promotes the desterification of phenolic acids from the arabinosyl
310
residues. Figure 4 shows that EtSn fractions present a decrease in the ratio 5-
13 Page 13 of 32
Araf/Aratotal from 44.4 to 30.5 at temperatures between 140 ºC and 180 ºC, to 16.2 at
312
190 ºC, and to 9.3 at 210 ºC. The same behaviour was observed for the Et70 but the
313
ratio 5-Araf/Aratotal was smaller than those observed for the EtSn fractions, ranging
314
between 9.2 and 0.8, for 140 ºC and 190 ºC, respectively. The presence of higher
315
contents of esterified phenolic acids should justify their solubility in the ethanol
316
solutions.
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The analysis of the relative amount of total xylose divided by the amount of
318
terminally-linked xylose offers a way to determine the degree of polymerization (DP) of
319
the AX (DP≥10) and AXOS (1
