Pressurized aqueous ethanol extraction of β-glucans and phenolic compounds from waxy barley

Food Research International 75 (2015) 252–259

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Pressurized aqueous ethanol extraction of β-glucans and phenolic compounds from waxy barley Óscar Benito-Román a,b, Víctor H. Alvarez a, Esther Alonso b, Maria J. Cocero b, Marleny D.A. Saldaña a,⁎ a b

Department of Agricultural, Food and Nutritional Science, Faculty of Agricultural, Life and Environmental Science, University of Alberta, Edmonton, AB T6G 2P5, Canada Department of Chemical Engineering and Environmental Technology, Escuela de Ingenierías Industriales, University of Valladolid, Valladolid, Spain

a r t i c l e

i n f o

Article history: Received 6 March 2015 Received in revised form 26 May 2015 Accepted 3 June 2015 Available online 6 June 2015 Keywords: β-Glucan Antioxidants Ethanol Pressurized hot water Extraction

a b s t r a c t Beta-glucans and phenolics were extracted from waxy barley using pressurized aqueous ethanol in a stirred batch reactor at 25 bar and 500 rpm. The effect of temperature (135–175 °C), extraction time (15–55 min) and ethanol content (5–20%) was evaluated. Temperature had an opposite effect on the extraction of both compounds. The higher the temperature, the lower the β-glucan extraction yield due to fragmentation, but a significant increase on the phenolic recovery was observed. Long extraction times favored the extraction of β-glucans at low temperatures and phenolics at any temperature. The ethanol content was not statistically significant on the β-glucan extraction, but helped to maintain the molecular weight of the extracted β-glucan. To obtain liquid extracts rich in high molecular weight β-glucans and phenolics, mild conditions of 151 °C, 21 min and 16% ethanol are needed, leading to 51% β-glucan extraction yield with a molecular weight of 500–600 kDa and 5 mg GAE/ g barley. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Barley (Hordeum vulgare) is one of the most spread crops worldwide. According to the 2013 Alberta Crop Report published by Alberta Agriculture and Rural Development, the province of Alberta produced 5.5 million metric tons of barley. Almost 90% of barley has been traditionally used in animal feeding and brewery (Liu & Yao, 2007). However, the presence of phenolic compounds, with proven in vitro and in vivo antioxidant activity (Bonoli, Marconi, & Caboni, 2004), and β-glucan, with prebiotic properties, has renewed the interest in barley. Beta-glucans are non-starchy polysaccharides found in the cell walls of different cereals, including barley. This biopolymer has been associated with the cholesterol and glucose level reduction in blood (Izydorczyk & Dexter, 2008), claimed by the FDA and EFSA. Phenolics are also present in barley, mainly as benzoic and cinnamic acid derivatives. Phenolics are mainly found in the outer layers of the grain (hull) and also in the aleurone (layer rich in β-glucan) and endosperm (Madhujith, Izydorczyk, & Shahidi, 2006) either in free or bound forms. Ferulic acid is the main free phenolic, while p-hydroxybenzoic acid is the main bound phenolic in barley seed (Naczk & Shahidi, 2006). These bound phenolics are strongly linked to other molecules, such as the hemicelluloses (Naczk & Shahidi, 2006), forming strong ester-to-ester

⁎ Corresponding author. E-mail address: [email protected] (M.D.A. Saldaña).

http://dx.doi.org/10.1016/j.foodres.2015.06.006 0963-9969/© 2015 Elsevier Ltd. All rights reserved.

bonds, though they can also form ether bonds with other components (Madhujith & Shahidi, 2009). There has been extensive research on β-glucan extraction from barley as summarized by Benito-Román, Alonso, and Lucas (2011). But, despite the use of different technologies, all authors concluded that βglucan recovery from cereals is complicated since there are mass transfer limitations to the extraction. Conventional extraction methods for free phenolics involve long extraction times up to 24 h in a controlled temperature stirred tank using methanol or acetone at concentrations up to 70%. But, the use of these solvents exhibits major drawback as they are not environmental friendly. To extract bound phenolics, it is necessary to break the bond between them and the polysaccharides they are linked to. This is only possible by using NaOH solutions up to 4 M (Madhujith & Shahidi, 2009) to successfully release them after sequential extractions (Rodriguez-Arcos, Smith, & Waldron, 2002). To overcome the mass transfer limitations and enhance the simultaneous extraction of β-glucans and phenolics, new processes are required. Among them, processes based on pressurized hot fluid (PHF) are promising. PHF term refers to the fluid in its liquid state above the boiling temperature by the application of pressure. Among the most common PHFs are pressurized hot water (PHW) and pressurized hot aqueous ethanol (PHAE) for phytochemical extraction (Saldaña & Valdivieso-Ramirez, 2015). The increase in temperature leads to important changes in the physico-chemical properties of water related to the reduction of the relative permittivity (εr), the viscosity and the surface tension or the increase of diffusivity. All these changes make the PHW

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a unique solvent with a solvent power that can be tuned by changing pressure and temperature. PHW also helps to weaken the hydrogen bonds formed between the different molecules that form the cereal matrix, releasing the target molecules. PHW has been used to extract βglucans from barley, either in batch (53.7% extraction yield, and molecular weight of 200 kDa) and semicontinuous mode (52.4% extraction yield, and molecular weight of 100–500 kDa) (Benito-Román, Alonso, & Cocero, 2013; Benito-Román, Alonso, Gairola, & Cocero, 2013). PHW has also been used to extract antioxidants from barley hull (Sarkar, Alvarez, & Saldaña, 2014), flax shives (Kim & Mazza, 2006), rice bran (Pourali, Asghari, & Yoshida, 2010), and potato peel (Alvarez, Cahyadi, Xu, & Saldaña, 2014; Singh & Saldaña, 2011). PHAE has been the preferred solvent to isolate antioxidants and carbohydrates from barley hull (Sarkar et al., 2014) at temperatures in the range of 120–180 °C with mixtures of ethanol–water (0–20%), static holding time (2–20 min) and flow rate (2–6 mL/min). The maximum amounts of total carbohydrates (450.3 mg/g barley hull) and total phenolics (80.3 mg/g barley hull) were obtained at 180 °C, 15 min of static holding time, 15 MPa using 12% ethanol concentration at 5 mL/min of flow rate. Even though concentrations of ethanol above 50% make β-glucans insoluble (Vasanthan & Temelli, 2008), low quantities of ethanol in the aqueous mixtures might improve the extraction of phenolics, without reducing significantly the amount of extracted β-glucans. Despite the efforts to isolate β-glucans or phenolics from cereals using PHF, there is a lack of a complete and systematic study for the extraction of both compounds. The presence of antioxidants and βglucans in one single product would complement and reinforce the health benefits associated to both molecules. Therefore, the main objective of this study was to evaluate the influence of temperature, time and ethanol concentration on the extraction of β-glucans and four types of phenolic compounds (hydroxybenzoic acids, hydroxycinnamic acids, a flavan and flavonols) and one ferulic acid derivative from waxy barley using pressurized hot aqueous ethanol as the solvent. A response surface methodology (RSM) from Box–Behnken experimental design was used to study the effect of temperature (135–175 °C), extraction time (15–55 min) and ethanol content in the mixture (5–20%) at 25 bar and 500 rpm of mechanical mixing on the target compounds in terms of extraction yield, molecular weight and β-glucan depolymerization. 2. Materials and methods 2.1. Raw material

further analysis. Free and bound phenolics extracted according to the described procedures were quantified by means of the Folin– Ciocalteau's reagent (Singleton & Rossi, 1965). The total phenolic content was expressed as milligrams of gallic acid equivalents per gram of barley. 2.2. Experimental set-up A 100 mL pressurized vessel was used in this study. The former SEPAREX unit (Champigneulles, France), located at the Agri-Food Discovery Place (University of Alberta, Edmonton, AB, Canada), was modified for fast heating/cooling of the vessel as shown in Fig. 1. In each experiment, 3 g of barley was suspended in 100 mL of the suitable water–ethanol mixture. A mechanical stirrer set at 500 rpm was used to provide homogeneous solution, where two impellers (1 in. each) were placed. The reactor was heated with a band heater (TruTemp, Edmonton, AB, Canada) and the temperature was controlled by a type K themopar and a PID controller (Watlow, Winona, MN). After the extraction time, stirring was stopped and the vessel was introduced inside a cold water bath to produce a sudden decrease in the temperature and stop the extraction process. Subsequently, the extraction vessel was emptied by means of a valve placed at the bottom and the solid–liquid mixture was centrifuged for 10 min at 5500 rpm. Then, the solid material was discarded and the supernatant (named “liquid extract”) was divided in three aliquots: two of them were frozen at − 18 °C and the other one was kept at 4 °C to be used in the analysis. 2.3. Analytical methods The content of β-glucan, total phenolics, antioxidant activity, total solids dissolved, brown color formation, pH of the extracts, sugar degradation products (HMF, furfural, glucose and xylose–arabinose), and molecular weight of the β-glucan was performed following procedures described below. 2.3.1. β-Glucan content β-Glucan in the liquid extract was analyzed by means of the “Mixed linkage beta glucan assay kit” from Megazyme (Wicklow, Ireland), following the modification “c” of the general procedure. Briefly, samples were mixed with salts of ammonium to avoid interferences with the ethanol present in the liquid extract. After 20 h at 4 °C, samples were centrifuged and supernatant discarded. The pellet was resuspended in

A dehulled waxy barley cultivar variety “fibre” was used in this study, which was provided by GrainFrac Inc. (Leduc, AB, Canada). 2.1.1. Proximate compositional analysis A complete analysis of the raw material was performed prior to the extraction process. Protein, moisture, β-glucan, starch, ash and phenolics (both free and bound) were analyzed. Protein content was measured using a nitrogen analyzer (Mississiauga, ON, Canada) where the nitrogen content was multiplied by 6.25 (Salo-Väänänen & Koivistoinen, 1996). βGlucan and starch contents were determined using the “Mixed linkage βglucan assay kit” (Megazyme, Wicklow, Ireland) and the “Total starch assay kit” (Megazyme, Wicklow, Ireland), respectively. Moisture of samples was determined by convective oven drying, according to the method SM2540 described in APHA-AWWA-WPCF (1992) and ash content was analyzed using the standard official method of the Association of Analytical Communities (AOAC) 967.04. To quantify free phenolics, 2 g finely milled barley was extracted with 40 mL of 70% methanol for 4 h at room temperature. The mixture was centrifuged and the supernatant was stored at 4 °C until further analysis. To quantify bound phenolics, 2 g barley was digested with 40 mL of NaOH 2 M for 4 h at 65 °C. The mixture was acidified to pH 2–3 by the addition of some drops of hydrochloric acid. The resultant acidified solution was centrifuged and the supernatant was stored until

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Fig. 1. Subcritical fluid extraction system.

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ethanol (50%, v/v) and centrifuged again. The supernatant was discarded and the pellet resuspended in 5 mL of phosphate buffer (20 mM, pH 6.5) and samples were incubated with lichenase. Then, the general procedure was followed: a second hydrolysis was performed with β-glucosidase and the glucose obtained after the two enzymatic hydrolysis was determined spectrophotometrically at 510 nm after having reacted with the GOPOD reagent, provided with the assay kit. The β-glucan extraction yield was calculated according to Eq. (1): ð1Þ Extraction yieldð%Þ β‐glucan content in liquidð%Þ  Extract volumeðmLÞ  100: ¼ barleyðgÞ  β‐glucan content in barleyð%Þ

2.3.2. Total solids dissolved and pH measurements The total solids dissolved in the extracts were measured by convective oven drying, according to the method SM2540 described in APHAAWWA-WPCF (1992). The pH of samples was measured at room temperature using the Fisher Scientific Accumet instrument (Ottawa, ON, Canada) with a resolution of ±10−2, an uncertainty in the experimental measurements of ±2 × 10−2 and an uncertainty in the temperatures of ±0.1 °C. 2.3.3. Total phenolic content Total phenolic content was measured by Folin–Ciocalteau method as previously reported (Alvarez et al., 2014; Sarkar et al., 2014). A sample of 0.04 mL was mixed with 3.16 mL of water and 0.2 mL of Folin–Ciocalteau's reagent. After vortexing the solution, 0.6 mL of sodium carbonate in aqueous solution (20% w/w sodium carbonate) was subsequently added. After shaking, the mixture was incubated at room temperature in a dark place for 2 h. The absorbance was measured at 765 nm using a Geneva spectrophotometer (Rochester, NY). Different concentrations of gallic acid were used to construct the standard curve and final results were expressed as milligrams of gallic acid equivalents per gram of barley used in the extraction (mg GAE/g barley). 2.3.4. Total antioxidant activity Total antioxidant activity was measured by ferric reducing antioxidant power (FRAP) (Benzie & Strain, 1996) according to a modified methodology reported earlier (Alvarez et al., 2014; Sarkar et al., 2014). The working FRAP solution was prepared by mixing buffer acetate (pH 3.6), 10 mM of 2, 4, 6-tripyridyl-s-triazine solution and 20 mM ferric chloride solutions in the ratio 10:1:1. Then, 3 mL of the working FRAP reagent was added to 0.1 mL of the sample and incubated at 37 °C for 30 min. Absorbance was read at 593 nm. As standard, a solution of FeSO4·7H2O (0.1 M) was used. Different concentrations of this solution were used for the calibration curve. Results were expressed in μmol of FeSO4·7H2O per gram of barley. 2.3.5. Brown color intensity A simple way to monitor the formation of sugar degradation compounds as a consequence of the non-enzymatic Maillard reactions is to measure the brown color intensity of the pressurized liquid extracts (Alvarez et al., 2014; Sarkar et al., 2014). To quantify the intermediates and final products of the browning reactions intensity, the absorbance of the extracts was measured at 294 and 420 nm (Genova spectrophotometer, Rochester, NY), respectively, using a 1.5 mL plastic cuvette. If required, samples were diluted. Results were calculated according to Eq. (2), reported by Smith, Hole, and Hanson (1990): AbsorbanceðmL=gÞ Absorbance of the extract  Volume of the extractðmLÞ : ¼ Weigth of the initial sampleðgÞ

ð2Þ

2.3.6. Sugar degradation compounds Sugars (glucose, arabinose and xylose), sugar degradation compounds (5-hydroxymethyl furfural and furfural) and organic acids (lactic, acrylic and formic acids) formed during the extraction process were determined by HPLC. The chromatography system consists of a gradient pump 1525 (Waters, Milford, MA), an automatic injector 717 (Waters, Milford, MA), a refractive index detector 2414 (Waters, Milford, MA) and an UV/Visible Detector 2489 (Waters, Milford, MA). The column and guard column were purchased from Shodex (SH-1011 and guard column SH-G; Showa Denko Europe Gmbh; Munich, Germany) and set at 60 °C. A solution of H2SO4 0.01 N was used as mobile phase and set at 0.8 mL·min−1. 2.3.7. Molecular weight Molecular weight of the β-glucan was determined by Gel Permeation Chromatography (GPC) using a chromatography system that consists of an isocratic pump 1515 (Waters, Milford, MA), an automatic injector 717 (Waters, Milford, MA), a guard column (Ultrahydrogel Guard Colum, Waters, Milford, MA), a GPC column (Ultrahydrogel 500, Waters, Milford, MA) and a differential refractive index detector 410 (Waters, Milford, MA). The column was kept at 35 °C, and flow rate of the mobile phase (0.1 M NaNO3 + 0.02% NaN3) was set at 0.4 mL·min−1. Five different MW β-glucan standards from Megazyme (Wicklow, Ireland) in the range of 40–359 kDa were dissolved in ultrapure water and used to obtain the calibration curve. 2.3.8. Identification and quantification of individual phenolics A solution of 1 mL of the extracted sample with 1 mL of anhydrous ethanol was very well mixed in a vortex for 5 min. Then, the solution was centrifuged at 7000 rpm for 10 min, producing a supernatant used for the analysis. HPLC analysis was performed using a Varian Prostar HPLC system (Palo Alto, CA) equipped with a 401 model autosampler, pumps, and a UV model 1305 detector. The column used was Luna RP-18 (150 mm × 4.6 mm i.d. × 5 μm) with a Phenomenex security guard column C18 (4 mm × 3 mm) (Irvine, CA). Identification of individual phenolics was performed using a slightly modified HPLC methodology reported in Singh and Saldaña (2011). The error of the analysis was lower than 1.5%. The mobile phase consisted of: (A) formic acid (0.5%; v/v) in water, and (B) formic acid (0.5%, v/v) in methanol. The elution profile consisted of an eight step linear gradient using formic acid 0.5% in water and methanol, rising from 16 to 19% of (B) in 15 min, 19 to 27% of (B) in 25 min, 27 to 41% of (B) in 26 min, followed by further increase to 65% of (B) in 36 min to 100% of (B) in 44 min and then back to the initial concentration in 45 min. The total run time was 50 min and the volume injected was 10 μL. The flow rate was 1 mL/min with detection at 280 nm. The phenolic compounds, including six hydroxybenzoic acids (Gal (Gallic Acid), Pro (Protocatechuic Acid), p-Hyd (p-Hydroxybenzoic Acid), Gen (Genistic Acid), Van (Vanillic Acid), and Syr (Syringic Acid)), seven hydroxycinnamic acids (Caf (Caffeic Acid), Chl (Chlorogenic Acid), pCou (p-Coumaric Acid), Fer (Ferulic Acid), Sin (Sinapinic Acid), 2Hyd (2Hydroxycinnamic Acid), and Cin (Cinnamic Acid)), one ferulic acid derivative (EF (Ethyl Ferulate)), one flavan (Cat (Catechin)), and four flavonols (Nar (Naringinin), Mor (Morin), Rut (Rutin),and Que (Quercetin)) were used as standards. 2.4. Experimental design Box Behnken experimental design was used in this study where three factors at three levels were considered: X1 temperature (135, 155 and 175 °C), X2 extraction time (15, 35 and 55 min) and X3 ethanol content in the solvent (5, 12.5 and 20%). The experimental plan consisted of 17 runs, including five repetitions of the central point of the experimental design, in order to evaluate the reproducibility of the experimental results and warrant the reliability of the results. All the experiments were randomized. From all the

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response parameters analyzed, in the optimization process, the maximization of the extraction yield (Eq. (1)) and FRAP antioxidant activity were primarily considered. Besides the maximization of those two parameters, the minimization of the absorbance measured at 420 nm was considered, to avoid extraction conditions that led to the formation of brown colored compounds that would indicate the degradation of the extracted β-glucan. All the calculations were done by means of Statgraphics Centurion Statistical Software. A generalized second-order polynomial model, as shown in Eq. (3), was used to fit the experimental results. Y ¼ β0 þ

Xk j¼1

β jX j þ

Xk j¼1

β j j X 2j þ

Xk Xk i¼1

β XX j¼1 i j i j

ð3Þ

In that polynomial model, Y is the response variable to be optimized, β0, βj, βjj and βij are the regression coefficients for intercept, linearity, square and interaction, respectively; Xj is the coded independent factor and the terms XiXj and X2j represent the interaction and quadratic terms, respectively. An analysis of variance (ANOVA) with 95% confidence level was done for each response variable to test the model significance and suitability. The significance of each factor was determined using the F-value test, at a 95% confidence level and reported as the p-value. To evaluate the effect that the addition of ethanol had on the extraction of β-glucan and phenolic compounds, blank experiments were run at the extraction conditions of the central conditions of the experimental design (155 °C and 35 min) using only pressurized hot water as a solvent. 3. Results and discussion 3.1. Proximate compositional analysis The proximate compositional analysis of the barley used in this study is shown in Table 1. 3.2. Pressurized hot aqueous-ethanol extraction of barley 3.2.1. β-Glucan extraction yield and fragmentation products Table 2 shows the experimental design used in this study, as well as the extraction results of β-glucan yield, total phenolics, total antioxidant activity, total solid content, final pH, absorbance readings at 294 and 420 nm in the liquid extracts, and β-glucan molecular weight. βglucans were successfully removed using PHAE with extraction yields in the range of 34.7–80.3% at 135–175 °C and extraction times up to 55 min. The mixture ethanol–water at high temperatures under pressure was a suitable solvent to weaken the hydrogen bonds between β-glucans and barley matrix, leading to their release. Previously, PHW was used for the extraction of β-glucans from barley within a batch system (Benito-Román, Alonso, & Cocero, 2013) where the best extraction conditions (155 °C and 18 min) led to an extraction yield of 53.7% and a molecular weight of 200 kDa. Fig. 2 presents the main effect diagram, showing the effect of temperature, time and ethanol content on the β-glucan extraction yield. Temperature had a dramatic effect on the extraction yield. The highest extraction yield was achieved at the lowest temperature studied (135 °C), with an extraction yield above 70%. When temperature increased up to 155 °C, a slight decrease in the extraction yield was Table 1 Proximate compositional analysis. Component

Units

Value

Moisture β-Glucan Starch Protein Ash Total phenolics

% % % % % mg GAE/g barley

9.33 ± 0.01 9.75 ± 0.17 48.70 ± 0.11 16.32 ± 0.07 1.90 ± 0.01 9.28 ± 0.60

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observed, and further an increase up to 175 °C, reduced dramatically the extraction yield to below 45%. This reduction on the β-glucan extraction yield can be explained by the fact that β-glucans exposed to high temperatures are depolymerized in agreement with findings reported previously by Benito-Román, Alonso, Gairola, et al. (2013). To support our findings, analyses of the liquid extracts were performed, revealing the presence of glucose (β-glucan constituent monomer) and in some samples 5-hydroxymethylfurfural (5-HMF, a glucose degradation product) as shown in Table 2. Analysis of variance (ANOVA) showed that temperature and extraction time were statistically significant parameters affecting β-glucan extraction yield. The ethanol content in the mixture was not statistically significant, and no significant interactions between the parameters were observed. Extraction time had a significant effect on the amount of β-glucan removed, showing a slight increase on the extraction yield when time increased (Fig. 2), contrarily to the findings of BenitoRomán, Alonso, and Cocero (2013). These authors found a dramatic reduction on the extraction yield in experiments longer than 45 min. The main difference between these two studies is the presence of ethanol in the solvent mixture and the use of a mechanical stirrer for mixing. Ethanol content in the solvent mixture did not have any effect on the extraction of β-glucans as no significant differences were observed in the extraction yield when ethanol content varied from 5 to 20%, being possible to obtain relatively high extraction yields, around 40%, even at high ethanol contents (20%) and high temperatures (175 °C), according to the results reported in Table 2. Buranov and Mazza (2012) reported much lower glucan recovery yields (b 25%) with aqueous solutions that contain up to 20% ethanol at 180 °C. In our study, 80% of β-glucan was obtained at 135 °C with 12.5% ethanol for 55 min. Therefore, there is a need to determine the role that low concentrations of ethanol play in the molecular weight of β-glucans extracted. In Fig. 3, contour plot diagrams for β-glucan extraction yield and its molecular weight against two operational parameters are presented, where a maximum β-glucan recovery of 80% was obtained after 55 min. The extraction of β-glucan is higher at temperatures lower than 155 °C and over 55 min of extraction (Fig. 3a). Interestingly, the increase of ethanol content increases the molecular weight (MW) of the β-glucan extracted (Fig. 3b). Temperatures over 155 °C and extraction times over 35 min decrease the MW of the extracted β-glucan due to its thermo-catalytic fragmentation. When milder conditions of temperature were used, MW of the β-glucans was above 300 kDa. In some cases, when temperature was 155 °C and ethanol content 20%, the MW of the extracted β-glucans was above the exclusion limit of the used GPC column (N500 kDa) and hence are not reported. It is recommended for future studies to perform this analysis using a GPC column with a wider operation range up to 1000 kDa and β-glucan standards that are above the 500 kDa molecular weight. A previous study using only PHW extraction (155 °C, 20 bar, 4 g/min) obtained lower yields of β-glucans with molecular weights of up to 500 kDa (Benito-Román, Alonso, Gairola, et al., 2013). In our study, a higher ethanol concentration in the mixture led to higher β-glucan MW. Ethanol has been traditionally used to deactivate endogenous β-glucanases (Irakli, Biliaderis, Izydorczyk, & Papadoyannis, 2004). Moreover, Benito-Román, Alonso, and Cocero (2013) reported that high temperatures also tend to deactivate endogenous β-glucanases, as these authors reported molecular weight of the β-glucan up to 200 kDa. Then, the use of HPAE produced β-glucan with higher molecular weight, deactivating β-glucanases when extraction time is controlled. According to this result, HPAE is a suitable solvent for the high MW β-glucan recovery from waxy barley. Fig. 1Sa in the supporting information shows an increase of the absorbance values at 420 nm with the increase of temperature and time, but a decrease of the absorbance values at 420 nm with the increase of ethanol content (Fig. 1Sb). The higher absorbances at 420 nm were observed at 175 °C and when the extraction time was longer than 35 min. The conditions of higher temperature and extraction time allow the fragmentation of sugars and protein, leading to compounds,

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Table 2 RSM-Box Behnken experimental design and main results. Run

Processing conditions T

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Mean Blank

t

Ethanol

(°C)

(min)

(%)

135 175 135 175 135 175 135 175 155 155 155 155 155 155 155 155 155 155 155

15 15 55 55 35 35 35 35 15 55 15 55 35 35 35 35 35 35 35

12.5 12.5 12.5 12.5 5 5 20 20 5 5 20 20 12.5 12.5 12.5 12.5 12.5 12.5 0

Liquid extracts εr

β-Glucan

TP-F

FRAP

Absorbance (g/g barley)

(%)

(mg GAE/g)

(μmol FS/g)

294 nm

420 nm

(%)

42 34 42 34 45 37 39 31 41 41 35 35 38 38 38 38 38 38 43

62.3 40.6 80.3 47.0 71.2 44.4 73.2 34.7 64.3 69.6 61.9 65.8 66.3 64.3 63.7 67.8 68.2 66.1 81.1

2.84 5.51 3.62 10.71 3.50 9.92 4.69 9.30 2.65 5.91 3.37 4.98 3.16 3.2 3.39 3.35 3.97 3.45 4.12

26.43 55.27 35.59 111.58 34.00 101.84 41.36 98.81 32.72 55.07 50.57 52.58 38.95 37.17 33.06 41.38 42.30 38.60 79.01

4.27 21.80 6.40 21.6 7.63 23.37 5.37 14.80 8.93 26.67 11.37 16.10 13.27 13.03 12.77 13.1 13.37 13.1 23.2

4.80 18.03 2.53 45.67 12.33 60.47 3.80 24.93 8.60 41.57 8.43 8.50 6.6 6.87 8.13 7.67 7.57 7.4 29.8

1.71 2.04 2.15 2.24 2.17 2.31 2.13 2.26 2.05 2.15 1.87 2.21 2.03 2.05 2.09 2.08 2.04 2.1 2.1

Solids

pH

5.45 5.31 5.54 4.52 5.51 4.58 5.37 5.09 5.5 5.22 5.58 5.46 5.55 5.54 5.52 5.49 5.48 5.5 5.6

MW

Glucose

X-A

HMF

Furfural

(kDa)

(ppm)

(ppm)

(ppm)

(ppm)

451 266 346 27 221 45 482 57 511 100 N N 500 N N 500 298 351 387 365 333 347 89

122.2 336.1 178.0 336.3 143.6 437.4 215.3 89.9 208.3 340.4 367.3 331.7 295.2 340.9 306.2 328.3 321.3 318.4 0

241.3 517.0 369.4 702.4 324.8 1020.3 319.0 1084.9 368.0 449.2 424.4 918.3 610.2 711.2 675.6 484.4 748.7 646.0 0

0 6.5 0 42.1 0 29.4 0 10.5 0.9 5.2 0.7 4.4 2.3 1.6 1.6 1.8 3 2.1 3.9

0 0 0 3.3 0 3.6 0 0.2 0 0 0 0 0 0 0 0 0 0 0

T: Temperature, t: time, εr: relative permittivity, TP-F: total phenolics by Folin-Ciocalteu method, FRAP: ferric reducing antioxidant power, X-A: xylose + arabinose, HMF: 5hydroxymethylfurfural. Mean: Average values for the central points (experiments 13–17) of the experimental design.

such as HMF (hexoses degradation product), and furfural (pentoses degradation product) as reported in Table 2. Both provided colored solutions and are, among other compounds, responsible for the brown color of the final liquid extracts. Table 2 also shows the presence of sugars in the extracts: glucose (as the monomer of polysaccharides, such as starch, β-glucan and cellulose) and arabinose + xylose (monomers of the hemicellulose arabinoxylan). According to Izydorczyk and Dexter (2008), arabinose + xylose is usually found in barley in concentrations up to 5.8%. These compounds were difficult to quantify as their peaks overlapped. These compounds were also reported when βglucans were extracted from barley using water at 40 °C for 30 min (Izydorczyk, Macri, & MacGregor, 1998). Under these conditions, the liquid extract had 1.4% of solid material (82.5% were β-glucans and 15.9% were arabinoxylans). Fig. 1Sc shows that the increase of temperature and ethanol content increases the amount of xylose–arabinose (aldopentose) extracted, indicating the depolymerization of hemicellulose. HMF and furfural were only present in some extracts and organic acids, such as acrylic, acetic or formic acids, were not detected in this study. Samples obtained after more severe extraction conditions (higher temperatures and longer extraction times) had more HMF (a maximum of 42 ppm) and furfural (3.6 ppm). In the case of HMF, it

was observed that its concentration increases with the increase of temperature and extraction time. Nevertheless, the ethanol content had no significant impact, ethanol content contributed to less formation of HMF (Fig. 1Sd). This is consistent with the fact that, at constant temperature, the higher ethanol content, the less glucose was detected, the less βglucan was extracted and the higher the molecular weight of the βglucan extracted. Ethanol helped to preserve the native molecular weight of the β-glucan, preventing its depolymerization and the formation of the monomer degradation products. Furfural at 3 ppm level was only detected when the working temperature was 175 °C. According to these results, the operational range selected in terms of temperature and time is suitable for the extraction of β-glucans, as in the liquid extracts only the monomers that form the main polysaccharides are found (glucose, xylose and arabinose), no organic acids are formed and only in the most severe conditions led to degradation products of hexoses and pentoses at ppm level (HMF and furfural, respectively). The blank center point from Table 2 shows that the addition of 20% ethanol to the extraction solvent reduces the amount of β-glucan extracted from 81.1 during 35 min to 65.8% during 55 min but in turn increases dramatically the molecular weight of β-glucans extracted from 89 to N500 kDa, indicating an efficient extraction.

Fig. 2. Main effects diagram for β-glucan extraction yield.

3.2.2. Phenolic compounds and antioxidant activity The amount of total phenolic compounds measured by the Folin– Ciocalteu method (TP-F) is higher than that measured by the HPLC method (TP-HPLC). This difference (dTP) is due to expected additive effects if quetones, aldehydes or enols are present in the HPAE extracts (Alvarez et al., 2014). Comparing the total phenolics obtained in this study (run 4 in Table 2, at 175 °C, 25 bar and 12.5% ethanol) against our previous study using barley hulls at 160 °C, 150 bar and 12% ethanol (Sarkar et al., 2014), the values from the HPLC method are similar but not the values from the Folin–Ciocalteu method. For the TP-HPLC, the values of this study and those from the previous study (Sarkar et al., 2014) are 3.3 mg/g barley and 3.4 mg phenolic/g barley hull, respectively, in agreement with 3.1 mg/g waxy barley analyzed by HPLC (GómezCaravaca, Verardo, Berardinelli, Marconi, & Caboni, 2014). For the TP-F, the values of this study and the previous one are 10.7 mg/g barley and 40.4 mg phenolic/g barley hull, respectively. The high values of the TPF in the study of Sarkar et al. (2014) are expected due to the higher pressure used that increased the yield of browning products and its additive

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(a) 55

45

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60.5

35

54.0

47.6

66.9

25

Fig. 4. Total phenolics, β-glucan and HMF against FRAP values.

15 135

145

155

165

175

(b) 20

447.5

15 361.5

189.6 103.6

10 275.6

5 135

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155

165

175

The dTP is due to the interference of oxidizable compounds, such as HMF and furfural. Against values of dTP, the absorbance at 420 nm and amount of HMF show a positive correlation but the pH shows a negative correlation (Fig. 2S). The pH affects the concentration of browning compounds, while the concentration of phenolics remains the same. A decreasing pH increases oxidation–reduction reactions because the oxidative power of free radicals increases. Also, longer times and higher temperatures increase the extraction of phenolics and the production of browning compounds (Fig. 1Sa). Our data show that temperature and the interaction temperature–time are significant factors at any content of ethanol for the high antioxidant activity and phenolics content in the extracts. Fig. 5 shows the main effects diagram for FRAP antioxidant. When temperature increased above 155 °C, a dramatic increase in the antioxidant activity of the liquid extract was observed. This result is in agreement with the results published earlier by Sarkar et al. (2014). Also, a change in the extract color was observed where the higher the temperature the darker the color (Singh & Saldaña, 2011). Therefore, the dark color is due to the presence of browning and Maillard reactions that influence the colorimetric analysis. Opposite trends for the FRAP antioxidant activity/total phenolics content compared to the β-glucan extraction yield were observed in Fig. 4. Fig. 6a shows a schematic drawing of extraction from waxy barley using PHAE where breaking of the crosslinked esther bonds to polysaccharides releases gallic and ferulic acids. High temperatures and ethanol content are needed to break the ether bonds of phenolics to lignin.

Fig. 3. β-Glucan extraction yield as a function of temperature and time at 12.5% ethanol (a). β-glucan molecular weight as a function of temperature and ethanol content for 35 min of extraction (b).

interferences to the Folin–Ciocalteu method. Moreover, similar values of total phenolics from the HPLC method indicate that the source of browning compounds is not the extracted phenolics but the carbohydrates and proteins present in barley. The amount of TP-HPLC and HMF shows a positive correlation against the FRAP values but the amount of β-glucan shows a negative correlation against the FRAP values (Fig. 4). This behavior can be explained as the use of high temperature facilitates the extraction of antioxidants but fragments the extracted β-glucan. There is a correlation between total phenolics and the brown color intensity (Fig. 4), but color formation cannot be explained only by this phenomena. From Fig. 4, brown color products from Maillard reactions have high antioxidant activity or additive interferences to the FRAP method (Alvarez et al., 2014; Singh & Saldaña, 2011).

Fig. 5. Main effects diagram for antioxidant activity (FRAP) and total phenolics extraction.

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(a)

(b)

Fig. 6. PHAE extraction of waxy barley (Adapted from Alvarez et al. [16]) (a). Concentration profile of antioxidants found in HPAE extracts of waxy barley (b).

Fig. 6b shows the concentration profiles of the different types of antioxidants studied. HPLC results show that three antioxidants were found in high amounts (gallic acid, ferulic acid, and catechin). Temperature is the main factor for the extraction of phenolics followed by its interactions with time and ethanol. The total amount of hydroxybenzoic acids (mainly gallic and syringic acids) was higher than that of hydroxycinnamic acids (mainly ferulic and p-coumaric acids), probably due to the recalcitrant bonds between hydroxycinnamic acids and lignin. Flavonoids are also extracted at 175 °C, with 12.5% of ethanol during 55 min with a higher amount of flavans than that of flavonols. Ethyl ferulate was extracted with high ethanolic content solutions (N 12.5%), indicating that ethanol content had a significant effect on the extraction. Then, the extraction efficiency of the HPAE process is related to the structure of the antioxidant or its type of interaction with other biomass components like proteins. The treatment of lignocellulosic biomass with PHF can originate high amounts of gallic acid due to the solubilization of hemicellulose and proteins (Alvarez et al., 2014; Singh & Saldaña, 2011). Moreover, the use of ethanol breaks the bonds of lignin-phenolics, facilitating lignin depolymerization, as shown in the release of ferulic acid and ethyl ferulate. The comparison between the center point of the factorial design at 155 °C for 35 min with and without (blank experiment) ethanol showed that the addition of ethanol reduced the amount of extracted gallic acid but extracted syringic acid, chlorogenic acid, naringenin, and ethyl ferulate. These results indicate that ethanol enhanced the solvent properties of PHW to extract wider families of antioxidant components. The

blank center point has lower values for TP-HPLC, and higher values for, TP-F and HMF contents, indicating that ethanol decreased the browning compounds, minimizing fragmentation reactions. Previous studies on commercial β-glucan samples of barley balance (25% β-glucan) and barley fibre rich fraction (28% β-glucan) obtained 0.8 and 1.0 mg GAE/g sample, respectively, using 70% ethanol in aqueous solution (Thondre, Ryan, & Henry, 2011). These results indicate that conventional extractions at room pressure should be improved by the use of HPAE methods to achieve high β-glucan (47.0%) and phenolics (10.7 mg GAE/g extract) (Table 2). The addition of ethanol in the HPF process should be considered if the target is the preservation of the molecular weight of the extracted β-glucan as there are various studies that suggest that high molecular weight of β−glucans is beneficial for human health. The obtained extracts are rich in high molecular weight β-glucans and in phenolic compounds. This process is therefore attractive to generate food additives/ ingredients to functional foods. Also, PHAE contains additional ionic species from ethanol in the solution with lower relative permittivity that allows the depolymerization of hemicelluloses and partially lignin, producing higher amount of phenolics than that obtained using only pressurized water. The relative permittivity of water at 25, 135, 155 and 175 °C is approximately 80, 47, 43 and 39, respectively, a reduction of about 50% (Dannhauser & Bahe, 1964). The relative permittivity of ethanol at 25, 135, and 175 °C is approximately 25, 11 and 8, respectively, a reduction of about 68% (Dannhauser & Bahe, 1964). The relative

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permittivity for solutions with 5 to 20% ethanol at temperatures from 135 to 175 °C was calculated by the equation provided by Fakhree, Delgado, Martínez, and Jouyban (2010) and reported in Table 2. The relative permittivity and the time were used as factors to design contour plots for the TP-HPLC and FRAP responses (Fig. 3S). The low relative permittivity, a significant factor, drives the extraction of phenolics (Fig. 3Sa,b) while high values of relative permittivity increased the extraction of β-glucan (Fig. 3Sc). Relative permittivity of phenolics (lignin, ~4) and polysaccharides (starch, ~20) indicates the degree of dissolution by the solvent with low and high relative permittivity for phenolics and polysaccharides, respectively (Sarkar et al., 2014). However, the relative permittivity is not a significant factor for the release of solids (Fig. 3Sd). Temperatures over 155 °C released high amounts of solids (Table 2), showing that temperature influences the mass transfer of the extraction mainly by the decrease of the viscosity and increase of the sublimation pressure of the solids found in barley. To find the conditions that maximized the β-glucan extracted with high molecular weight, specific phenolic compounds extracted and minimized the brown color in the liquid extract, the statistical software Statgraphics was used. The processing conditions were 151 °C, 21 min and 16.8% of ethanol in the solvent mixture, and β-glucan extraction yield was 51% and phenolics content was 5 mg GAE/g barley. Under these working conditions, a molecular weight of the β-glucan in the range of 500–600 kDa is expected, based on the experimental results obtained in this study. 4. Conclusions In this study, mixtures of ethanol–water (5–20% v/v) were used at high temperatures (135–175 °C) to extract β-glucan and phenolic compounds, in relatively short extraction times (15–55 min). This new approach allowed obtaining concentrated solutions rich in βglucan and antioxidants to be used as food additives/ingredients. Based on our findings, a careful selection of the temperature and ethanol content must be done, since β-glucans and phenolic compounds behave in a completely opposite trend during the extraction. High temperatures and long extraction times (N35 min) result in low concentrations of βglucan and high phenolic content in the liquid extract. High temperatures tend to degrade the β-glucan, indicated by the low extraction yields with low molecular weight and the presence of sugar degradation compounds like HMF. However, mixtures of pressurized aqueous ethanol at high temperatures were useful to extract hydroxycinnamic acid, that are strongly bounded to lignin. The amount of ethanol used in this study was not statistically significant, as did not affect the extraction of any of the target compounds but decreased the fragmentation of βglucan. To maximize both β-glucan extraction yield and phenolic content, it is necessary to work at mild conditions of 151 °C, 21 min and 16.8% of ethanol in the mixture in the range of process parameters evaluated in this study. Acknowledgments We are grateful to the Natural Sciences and Engineering Research Council of Canada (NSERC #05356) for funding this project, and University of Valladolid for the economic support to Benito-Román during his stay at the University of Alberta, in the frame of “Plan de Ayudas para la Movilidad del Personal Investigador 2013”. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.foodres.2015.06.006.

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